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1.  BIN1 Localizes the L-Type Calcium Channel to Cardiac T-Tubules 
PLoS Biology  2010;8(2):e1000312.
Cardiac tubular-like membrane invaginations contain the membrane scaffolding protein BIN1, which tethers dynamic microtubules that deliver calcium channels directly to T-tubule membrane.
The BAR domain protein superfamily is involved in membrane invagination and endocytosis, but its role in organizing membrane proteins has not been explored. In particular, the membrane scaffolding protein BIN1 functions to initiate T-tubule genesis in skeletal muscle cells. Constitutive knockdown of BIN1 in mice is perinatal lethal, which is associated with an induced dilated hypertrophic cardiomyopathy. However, the functional role of BIN1 in cardiomyocytes is not known. An important function of cardiac T-tubules is to allow L-type calcium channels (Cav1.2) to be in close proximity to sarcoplasmic reticulum-based ryanodine receptors to initiate the intracellular calcium transient. Efficient excitation-contraction (EC) coupling and normal cardiac contractility depend upon Cav1.2 localization to T-tubules. We hypothesized that BIN1 not only exists at cardiac T-tubules, but it also localizes Cav1.2 to these membrane structures. We report that BIN1 localizes to cardiac T-tubules and clusters there with Cav1.2. Studies involve freshly acquired human and mouse adult cardiomyocytes using complementary immunocytochemistry, electron microscopy with dual immunogold labeling, and co-immunoprecipitation. Furthermore, we use surface biotinylation and live cell confocal and total internal fluorescence microscopy imaging in cardiomyocytes and cell lines to explore delivery of Cav1.2 to BIN1 structures. We find visually and quantitatively that dynamic microtubules are tethered to membrane scaffolded by BIN1, allowing targeted delivery of Cav1.2 from the microtubules to the associated membrane. Since Cav1.2 delivery to BIN1 occurs in reductionist non-myocyte cell lines, we find that other myocyte-specific structures are not essential and there is an intrinsic relationship between microtubule-based Cav1.2 delivery and its BIN1 scaffold. In differentiated mouse cardiomyocytes, knockdown of BIN1 reduces surface Cav1.2 and delays development of the calcium transient, indicating that Cav1.2 targeting to BIN1 is functionally important to cardiac calcium signaling. We have identified that membrane-associated BIN1 not only induces membrane curvature but can direct specific antegrade delivery of microtubule-transported membrane proteins. Furthermore, this paradigm provides a microtubule and BIN1-dependent mechanism of Cav1.2 delivery to T-tubules. This novel Cav1.2 trafficking pathway should serve as an important regulatory aspect of EC coupling, affecting cardiac contractility in mammalian hearts.
Author Summary
Calcium plays a primary role in regulating heart function. During each heartbeat, calcium ions cross the membrane of individual cardiac muscle cells and trigger a rapid increase of calcium within the cell (called the calcium transient). Calcium causes the muscle cells to contract and determines the strength of the overall heartbeat. Each cardiac muscle cell has many small tubular-like membrane invaginations known as T-tubules where calcium channels localize, allowing calcium ions to enter and immediately encounter intracellular calcium release organelles. While this organization is well described, it is not known how calcium channels localize to T-tubule membrane. Here we show that in human and mouse heart cells, a membrane scaffolding protein known as BIN1 is localized together with calcium channels at T-tubules. Using high-resolution live cell microscopy, we found that microtubules, which are necessary for calcium channel delivery to the membrane, are also tethered by BIN1. Loss of BIN1 in cardiac cells impairs delivery of calcium channels to the membrane and diminishes the intracellular calcium transient. According to this model, microtubules function as highways that carry newly synthesized calcium channels to BIN1-containing membrane. Once tethered to T-tubules by BIN1, the microtubules can deliver their calcium channel cargo. We postulate that this calcium channel delivery pathway is important to the regulation of cardiac calcium signaling and beat-to-beat cardiac function.
doi:10.1371/journal.pbio.1000312
PMCID: PMC2821894  PMID: 20169111
2.  Economics of membrane occupancy and respiro-fermentation 
The authors propose that prokaryotic metabolism is fundamentally constrained by the cytoplasmic membrane surface area available for protein expression, and show that this constraint can explain previously puzzling physiological phenomena, including respiro-fermentation.
We propose that prokaryotic cellular metabolism is fundamentally constrained by the finite cytoplasmic membrane surface area available for protein expression.A metabolic model of Escherichia coli updated to include a cytoplasmic membrane constraint is capable of predicting a variety of puzzling phenomena in this organism, including the respiro-fermentation phenomenon.Because the surface area to volume ratio is directly related to the morphology of the cell, this constraint provides a direct link between prokaryotic morphology and physiology.The potential relevance of this constraint to eukaryotes is discussed.
Many heterotrophs can produce ATP through both respiratory and fermentative pathways, allowing them to survive with or without oxygen. Since the molar ATP yield (molar ATP yield: mole of ATP produced/mole of substrate consumed) from respiration is about 15-fold higher than that from fermentation, ATP production via respiration is more efficient. Surprisingly, at high catabolic rate, many facultative aerobic organisms employ fermentative pathways simultaneously with respiration, even in the presence of abundant oxygen to produce ATP (Pfeiffer et al, 2001; Vemuri et al, 2006; Molenaar et al, 2009). This leads to an observable tradeoff between the ATP yield and the catabolic rate (Pfeiffer et al, 2001; Vemuri et al, 2006). This respiro-fermentation physiology is commonly observed in microorganisms, including Escherichia coli, Bacillus subtilis, Saccharomyces cerevisiae (Molenaar et al, 2009), as well as cancer cells (Vander Heiden et al, 2009). Despite extensive research, existing theories (Majewski and Domach, 1990; Varma and Palsson, 1994; Pfeiffer et al, 2001; Vazquez et al, 2008; Molenaar et al, 2009) cannot fully explain the respiro-fermentation phenomenon.
The membrane economics theory
We propose the hypothesis that the prokaryotic metabolism is fundamentally constrained by the finite cytoplasmic surface area available for protein expression—in order to maximize fitness, prokaryotic organisms such as E. coli must economically manage the expression of membrane proteins based on the membrane cost and the fitness benefit of the proteins. This hypothesis is proposed based on theoretical considerations (in this work), numerical analysis (Phillips and Milo, 2009), and experimental observation that the overexpression of non-respiratory membrane protein significantly reduces the oxygen consumption rate and induces aerobic fermentation (Wagner et al, 2007). Such a constraint on transmembrane protein expression may have significant physiological consequences in prokaryotes, such as E. coli, at higher catabolic rates. First, since both substrate transporters and respiratory enzymes are localized on the cytoplasmic membrane in prokaryotes, increased substrate uptake rates necessitates a decrease in the respiratory rate. This decrease in the respiratory rate, forces prokaryotes to process the additional substrate through the fermentative pathways, which are not catalyzed by transmembrane proteins, for continued ATP production. Furthermore, since the membrane requirement of an enzyme is inversely related to its turnover rate (see Materials and methods section in the manuscript), the faster and inefficient respiratory enzymes (such as Cyd-I and Cyd-II in E. coli) might be preferred over the slower and efficient enzymes (such as Cyo in E. coli), leading to an altered respiratory stoichiometry at higher catabolic rates. Finally, the absence of the respiratory enzymes under anaerobic conditions explains why the maximum glucose uptake rate (GUR) of E. coli is much higher.
Applying membrane economics theory to E. coli
To illustrate that the ‘membrane economics' theory could satisfactorily explain the physiological changes associated with the respiro-fermentation phenomenon in E. coli, we modified the genome-scale metabolic model of E. coli (Feist et al, 2007) to include a cytoplasmic membrane occupancy constraint. Using ‘relative membrane costs' calculated from experimental data, the new modeling framework—FBA with membrane economics (FBAME)—predicted that wild-type E. coli has a GUR of 10.7 mmol/gdw/h, an oxygen uptake rate (OUR) of 15.8 mmol/gdw/h, and a specific growth rate of 0.69 per hour during aerobic growth with excess glucose. FBAME also predicted that under the same growth condition, an E. coli knockout strain with no cytochromes has a GUR of 18 mmol/gdw/h and growth rate of 0.42. These values agree very well with the reported experimental values for E. coli grown in batch cultures (Vemuri et al, 2006; Portnoy et al, 2008), which supports our hypothesis that the higher GUR of E. coli during glucose-excess anaerobiosis than under aerobic conditions is due to the absence of the respiratory enzymes. We also simulated the aerobic growth of E. coli in glucose-limited chemostat using both conventional FBA and FBAME. FBAME successfully predicted the growth rate and yield changes with respect to increasing GUR (Figure 2A and B), as well as the aerobic production of acetate (Figure 2C) and concomitant repression of oxygen uptake (Figure 2D). On the other hand, traditional FBA significantly overestimated the growth rate and yield at higher GURs (this overestimation cannot be explained by varying the growth-associated maintenance (GAM) energy parameter; Figure 2A), and failed to predict the decrease in yield independent of acetate overflow and reduction in oxygen uptake at higher GURs (Figure 2). In addition, FBAME was able to predict the reduction of the TCA cycle activities at higher uptake rates (Figure 3C and D) as well as the selective expression of Cyo and Cyd-II at lower uptake rates (Figure 3A and B), whereas conventional FBA cannot predict the expression of inefficient Cyd-II. These predictions agree with the gene expression data from glucose-limited chemostat (Figure 3). Given the simplicity of the constraint we imposed, our model predictions agree surprisingly well with experimental observations, lending strong credibility to the membrane economics hypothesis.
Concluding remarks
Although it has been long suggested that cellular evolution are governed by non-adjustable mechanistic constraints (Palsson, 2000; Papin et al, 2005; Novak et al, 2006), to date, most metabolic models rely on empirically derived parameters such as glucose and OUR. In this article, we showed that complex phenomena, such as the respiro-fermentation in E. coli, could be satisfactorily explained and accurately predicted by using constraint-based optimization by introducing a simple mechanistic constraint on membrane enzyme occupancy. Given that the cytoplasmic membrane occupancy constraint is directly related to the surface area to volume (S/V) ratio of the cell, it is possible that this constraint resulted in the evolution of mitochondria in eukaryotes as mitochondria allows for a significantly increased S/V ratio. Further efforts to elucidate such fundamental cellular constraints as well as the underlying design principles could significantly improve our understanding of the regulation and evolution of metabolism.
The simultaneous utilization of efficient respiration and inefficient fermentation even in the presence of abundant oxygen is a puzzling phenomenon commonly observed in bacteria, yeasts, and cancer cells. Despite extensive research, the biochemical basis for this phenomenon remains obscure. We hypothesize that the outcome of a competition for membrane space between glucose transporters and respiratory chain (which we refer to as economics of membrane occupancy) proteins influences respiration and fermentation. By incorporating a sole constraint based on this concept in the genome-scale metabolic model of Escherichia coli, we were able to simulate respiro-fermentation. Further analysis of the impact of this constraint revealed differential utilization of the cytochromes and faster glucose uptake under anaerobic conditions than under aerobic conditions. Based on these simulations, we propose that bacterial cells manage the composition of their cytoplasmic membrane to maintain optimal ATP production by switching between oxidative and substrate-level phosphorylation. These results suggest that the membrane occupancy constraint may be a fundamental governing constraint of cellular metabolism and physiology, and establishes a direct link between cell morphology and physiology.
doi:10.1038/msb.2011.34
PMCID: PMC3159977  PMID: 21694717
constraint-based modeling; flux balance analysis; membrane occupancy; overflow metabolism; respiro-fermentation
3.  Protein localization as a principal feature of the etiology and comorbidity of genetic diseases 
Proteins localized within the same subcellular compartment tend to be functionally associated. This study shows that subcellular localization and network distance between disease-associated proteins provide complementary information explaining patterns of disease comorbidity.
A positive correlation was found between subcellular localization of disease-associated protein pairs and measures of comorbidity.A higher comorbidity tendency was found for disease-associated protein pairs that are positioned within a shorter distance in the protein interaction network.The integration of subcellular localization information with protein interaction network sheds light onto the potential molecular connections underlying comorbidity patterns and will help to understand the mechanisms of human disease.
It was shown that the emergence of phenotypically similar diseases are triggered as a result of molecular connections between disease-causing genes (Oti and Brunner, 2007; Zaghloul and Katsanis, 2010). From a genetics, perspective diseases are associated with certain genes (Goh et al, 2007; Feldman et al, 2008), whereas from a proteomics perspective phenotypically similar diseases are connected via biological modules such as protein–protein interactions (PPIs) or molecular pathways (Lage et al, 2007; Jiang et al, 2008; Wu et al, 2008; Linghu et al, 2009; Suthram et al, 2010). These molecular connections between diseases were observed on the population level as well: diseases connected through molecular connections such as shared genes, PPIs, and metabolic pathways tend to show elevated comorbidity (Rzhetsky et al, 2007; Lee et al, 2008; Zhernakova et al, 2009; Park et al, 2009a, 2009b). While these findings constitute a step toward improving our understanding of the mechanism of disease progression, there are still many more molecule-level connections between disease pairs that need to be explored in order to establish a firmer comorbidity association.
Subcellular localization provides spatial information of proteins in the cell; proteins target subcellular localizations to interact with appropriate partners and form functional complexes in signaling pathways and metabolic processes (Au et al, 2007). Abnormal protein localizations are known to lead to the loss of functional effects in diseases (Luheshi et al, 2008; Laurila and Vihinen, 2009). For example, mis-localizations of nuclear/cytoplasmic transport have been detected in many types of carcinoma cells (Kau et al, 2004). A proper identification of protein subcellular localization can hence be useful in discovering disease-associated proteins (Giallourakis et al, 2005; Calvo and Mootha, 2010). With this understanding, we postulate that disease-associated proteins connected by subcellular localizations could also explain the phenotypic similarities between diseases. Furthermore, such connections may also couple to disease progressions that contribute to multiple disease manifestation, that is, comorbidity.
Protein subcellular localization has been extensively studied through various methods to determine a variety of protein functions. To the best of our knowledge, the connection between diseases and subcellular localizations are yet to be studied systematically. To resolve this we constructed, for the first time, a human Disease-associated Protein and subcellular Localization (DPL) matrix (top panel in Box 1). Our DPL matrix provides the ‘cellular localization map of diseases' that represents the spatial index of diseases in the cell. We found that each disease shows unique characteristics of subcellular localization profile in the DPL matrix. We were interested in determining whether subsets of 1284 human diseases exhibit distinct enrichment profiles across subcellular localizations. We calculated pairwise correlations and performed a hierarchical clustering of the enrichments of the 1284 diseases across 10 different subcellular localizations.
Our DPL matrix revealed that 778 diseases (∼62%, P=1.40 × 10−3) are enriched in a single localization and 273 diseases (∼21%, P=3.45 × 10−3) are enriched in dual localizations. In the DPL matrix, certain disease-associated proteins are likely to be found in membrane-bounded organelles such as mitochondria, lysosome, and peroxisome, indicating that the mutations of proteins localized to these compartments are connected to the pathophysiological conditions of those organelles. Meanwhile, certain disease-associated proteins in the DPL matrix are enriched in dual localizations, such as extracellular/plasma membrane or endoplasmic reticulum/Golgi. Although these two pairs of subcellular localizations appear to be distinct compartments at first, they are functionally related compartments in close proximity during protein translocation process in the cell, and thus are likely to share interacting protein partners (Gandhi et al, 2006).
Comorbidity represents the co-occurrence of multiple diseases in the same individual (Lee et al, 2008; Hidalgo et al, 2009; Park et al, 2009a). Many comorbid disease pairs have been shown to share common genes in the human disease network. For example, Diabetes and Alzheimer's disease share a risk factor in angiotensin I converting enzyme, and frequently occur together in an individual. In such instances, comorbidity can be partially attributed to the disease connections on the molecular level. To explore the impact of protein subcellular localization on comorbidity, we hypothesized that certain disease pairs could also be connected via subcellular localization by the molecular connections between the disease-associated proteins (bottom panel in Box 1).
We found a positive correlation between subcellular localization similarity and relative risk (Figure 3B, Pearson's correlation coefficient between relative risk and subcellular localization similarity=0.81, P=2.96 × 10−5). The subcellular localization similarity represents the correlation of subcellular localization profiles between disease pairs. To our surprise, when we compared the relative risk of disease pairs linked via various molecular connections, we found that disease pairs connected by subcellular localization showed a near three-fold higher comorbidity tendency (with link distances equal to 2 or 3) when compared with random pairs (Figure 3E).
We then assessed quantitatively the impact of network distances and subcellular localizations on the comorbidity tendency of disease pairs. We expected the proteins associated with comorbid disease pairs to be located closely in the protein interaction network via fewer links compared with random disease pairs. Indeed, a higher comorbidity tendency was found when two disease-associated proteins were positioned within a shorter distance (gray plots in Figure 3F). Moreover, when subcellular localization information was combined with small network distances, the comorbidity tendency increased dramatically (orange plots in Figure 3F). It suggests that subcellular localization and close network distances, two conceptually distinct molecular connections, contributed synergistically to the comorbidity tendency.
Disease progression is not restricted to the mutation of disease-causing genes, but also affected by molecular connections in ‘disease modules,' resulting in comorbidity (Fraser, 2006; Lee et al, 2008). In this study, for the first time we applied subcellular localization information to elucidate the molecular connections between comorbid diseases. We believe that, based on our finding, our approach helps to define the boundaries of ‘disease modules.' Taken together, integration of diverse molecular connections should improve the molecular level understanding of hitherto unexplained comorbid disease pairs and help us in expanding the scope of our knowledge of the mechanism of human disease progression.
Proteins targeting the same subcellular localization tend to participate in mutual protein–protein interactions (PPIs) and are often functionally associated. Here, we investigated the relationship between disease-associated proteins and their subcellular localizations, based on the assumption that protein pairs associated with phenotypically similar diseases are more likely to be connected via subcellular localization. The spatial constraints from subcellular localization significantly strengthened the disease associations of the proteins connected by subcellular localizations. In particular, certain disease types were more prevalent in specific subcellular localizations. We analyzed the enrichment of disease phenotypes within subcellular localizations, and found that there exists a significant correlation between disease classes and subcellular localizations. Furthermore, we found that two diseases displayed high comorbidity when disease-associated proteins were connected via subcellular localization. We newly explained 7584 disease pairs by using the context of protein subcellular localization, which had not been identified using shared genes or PPIs only. Our result establishes a direct correlation between protein subcellular localization and disease association, and helps to understand the mechanism of human disease progression.
doi:10.1038/msb.2011.29
PMCID: PMC3130560  PMID: 21613983
cellular networks; comorbidity; human disease; subcellular localization
4.  Differential Use of Signal Peptides and Membrane Domains Is a Common Occurrence in the Protein Output of Transcriptional Units 
PLoS Genetics  2006;2(4):e46.
Membrane organization describes the orientation of a protein with respect to the membrane and can be determined by the presence, or absence, and organization within the protein sequence of two features: endoplasmic reticulum signal peptides and alpha-helical transmembrane domains. These features allow protein sequences to be classified into one of five membrane organization categories: soluble intracellular proteins, soluble secreted proteins, type I membrane proteins, type II membrane proteins, and multi-spanning membrane proteins. Generation of protein isoforms with variable membrane organizations can change a protein's subcellular localization or association with the membrane. Application of MemO, a membrane organization annotation pipeline, to the FANTOM3 Isoform Protein Sequence mouse protein set revealed that within the 8,032 transcriptional units (TUs) with multiple protein isoforms, 573 had variation in their use of signal peptides, 1,527 had variation in their use of transmembrane domains, and 615 generated protein isoforms from distinct membrane organization classes. The mechanisms underlying these transcript variations were analyzed. While TUs were identified encoding all pairwise combinations of membrane organization categories, the most common was conversion of membrane proteins to soluble proteins. Observed within our high-confidence set were 156 TUs predicted to generate both extracellular soluble and membrane proteins, and 217 TUs generating both intracellular soluble and membrane proteins. The differential use of endoplasmic reticulum signal peptides and transmembrane domains is a common occurrence within the variable protein output of TUs. The generation of protein isoforms that are targeted to multiple subcellular locations represents a major functional consequence of transcript variation within the mouse transcriptome.
Synopsis
Many genes produce only a single protein; however, others are known to produce a number of proteins with different functions in the cell. The function of a protein within the cell is influenced by its location; for example, proteins that are secreted can act as messengers, whereas proteins embedded in the membrane may act as receptors or channels. Features that determine the eventual location of a protein are found in the protein sequence. The authors identified two such features, the signal peptide that targets a protein for secretion, and the transmembrane domain that embeds a protein in the membrane, predicting their occurrence in mouse protein sequences. The authors then searched the entire mouse genome for genes that vary in the use of these features in protein isoforms. They found a large number of genes that produce proteins with variation in these features; for example, they identified genes producing proteins that are both secreted and intracellular, and genes producing proteins that are both membrane bound and soluble. This process is likely to be a major source of functional variation in the output of mammalian genes.
doi:10.1371/journal.pgen.0020046
PMCID: PMC1449889  PMID: 16683029
5.  MemPype: a pipeline for the annotation of eukaryotic membrane proteins 
Nucleic Acids Research  2011;39(Web Server issue):W375-W380.
MemPype is a Python-based pipeline including previously published methods for the prediction of signal peptides (SPEP), glycophosphatidylinositol (GPI) anchors (PredGPI), all-alpha membrane topology (ENSEMBLE), and a recent method (MemLoci) that specifically discriminates the localization of eukaryotic membrane proteins in: ‘cell membrane’, ‘internal membranes’, ‘organelle membranes’. MemLoci scores with accuracy of 70% and generalized correlation coefficient (GCC) of 0.50 on a rigorous homology-unbiased validation set and overpasses other predictors for subcellular localization. The annotation process is based both on inheritance through homology and computational methods. Each submitted protein first retrieves, when available, up to 25 similar proteins (with sequence identity ≥50% and alignment coverage ≥50% on both sequences). This helps the identification of membrane-associated proteins and detailed localization tags. Each protein is also filtered for the presence of a GPI anchor [0.8% false positive rate (FPR)]. A positive score of GPI anchor prediction labels the sequence as exposed to ‘Cell surface’. Concomitantly the sequence is analysed for the presence of a signal peptide and classified with MemLoci into one of three discriminated classes. Finally the sequence is filtered for predicting its putative all-alpha protein membrane topology (FPR <1%). The web server is available at: http://mu2py.biocomp.unibo.it/mempype.
doi:10.1093/nar/gkr282
PMCID: PMC3125734  PMID: 21543452
6.  High-accuracy prediction of transmembrane inter-helix contacts and application to GPCR 3D structure modeling 
Bioinformatics  2013;29(20):2579-2587.
Motivation: Residue–residue contacts across the transmembrane helices dictate the three-dimensional topology of alpha-helical membrane proteins. However, contact determination through experiments is difficult because most transmembrane proteins are hard to crystallize.
Results: We present a novel method (MemBrain) to derive transmembrane inter-helix contacts from amino acid sequences by combining correlated mutations and multiple machine learning classifiers. Tested on 60 non-redundant polytopic proteins using a strict leave-one-out cross-validation protocol, MemBrain achieves an average accuracy of 62%, which is 12.5% higher than the current best method from the literature. When applied to 13 recently solved G protein-coupled receptors, the MemBrain contact predictions helped increase the TM-score of the I-TASSER models by 37% in the transmembrane region. The number of foldable cases (TM-score >0.5) increased by 100%, where all G protein-coupled receptor templates and homologous templates with sequence identity >30% were excluded. These results demonstrate significant progress in contact prediction and a potential for contact-driven structure modeling of transmembrane proteins.
Availability: www.csbio.sjtu.edu.cn/bioinf/MemBrain/
Contact: hbshen@sjtu.edu.cn or zhng@umich.edu
Supplementary information: Supplementary data are available at Bioinformatics online.
doi:10.1093/bioinformatics/btt440
PMCID: PMC3789543  PMID: 23946502
7.  Tumor Imaging and Targeting Potential of an Hsp70-Derived 14-Mer Peptide 
PLoS ONE  2014;9(8):e105344.
Background
We have previously used a unique mouse monoclonal antibody cmHsp70.1 to demonstrate the selective presence of a membrane-bound form of Hsp70 (memHsp70) on a variety of leukemia cells and on single cell suspensions derived from solid tumors of different entities, but not on non-transformed cells or cells from corresponding ’healthy‘ tissue. This antibody can be used to image tumors in vivo and target them for antibody-dependent cellular cytotoxicity. Tumor-specific expression of memHsp70 therefore has the potential to be exploited for theranostic purposes. Given the advantages of peptides as imaging and targeting agents, this study assessed whether a 14-mer tumor penetrating peptide (TPP; TKDNNLLGRFELSG), the sequence of which is derived from the oligomerization domain of Hsp70 which is expressed on the cell surface of tumor cells, can also be used for targeting membrane Hsp70 positive (memHsp70+) tumor cells, in vitro.
Methodology/Principal Findings
The specificity of carboxy-fluorescein (CF-) labeled TPP (TPP) to Hsp70 was proven in an Hsp70 knockout mammary tumor cell system. TPP specifically binds to different memHsp70+ mouse and human tumor cell lines and is rapidly taken up via endosomes. Two to four-fold higher levels of CF-labeled TPP were detected in MCF7 (82% memHsp70+) and MDA-MB-231 (75% memHsp70+) cells compared to T47D cells (29% memHsp70+) that exhibit a lower Hsp70 membrane positivity. After 90 min incubation, TPP co-localized with mitochondrial membranes in memHsp70+ tumors. Although there was no evidence that any given vesicle population was specifically localized, fluorophore-labeled cmHsp70.1 antibody and TPP preferentially accumulated in the proximity of the adherent surface of cultured cells. These findings suggest a potential association between membrane Hsp70 expression and cytoskeletal elements that are involved in adherence, the establishment of intercellular synapses and/or membrane reorganization.
Conclusions/Significance
This study demonstrates the specific binding and rapid internalization of TPP by tumor cells with a memHsp70+ phenotype. TPP might therefore have potential for targeting and imaging the large proportion of tumors (∼50%) that express memHsp70.
doi:10.1371/journal.pone.0105344
PMCID: PMC4148261  PMID: 25165986
8.  Aggregation Propensity of the Human Proteome 
PLoS Computational Biology  2008;4(10):e1000199.
Formation of amyloid-like fibrils is involved in numerous human protein deposition diseases, but is also an intrinsic property of polypeptide chains in general. Progress achieved recently now allows the aggregation propensity of proteins to be analyzed over large scales. In this work we used a previously developed predictive algorithm to analyze the propensity of the 34,180 protein sequences of the human proteome to form amyloid-like fibrils. We show that long proteins have, on average, less intense aggregation peaks than short ones. Human proteins involved in protein deposition diseases do not differ extensively from the rest of the proteome, further demonstrating the generality of protein aggregation. We were also able to reproduce some of the results obtained with other algorithms, demonstrating that they do not depend on the type of computational tool employed. For example, proteins with different subcellular localizations were found to have different aggregation propensities, in relation to the various efficiencies of quality control mechanisms. Membrane proteins, intrinsically disordered proteins, and folded proteins were confirmed to have very different aggregation propensities, as a consequence of their different structures and cellular microenvironments. In addition, gatekeeper residues at strategic positions of the sequences were found to protect human proteins from aggregation. The results of these comparative analyses highlight the existence of intimate links between the propensity of proteins to form aggregates with β-structure and their biology. In particular, they emphasize the existence of a negative selection pressure that finely modulates protein sequences in order to adapt their aggregation propensity to their biological context.
Author Summary
Amyloid-like fibrils are insoluble proteinaceous fibrillar aggregates with a characteristic structure (the cross-β core) that form and deposit in more than 40 pathological conditions in humans. These include Alzheimer's disease, Parkinson's disease, type II diabetes, and the spongiform encephalopathies. A number of proteins not involved in any disease can also form amyloid-like fibrils in vitro, suggesting that amyloid fibril formation is an intrinsic property of proteins in general. Recent efforts in understanding the physico-chemical grounds of amyloid fibril formation has led to the development of several algorithms, capable of predicting a number of aggregation-related parameters of a protein directly from its amino acid sequence. In order to study the predicted aggregation behavior of the human proteome, we have run one of these algorithms on the 34,180 human protein sequences. Our results demonstrate that molecular evolution has acted on protein sequences to finely modulate their aggregation propensities, depending on different parameters related to their in vivo environment. Together with cellular control mechanisms, this natural selection protects proteins from aggregation during their lifetime.
doi:10.1371/journal.pcbi.1000199
PMCID: PMC2557143  PMID: 18927604
9.  Spectrum of Cav1.4 dysfunction in congenital stationary night blindness type 2☆ 
Biochimica et Biophysica Acta  2014;1838(8):2053-2065.
Defective retinal synaptic transmission in patients affected with congenital stationary night blindness type 2 (CSNB2) can result from different dysfunction phenotypes in Cav1.4 L-type calcium channels. Here we investigated two prototypical Cav1.4 variants from either end of the functional spectrum. Using whole-cell and single-channel patch-clamp techniques, we provide analysis of the biophysical characteristics of the point mutation L860P and the C-terminal truncating mutation R1827X. L860P showed a typical loss-of-function phenotype attributed to a reduced number of functional channels expressed at the plasma membrane as implied by gating current and non-stationary noise analyses. This phenotype can be rationalized, because the inserted proline is predicted to break an amphipatic helix close to the transmembrane segment IIIS1 and thus to reduce channel stability and promote misfolding. In fact, L860P was subject to an increased turnover. In contrast, R1827X displayed an apparent gain-of-function phenotype, i.e., due to a hyperpolarizing shift of the IV-curve and increased single-channel activity. However, truncation also resulted in the loss of functional C-terminal modulation and thus unmasked calcium-dependent inactivation. Thus R1827X failed to support continuous calcium influx. Current inactivation curtails the dynamic range of photoreceptors (e.g., when adapting to variation in illumination). Taken together, the analysis of two representative mutations that occur in CSNB2 patients revealed fundamental differences in the underlying defect. These may explain subtle variations in the clinical manifestation and must be taken into account, if channel function is to be restored by pharmacochaperones or related approaches.
Graphical abstract
Highlights
•Mutation L860P resulted in accelerated degradation of Cav1.4 channel protein.•Mutation R1827X failed to support continuous calcium influx.•Underlying defects require distinct approaches to restore Cav1.4 function in CSNB2.
doi:10.1016/j.bbamem.2014.04.023
PMCID: PMC4065569  PMID: 24796500
L-type calcium channel; Cav1.4; Calcium channelopathy; Congenital stationary night blindness type 2
10.  The S218L familial hemiplegic migraine mutation promotes deinhibition of Cav2.1 calcium channels during direct G-protein regulation 
Pflugers Archiv   2008;457(2):315-326.
Familial hemiplegic migraine type 1 (FHM-1) is caused by mutations in CACNA1A; the gene encoding for the Cav2.1 subunit of voltage-gated calcium channels. Although various studies attempted to determine biophysical consequences of these mutations on channel activity, it remains unclear exactly how mutations can produce a FHM-1 phenotype. A lower activation threshold of mutated channels resulting in increased channel activity has been proposed. However, hyper-activity may also be caused by a reduction of the inhibitory pathway carried by G-protein coupled receptor activation. The aim of this study is to determine functional consequences of the FHM-1 S218L mutation on direct G-protein regulation of Cav2.1 channels. In HEK 293 cells, DAMGO activation of human μ-opioid receptors induced a 55% Ba2+ current inhibition through both wild-type and S218L mutant Cav2.1 channels. In contrast, this mutation considerably accelerates the kinetic of current deinhibition following channel activation by 1.7- to 2.3-fold depending on membrane potential values. Taken together, these data suggest that the S218L mutation does not affect G-proteins association onto channel in the closed state, but promotes its dissociation from the activated channel thereby decreasing the inhibitory G-protein pathway. Similar results were obtained with the R192Q FHM-1 mutation, although of lesser amplitude, which seems in line with the less severe associated clinical phenotype in patients. Functional consequences of FHM-1 mutations appear thus as the consequence of the alteration of both intrinsic biophysical properties and of the main inhibitory G-protein pathway of Cav2.1 channels. The present study furthers molecular insight in the physiopathology of FHM-1.
doi:10.1007/s00424-008-0541-2
PMCID: PMC2793404  PMID: 18581134
Animals; Calcium Channels; genetics; metabolism; Calcium Channels, N-Type; genetics; metabolism; Cell Line; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; pharmacology; GTP-Binding Protein alpha Subunits, Gi-Go; metabolism; Genotype; Humans; Ion Channel Gating; drug effects; Kinetics; Membrane Potentials; Migraine with Aura; genetics; metabolism; Mutation; Phenotype; Rats; Receptors, Opioid, mu; agonists; metabolism; Transfection; Familial hemiplegic migraine; S218L mutation; R192Q mutation; Cav2.1 type calcium channel; Cav2.1 subunit; P/Q current; G-protein; G-protein coupled receptor; μ-opioid receptor; β-subunit.
11.  Human ClC-6 Is a Late Endosomal Glycoprotein that Associates with Detergent-Resistant Lipid Domains 
PLoS ONE  2007;2(5):e474.
Background
The mammalian CLC protein family comprises nine members (ClC-1 to -7 and ClC-Ka, -Kb) that function either as plasma membrane chloride channels or as intracellular chloride/proton antiporters, and that sustain a broad spectrum of cellular processes, such as membrane excitability, transepithelial transport, endocytosis and lysosomal degradation. In this study we focus on human ClC-6, which is structurally most related to the late endosomal/lysomal ClC-7.
Principal Findings
Using a polyclonal affinity-purified antibody directed against a unique epitope in the ClC-6 COOH-terminal tail, we show that human ClC-6, when transfected in COS-1 cells, is N-glycosylated in a region that is evolutionary poorly conserved between mammalian CLC proteins and that is located between the predicted helices K and M. Three asparagine residues (N410, N422 and N432) have been defined by mutagenesis as acceptor sites for N-glycosylation, but only two of the three sites seem to be simultaneously N-glycosylated. In a differentiated human neuroblastoma cell line (SH-SY5Y), endogenous ClC-6 colocalizes with LAMP-1, a late endosomal/lysosomal marker, but not with early/recycling endosomal markers such as EEA-1 and transferrin receptor. In contrast, when transiently expressed in COS-1 or HeLa cells, human ClC-6 mainly overlaps with markers for early/recycling endosomes (transferrin receptor, EEA-1, Rab5, Rab4) and not with late endosomal/lysosomal markers (LAMP-1, Rab7). Analogously, overexpression of human ClC-6 in SH-SY5Y cells also leads to an early/recycling endosomal localization of the exogenously expressed ClC-6 protein. Finally, in transiently transfected COS-1 cells, ClC-6 copurifies with detergent-resistant membrane fractions, suggesting its partitioning in lipid rafts. Mutating a juxtamembrane string of basic amino acids (amino acids 71–75: KKGRR) disturbs the association with detergent-resistant membrane fractions and also affects the segregation of ClC-6 and ClC-7 when cotransfected in COS-1 cells.
Conclusions
We conclude that human ClC-6 is an endosomal glycoprotein that partitions in detergent resistant lipid domains. The differential sorting of endogenous (late endosomal) versus overexpressed (early and recycling endosomal) ClC-6 is reminiscent of that of other late endosomal/lysosomal membrane proteins (e.g. LIMP II), and is consistent with a rate-limiting sorting step for ClC-6 between early endosomes and its final destination in late endosomes.
doi:10.1371/journal.pone.0000474
PMCID: PMC1868598  PMID: 17534424
12.  Emergence of tissue polarization from synergy of intracellular and extracellular auxin signaling 
Here, we provide a novel mechanistic framework for cell polarization during auxin-driven plant development that combines intracellular auxin signaling for regulation of expression of PINFORMED (PIN) auxin efflux transporters and the theoretical assumption of extracellular auxin signaling for regulation of PIN subcellular dynamics.The competitive utilization of auxin signaling component in the apoplast might account for the elusive mechanism for cell-to-cell communication for tissue polarization.Computer model simulations faithfully and robustly recapitulate experimentally observed patterns of tissue polarity and asymmetric auxin distribution during formation and regeneration of vascular systems, and during the competitive regulation of shoot branching by apical dominance.Our model generated new predictions that could be experimentally validated, highlighting a mechanistically conceivable explanation for the PIN polarization and canalization of the auxin flow in plants.
A key question of developmental biology relates to a fundamental issue in cell and tissue polarities, namely, how an individual cell in a polarized tissue senses the polarities of its neighbors and its position within tissue. In plant development, this issue is of pronounced importance, because plants have a remarkable ability to redefine cell and tissue polarities in different developmental programs, such as embryogenesis, postembryonic organogenesis, vascular tissue formation, and tissue regeneration (Kleine-Vehn and Friml, 2008).
A polar, cell-to-cell transport of the small signaling molecule auxin in conjunction with local auxin biosynthesis determines auxin gradients during embryonic and postembryonic development, giving positional cues for primordia formation, organ patterning, and tropistic growth (Friml et al, 2002; Benková et al, 2003; Reinhardt et al, 2003; Heisler et al, 2005; Scarpella et al, 2006; Dubrovsky et al, 2008). Over the past decades, theoretical models proposed that auxin acts as a polarizing cue in the center of a positive feedback mechanisms for auxin transport that has a key role in synchronized polarity rearrangements. However, the mechanistic basis for such a feedback loop between auxin and its own transport remains to a large extent elusive.
The direction of auxin transport largely depends on the polar subcellular localization of PINFORMED (PIN) proteins at the plasma membrane (Petrášek et al, 2006; Wiśniewska et al, 2006). These proteins recycle between the plasma membrane and intracellular endosomal compartments (Geldner et al, 2001; Dhonukshe et al, 2007), and their recycling modulates PIN-dependent auxin efflux rates and enable rapid changes in PIN polarity (Dubrovsky et al, 2008; Kleine-Vehn et al, 2008a). Nevertheless, the molecular basis for PIN polarization in plants remains unknown.
To gain new mechanistic insights in the hypothetical feedback mechanisms governing PIN polarization, several theoretical studies (Mitchison, 1980; Sachs, 1981; Rolland-Lagan and Prusinkiewicz, 2005; Jönsson et al, 2006; Smith et al, 2006; Merks et al, 2007; Bayer et al, 2009; Kramer, 2009) have been carried out. These models suggest that auxin promotes its own transport by modulating the amount of PIN proteins at the plasma membrane by incorporating either not yet identified flux gradient-based component (Mitchison, 1980; Rolland-Lagan and Prusinkiewicz, 2005; Bayer et al, 2009; Kramer, 2009) or an unknown short-range intercellular signal-transmitting auxin concentrations of its direct neighbors (Jönsson et al, 2006; Smith et al, 2006; Merks et al, 2007; Bayer et al, 2009; Sahlin et al, 2009).
Here, we propose a feedback driven, biologically plausible model for PIN polarization and auxin transport that introduces the combination of intracellular and extracellular auxin signaling pathways as a unified approach for tissue polarization in plants. Our computer model is based on chemiosmotic hypothesis (Goldsmith et al, 1981; Figure 1A) and integrates up-to-date experimental data, such as auxin feedback on PIN expression (Peer et al, 2004; Heisler et al, 2005) via a nuclear auxin signaling pathway (Chapman and Estelle, 2009; Figure 1B), auxin carrier recycling auxin (Dubrovsky et al, 2008; Kleine-Vehn et al, 2008a; Figure 1C), and auxin feedback on PIN endocytosis (Paciorek et al, 2005) via novel hypothetical, yet plausible, assumption of extracellular auxin perception (Figure 1D).
The heart of our extracellular receptor-based polarization (ERP) mechanism is the competitive utilization of auxin receptors in the intercellular space that allows a direct and simple cell-to-cell communication scheme. In our model, auxin binds to its extracellular receptor in the concentration-dependent manner and induces signal to modulate PIN protein abundance at the plasma membrane (Figure 1D). The direct mode of the signal transfer involves temporal immobilization of recruited receptors to the plasma membrane, which is reflected by reduced diffusion of receptors involved in auxin signaling (Figure 1D). This competitive utilization mechanism enables cell-to-cell communication in our model, leading to receptor enrichment at the site of higher auxin concentration (Figure 1D). The PIN polarization and polar auxin transport in our model both depend on and contribute to the establishment of differential auxin signaling in the cell wall. This feedback loop leads ultimately to the alignment of PIN polarization within a tissue.
We demonstrated the plausibility of the ERP model for various processes, including de novo vascularization, venation patterning, and tissue regeneration in computer simulations performed with only minimal initial assumptions, a discrete auxin source, and a distal sink. The ERP model reproduces the very detailed PIN polarization events that occur during primary vein initiation (Scarpella et al, 2006), such as basal PIN1 polarity in provascular cells, transient adverse PIN1 polarization in neighboring cells during the alignment of tissue polarization, and inner-lateral polarity displayed by the tissues surrounding a conductive auxin channel (Figure 3). Additionally, the ERP model generates high auxin concentration and high auxin flux simultaneously in emerging veins, revising the classical canalization models (Mitchison, 1980; Rolland-Lagan and Prusinkiewicz, 2005). Importantly, all our model simulations support the claim that the ERP model represents the first single approach that faithfully reproduces PIN polarization, both with the auxin gradient (basal PIN1 polarity in provascular cells) and against the auxin gradient (transient adverse PIN1 polarization in neighboring cells surrounding the provascular bundle), as well as producing the corresponding auxin distribution patterns during auxin canalization.
The proposed model introduces the extracellular auxin signaling pathway, which is crucial to account for coordinated PIN polarization and auxin distribution during venation patterning in plants. The putative candidate for extracellular auxin receptor is auxin-binding protein 1 (ABP1), which resides in the lumen of the endoplasmic reticulum and is secreted to the cell wall (Napier et al, 2002; Tromas et al, 2009) where it is physiologically active (Leblanc et al, 1999; Steffens et al, 2001). Additionally, auxin inhibits clathrin-dependent PIN internalization via binding to ABP1 (Robert et al, 2010). Thus, we speculate that the extracellular fraction of ABP1 (or additionally yet to be identified ABPs) could correspond to the common pool of extracellular auxin receptors in the ERP model. A future challenge will be to test whether the ERP model unifies complex PIN polarization and auxin distribution patterns in embryogenesis, root system maintenance, and de novo organ formation.
Plant development is exceptionally flexible as manifested by its potential for organogenesis and regeneration, which are processes involving rearrangements of tissue polarities. Fundamental questions concern how individual cells can polarize in a coordinated manner to integrate into the multicellular context. In canalization models, the signaling molecule auxin acts as a polarizing cue, and feedback on the intercellular auxin flow is key for synchronized polarity rearrangements. We provide a novel mechanistic framework for canalization, based on up-to-date experimental data and minimal, biologically plausible assumptions. Our model combines the intracellular auxin signaling for expression of PINFORMED (PIN) auxin transporters and the theoretical postulation of extracellular auxin signaling for modulation of PIN subcellular dynamics. Computer simulations faithfully and robustly recapitulated the experimentally observed patterns of tissue polarity and asymmetric auxin distribution during formation and regeneration of vascular systems and during the competitive regulation of shoot branching by apical dominance. Additionally, our model generated new predictions that could be experimentally validated, highlighting a mechanistically conceivable explanation for the PIN polarization and canalization of the auxin flow in plants.
doi:10.1038/msb.2010.103
PMCID: PMC3018162  PMID: 21179019
auxin; canalization; cell polarity; PIN proteins
13.  Smooth muscle cell α2δ-1 subunits are essential for vasoregulation by CaV1.2 channels 
Circulation research  2009;105(10):948-955.
Rationale
Voltage-dependent L-type (CaV1.2) Ca2+ channels are a heteromeric complex formed from pore forming α1 and auxiliary α2δ and β subunits. CaV1.2 channels are the principal Ca2+ influx pathway in arterial myocytes and regulate multiple physiological functions, including contraction. The macromolecular composition of arterial myocyte CaV1.2 channels remains poorly understood, with no studies having examined the molecular identity or physiological functions of α2δ subunits.
Objective
Investigate the functional significance of α2δ subunits in myocytes of resistance-size (100–200 μm diameter) cerebral arteries.
Methods and Results
α2δ-1 was the only α2δ isoform expressed in cerebral artery myocytes. Pregabalin, an α2δ-1/-2 ligand, and an α2δ-1 antibody, inhibited CaV1.2 currents in isolated myocytes. Acute pregabalin application reversibly dilated pressurized arteries. Using a novel application of surface biotinylation, data indicated that >95 % of CaV1.2 α1 and α2δ-1 subunits are present in the arterial myocyte plasma membrane. α2δ-1 knockdown using shRNA reduced plasma membrane-localized CaV1.2 α1 subunits, caused a corresponding elevation in cytosolic CaV1.2 α1 subunits, decreased intracellular Ca2+ concentration, inhibited pressure-induced vasoconstriction (“myogenic tone”), and attenuated pregabalin-induced vasodilation. Prolonged (24 hour) pregabalin exposure did not alter total α2δ-1 or CaV1.2 α1 proteins, but decreased plasma membrane expression of each subunit, which reduced myogenic tone.
Conclusions
α2δ-1 is essential for plasma membrane expression of arterial myocyte CaV1.2 α1 subunits. α2δ-1 targeting can block CaV1.2 channels directly and inhibit surface expression of CaV1.2 α1 subunits, leading to vasodilation. These data identify α2δ-1 as a novel molecular target in arterial myocytes, manipulation of which regulates contractility.
doi:10.1161/CIRCRESAHA.109.203620
PMCID: PMC2783418  PMID: 19797702
L-type Ca2+ channels; arterial contractility
14.  Characterization of the Molecular Architecture of Human Caveolin-3 and Interaction with the Skeletal Muscle Ryanodine Receptor* 
The Journal of Biological Chemistry  2012;287(48):40302-40316.
Background: Caveolin-3 facilitates both caveolae formation and a range of cell signaling pathways, including Ca2+ homeostasis.
Results: Caveolin-3 forms a disc-shaped nonamer that binds the Ca2+-release channel, RyR1.
Conclusion: Multiple caveolin-3 nonamers bind to a single RyR1 homotetramer.
Significance: First three-dimensional structural insights into caveolin-3 assembly, interactions with RyR1 suggest a novel role in muscle contraction and/or for channel localization within the membrane.
Caveolin-3 (cav-3), an integral membrane protein, is a building block of caveolae as well as a regulator of a number of physiological processes by facilitating the formation of multiprotein signaling complexes. We report that the expression of cav-3 in insect (Sf9) cells induces caveola formation, comparable in size with those observed in native tissue. We have also purified the recombinant cav-3 determining that it forms an oligomer of ∼220 kDa. We present the first three-dimensional structure for cav-3 (using transmission electron microscopy and single particle analysis methods) and show that nine cav-3 monomers assemble to form a complex that is toroidal in shape, ∼16.5 nm in diameter and ∼ 5.5 nm in height. Labeling experiments and reconstitution of the purified cav-3 into liposomes have allowed a proposal for the orientation of the protein with respect to the membrane. We have identified multiple caveolin-binding motifs within the ryanodine receptor (RyR1) sequence employing a bioinformatic analysis. We have then shown experimentally that there is a direct interaction between recombinant cav-3 nonamers and purified RyR1 homotetramers that would imply that at least one of the predicted cav-3-binding sites is exposed within the fully assembled RyR1 structure. The cav-3 three-dimensional model provides new insights as to how a cav-3 oligomer can bind multiple partners in close proximity to form signaling complexes. Furthermore, a direct interaction with RyR1 suggests a possible role for cav-3 as a modifier of muscle excitation-contraction coupling and/or for localization of the receptor to regions of the sarcoplasmic reticulum.
doi:10.1074/jbc.M112.377085
PMCID: PMC3504746  PMID: 23071107
Caveolae; Caveolin; Electron Microscopy (EM); Excitation-Contraction Coupling; Membrane Proteins; Muscle; Ryanodine Receptor; Sarcoplasmic Reticulum (SR); Scaffold Proteins; Single Particle Analysis
15.  Caveolin-1 Contributes to Assembly of Store-operated Ca2+ Influx Channels by Regulating Plasma Membrane Localization of TRPC1 
The Journal of biological chemistry  2003;278(29):27208-27215.
TRPC1, a component of store-operated Ca2+ entry (SOCE) channels, is assembled in a complex with caveolin- 1 (Cav1) and key Ca2+ signaling proteins. This study examines the role of Cav1 in the function of TRPC1. TRPC1 and Cav1 were colocalized in the plasma membrane region of human submandibular gland and Madin-Darby canine kidney cells. Full-length Cav1 bound to both the N and C termini of TRPC1. Amino acids 271–349, which includes a Cav1 binding motif (amino acids 322–349), was identified as the Cav1 binding domain in the TRPC1 N terminus. Deletion of amino acids 271–349 or 322–349 prevented plasma membrane localization of TRPC1. Importantly, TRPC1Δ271–349 induced a dominant suppression of SOCE and was associated with wild-type TRPC1. Although the role of the C-terminal Cav1 binding domain is not known, its deletion did not affect localization of TRPC1 (Singh, B. B., Liu, X., and Ambudkar, I. S. (2000) J. Biol. Chem. 275, 36483–36486). Further, expression of a truncated Cav1 (Cav1Δ51–169), but not full-length Cav1, similarly disrupted plasma membrane localization of endogenously and exogenously expressed TRPC1 in human Submandibular gland and Madin-Darby canine kidney cells. Cav1Δ51–169 also suppressed thapsigargin- and carbachol-stimulated Ca2+ influx and increased the detergent solubility of TRPC1, although plasma membrane lipid raft domains were not disrupted. These data demonstrate that plasma membrane localization of TRPC1 depends on an interaction between its N terminus and Cav1. Thus, our data suggest that Cav1 has an important role in the assembly of SOCE channel(s).
doi:10.1074/jbc.M301118200
PMCID: PMC3621139  PMID: 12732636
16.  Quantitative Localization of Cav2.1 (P/Q-Type) Voltage-Dependent Calcium Channels in Purkinje Cells: Somatodendritic Gradient and Distinct Somatic Coclustering with Calcium-Activated Potassium Channels 
P/Q-type voltage-dependent calcium channels play key roles in transmitter release, integration of dendritic signals, generation of dendritic spikes, and gene expression. High intracellular calcium concentration transient produced by these channels is restricted to tens to hundreds of nanometers from the channels. Therefore, precise localization of these channels along the plasma membrane was long sought to decipher how each neuronal cell function is controlled. Here, we analyzed the distribution of Cav2.1 subunit of the P/Q-type channel using highly sensitive SDS-digested freeze-fracture replica labeling in the rat cerebellar Purkinje cells. The labeling efficiency was such that the number of immunogold particles in each parallel fiber active zone was comparable to that of functional channels calculated from previous reports. Two distinct patterns of Cav2.1 distribution, scattered and clustered, were found in Purkinje cells. The scattered Cav2.1 had a somatodendritic gradient with the density of immunogold particles increasing 2.5-fold from soma to distal dendrites. The other population with 74-fold higher density than the scattered particles was found within clusters of intramembrane particles on the P-face of soma and primary dendrites. Both populations of Cav2.1 were found as early as P3 and increased in the second postnatal week to a mature level. Using double immunogold labeling, we found that virtually all of the Cav2.1 clusters were colocalized with two types of calcium-activated potassium channels, BK and SK2, with the nearest neighbor distance of ~40 nm. Calcium nanodomain created by the opening of Cav2.1 channels likely activates the two channels that limit the extent of depolarization.
doi:10.1523/JNEUROSCI.2921-12.2013
PMCID: PMC4031662  PMID: 23426693
17.  On the Role of Aggregation Prone Regions in Protein Evolution, Stability, and Enzymatic Catalysis: Insights from Diverse Analyses 
PLoS Computational Biology  2013;9(10):e1003291.
The various roles that aggregation prone regions (APRs) are capable of playing in proteins are investigated here via comprehensive analyses of multiple non-redundant datasets containing randomly generated amino acid sequences, monomeric proteins, intrinsically disordered proteins (IDPs) and catalytic residues. Results from this study indicate that the aggregation propensities of monomeric protein sequences have been minimized compared to random sequences with uniform and natural amino acid compositions, as observed by a lower average aggregation propensity and fewer APRs that are shorter in length and more often punctuated by gate-keeper residues. However, evidence for evolutionary selective pressure to disrupt these sequence regions among homologous proteins is inconsistent. APRs are less conserved than average sequence identity among closely related homologues (≥80% sequence identity with a parent) but APRs are more conserved than average sequence identity among homologues that have at least 50% sequence identity with a parent. Structural analyses of APRs indicate that APRs are three times more likely to contain ordered versus disordered residues and that APRs frequently contribute more towards stabilizing proteins than equal length segments from the same protein. Catalytic residues and APRs were also found to be in structural contact significantly more often than expected by random chance. Our findings suggest that proteins have evolved by optimizing their risk of aggregation for cellular environments by both minimizing aggregation prone regions and by conserving those that are important for folding and function. In many cases, these sequence optimizations are insufficient to develop recombinant proteins into commercial products. Rational design strategies aimed at improving protein solubility for biotechnological purposes should carefully evaluate the contributions made by candidate APRs, targeted for disruption, towards protein structure and activity.
Author Summary
Biotechnology requires the large-scale expression, yield, and storage of recombinant proteins. Each step in protein production has the potential to cause aggregation as proteins, not evolved to exist outside the cell, endure the various steps involved in commercial manufacturing processes. Mechanistic studies into protein aggregation have revealed that certain sequence regions contribute more to the aggregation propensity of a protein than other sequence regions do. Efforts to disrupt these regions have thus far indicated that rational sequence engineering is a useful technique to reduce the aggregation of biotechnologically relevant proteins. To improve our ability to rationally engineer proteins with enhanced expression, solubility, and shelf-life we conducted extensive analyses of aggregation prone regions (APRs) within protein sequences to characterize the various roles these regions play in proteins. Findings from this work indicate that protein sequences have evolved by minimizing their aggregation propensities. However, we also found that many APRs are conserved in protein families and are essential to maintain protein stability and function. Therefore, the contributions that APRs, targeted for disruption, make towards protein stability and function should be carefully evaluated when improving protein solubility via rational design.
doi:10.1371/journal.pcbi.1003291
PMCID: PMC3798281  PMID: 24146608
18.  Rational Mutational Analysis of a Multidrug MFS Transporter CaMdr1p of Candida albicans by Employing a Membrane Environment Based Computational Approach 
PLoS Computational Biology  2009;5(12):e1000624.
CaMdr1p is a multidrug MFS transporter of pathogenic Candida albicans. An over-expression of the gene encoding this protein is linked to clinically encountered azole resistance. In-depth knowledge of the structure and function of CaMdr1p is necessary for an effective design of modulators or inhibitors of this efflux transporter. Towards this goal, in this study, we have employed a membrane environment based computational approach to predict the functionally critical residues of CaMdr1p. For this, information theoretic scores which are variants of Relative Entropy (Modified Relative Entropy REM) were calculated from Multiple Sequence Alignment (MSA) by separately considering distinct physico-chemical properties of transmembrane (TM) and inter-TM regions. The residues of CaMdr1p with high REM which were predicted to be significantly important were subjected to site-directed mutational analysis. Interestingly, heterologous host Saccharomyces cerevisiae, over-expressing these mutant variants of CaMdr1p wherein these high REM residues were replaced by either alanine or leucine, demonstrated increased susceptibility to tested drugs. The hypersensitivity to drugs was supported by abrogated substrate efflux mediated by mutant variant proteins and was not attributed to their poor expression or surface localization. Additionally, by employing a distance plot from a 3D deduced model of CaMdr1p, we could also predict the role of these functionally critical residues in maintaining apparent inter-helical interactions to provide the desired fold for the proper functioning of CaMdr1p. Residues predicted to be critical for function across the family were also found to be vital from other previously published studies, implying its wider application to other membrane protein families.
Author Summary
Membrane proteins belonging to the Major Facilitator Superfamily (MFS) transport molecules, including drugs, across the membrane and are known to be associated with drug resistance. CaMdr1p is one such MFS major multidrug efflux pump whose over-expression is linked to frequently encountered azole resistance in hospital isolates of C. albicans. Amino acid residues critical for a protein's function are conserved across members of the protein family. However, the traditional measure of conservation is not a useful parameter in mapping a functionally important residue in membrane proteins e.g., hydrophobically conserved stretches form helical transmembrane regions of the protein and are responsible for membrane localization, which individually have limited effect on binding and transport. We have developed a method that uses information theory to score the conservation of a residue relative to its context within the membrane and hypothesize that these residues would be critical for the protein's function. The relevance of predicted residues in the functioning of MFS is validated on CaMdr1p.
doi:10.1371/journal.pcbi.1000624
PMCID: PMC2789324  PMID: 20041202
19.  Conformational Changes during Pore Formation by the Perforin-Related Protein Pleurotolysin 
PLoS Biology  2015;13(2):e1002049.
Membrane attack complex/perforin-like (MACPF) proteins comprise the largest superfamily of pore-forming proteins, playing crucial roles in immunity and pathogenesis. Soluble monomers assemble into large transmembrane pores via conformational transitions that remain to be structurally and mechanistically characterised. Here we present an 11 Å resolution cryo-electron microscopy (cryo-EM) structure of the two-part, fungal toxin Pleurotolysin (Ply), together with crystal structures of both components (the lipid binding PlyA protein and the pore-forming MACPF component PlyB). These data reveal a 13-fold pore 80 Å in diameter and 100 Å in height, with each subunit comprised of a PlyB molecule atop a membrane bound dimer of PlyA. The resolution of the EM map, together with biophysical and computational experiments, allowed confident assignment of subdomains in a MACPF pore assembly. The major conformational changes in PlyB are a ∼70° opening of the bent and distorted central β-sheet of the MACPF domain, accompanied by extrusion and refolding of two α-helical regions into transmembrane β-hairpins (TMH1 and TMH2). We determined the structures of three different disulphide bond-trapped prepore intermediates. Analysis of these data by molecular modelling and flexible fitting allows us to generate a potential trajectory of β-sheet unbending. The results suggest that MACPF conformational change is triggered through disruption of the interface between a conserved helix-turn-helix motif and the top of TMH2. Following their release we propose that the transmembrane regions assemble into β-hairpins via top down zippering of backbone hydrogen bonds to form the membrane-inserted β-barrel. The intermediate structures of the MACPF domain during refolding into the β-barrel pore establish a structural paradigm for the transition from soluble monomer to pore, which may be conserved across the whole superfamily. The TMH2 region is critical for the release of both TMH clusters, suggesting why this region is targeted by endogenous inhibitors of MACPF function.
Author Summary
Animals, plants, fungi, and bacteria all use pore-forming proteins of the membrane attack complex-perforin (MACPF) family as lethal, cell-killing weapons. These proteins are able to insert into the plasma membranes of target cells, creating large pores that short circuit the natural separation between the intracellular and extracellular milieu, with catastrophic results. However, the pore-forming proteins must undergo a substantial transformation from soluble precursors to a large barrel-shaped transmembrane complex as they punch their way into cells. Using a combination of X-ray crystallography and cryo electron microscopy, we have visualized, for the first time, the mechanism of action of one of these pore-forming proteins—pleurotolysin, a MACPF protein from the edible oyster mushroom. This enabled us to propose a model of the pleurotolysin pore by fitting the crystallographic structures of the pore proteins into a three-dimensional map of the pore obtained by cryo electron microscopy. We then designed a set of double mutants that allowed us to chemically trap intermediate states along the trajectory of the pore formation process, and to determine their structures too. By combining these data we proposed a detailed molecular mechanism for pore formation. The pleurotolysin first assembles into rings of 13 subunits, each of which then opens up by about 70° during pore formation. This process is accompanied by refolding and extrusion of two compact regions from each subunit into long hairpins that then zipper together to form an 80-Å wide barrel-shaped channel through the membrane.
A combination of structural methods reveals the complex process by which the perforin-like fungal toxin Pleurotolysin rearranges its structure to form a pore that punches a hole in target cell membranes.
doi:10.1371/journal.pbio.1002049
PMCID: PMC4318580  PMID: 25654333
20.  A Fully Atomistic Model of the Cx32 Connexon 
PLoS ONE  2008;3(7):e2614.
Connexins are plasma membrane proteins that associate in hexameric complexes to form channels named connexons. Two connexons in neighboring cells may dock to form a “gap junction” channel, i.e. an intercellular conduit that permits the direct exchange of solutes between the cytoplasm of adjacent cells and thus mediate cell–cell ion and metabolic signaling. The lack of high resolution data for connexon structures has hampered so far the study of the structure–function relationships that link molecular effects of disease–causing mutations with their observed phenotypes. Here we present a combination of modeling techniques and molecular dynamics (MD) to infer side chain positions starting from low resolution structures containing only Cα atoms. We validated this procedure on the structure of the KcsA potassium channel, which is solved at atomic resolution. We then produced a fully atomistic model of a homotypic Cx32 connexon starting from a published model of the Cα carbons arrangement for the connexin transmembrane helices, to which we added extracellular and cytoplasmic loops. To achieve structural relaxation within a realistic environment, we used MD simulations inserted in an explicit solvent–membrane context and we subsequently checked predictions of putative side chain positions and interactions in the Cx32 connexon against a vast body of experimental reports. Our results provide new mechanistic insights into the effects of numerous spontaneous mutations and their implication in connexin-related pathologies. This model constitutes a step forward towards a structurally detailed description of the gap junction architecture and provides a structural platform to plan new biochemical and biophysical experiments aimed at elucidating the structure of connexin channels and hemichannels.
doi:10.1371/journal.pone.0002614
PMCID: PMC2481295  PMID: 18648547
21.  CaV1.2 I-II linker structure and Timothy syndrome 
Channels  2012;6(6):468-472.
CaV channels are multi-subunit protein complexes that enable inward cellular Ca2+ currents in response to membrane depolarization. We recently described structure-function studies of the intracellular α1 subunit domain I-II linker, directly downstream of domain IS6. The results show the extent of the linker’s helical structure to be subfamily dependent, as dictated by highly conserved primary sequence differences. Moreover, the difference in structure confers different biophysical properties, particularly the extent and kinetics of voltage and calcium-dependent inactivation. Timothy syndrome is a human genetic disorder due to mutations in the CaV1.2 gene. Here, we explored whether perturbation of the I-II linker helical structure might provide a mechanistic explanation for a Timothy syndrome mutant’s (human CaV1.2 G406R equivalent) biophysical effects on inactivation and activation. The results are equivocal, suggesting that a full mechanistic explanation for this Timothy syndrome mutation requires further investigation.
doi:10.4161/chan.22078
PMCID: PMC3536733  PMID: 22990809
Timothy syndrome; voltage-dependent calcium channels; voltage-dependent inactivation; α-helix
22.  Characterization of the gating brake in the I–II loop of CaV3 T-type calcium channels 
Channels  2010;4(6):453-458.
Our interest was drawn to the I–II loop of CaV3 channels for two reasons: (1) transfer of the I–II loop from a high voltage-activated channel (CaV2.2) to a low voltage-activated channel (CaV3.1) unexpectedly produced an ultra-low voltage activated channel; and (2) sequence variants of the I–II loop found in childhood absence epilepsy patients altered channel gating and increased surface expression of CaV3.2 channels. To determine the roles of this loop we have studied the structure of the loop and the biophysical consequences of altering its structure. Deletions localized the gating brake to the first 62 amino acids after IS6 in all three CaV3 channels, establishing the evolutionary conservation of this region and its function. Circular dichroism was performed on a purified fragment of the I–II loop from CaV3.2 to reveal a high α-helical content. De novo computer modeling predicted the gating brake formed a helix-loop-helix structure. This model was tested by replacing the helical regions with poly-proline-glycine (PGPGPG), which introduces kinks and flexibility. These mutations had profound effects on channel gating, shifting both steady-state activation and inactivation curves, as well as accelerating channel kinetics. Mutations designed to preserve the helical structure (poly-alanine, which forms α-helices) had more modest effects. Taken together, we conclude the second helix of the gating brake establishes important contacts with the gating machinery, thereby stabilizing a closed state of T-channels, and that this interaction is disrupted by depolarization, allowing the S6 segments to spread open and Ca2+ ions to flow through.
doi:10.4161/chan.4.6.12889
PMCID: PMC3052245  PMID: 21099341
calcium channels; epilepsy; ion channel gating; membrane potentials; molecular sequence data; mutation; protein structure
23.  An Amphipathic α-Helix Controls Multiple Roles of Brome Mosaic Virus Protein 1a in RNA Replication Complex Assembly and Function 
PLoS Pathogens  2009;5(3):e1000351.
Brome mosaic virus (BMV) protein 1a has multiple key roles in viral RNA replication. 1a localizes to perinuclear endoplasmic reticulum (ER) membranes as a peripheral membrane protein, induces ER membrane invaginations in which RNA replication complexes form, and recruits and stabilizes BMV 2a polymerase (2aPol) and RNA replication templates at these sites to establish active replication complexes. During replication, 1a provides RNA capping, NTPase and possibly RNA helicase functions. Here we identify in BMV 1a an amphipathic α-helix, helix A, and use NMR analysis to define its structure and propensity to insert in hydrophobic membrane-mimicking micelles. We show that helix A is essential for efficient 1a–ER membrane association and normal perinuclear ER localization, and that deletion or mutation of helix A abolishes RNA replication. Strikingly, mutations in helix A give rise to two dramatically opposite 1a function phenotypes, implying that helix A acts as a molecular switch regulating the intricate balance between separable 1a functions. One class of helix A deletions and amino acid substitutions markedly inhibits 1a–membrane association and abolishes ER membrane invagination, viral RNA template recruitment, and replication, but doubles the 1a-mediated increase in 2aPol accumulation. The second class of helix A mutations not only maintains efficient 1a–membrane association but also amplifies the number of 1a-induced membrane invaginations 5- to 8-fold and enhances viral RNA template recruitment, while failing to stimulate 2aPol accumulation. The results provide new insights into the pathways of RNA replication complex assembly and show that helix A is critical for assembly and function of the viral RNA replication complex, including its central role in targeting replication components and controlling modes of 1a action.
Author Summary
Positive-strand RNA viruses (one-third of all virus genera) transfer their genetic material between host cells as RNA of mRNA polarity, which are translated into proteins immediately upon entry. One immediate function of these proteins is to establish RNA replication compartments on intracellular membranes to copy the incoming viral RNA. Although much is known about the viral protein and RNA components in such replication complexes, little is understood about how the multiple protein–membrane–RNA interactions required for replication complex assembly are regulated. To study this, we used a well-established model virus that encodes only two replication proteins: an RNA polymerase enzyme that copies the viral RNA and an assembly-coordinating protein that guides the rearrangement of intracellular membranes to form replication compartments and recruits the viral RNA template and polymerase to these sites. We identified a small helix in this guiding replication protein that is essential for efficient association with and rearrangement of the correct intracellular membrane type and for regulating a switch between at least two different functional states of the replication guide protein. Mutations in this small helix interfere with separable guide protein functions, revealing new insights into the sequential steps in positive-strand RNA virus RNA replication complex formation.
doi:10.1371/journal.ppat.1000351
PMCID: PMC2654722  PMID: 19325881
24.  Caveolin-1 regulates VEGF-stimulated angiogenic activities in prostate cancer and endothelial cells 
Cancer biology & therapy  2009;8(23):2286-2296.
Caveolin-1 (cav-1) is a multifunctional protein and major component of caveolae membranes serving important functions related to signal transduction, endocytosis, transcytosis, and molecular transport. We previously showed that cav-1 is overexpressed and secreted by metastatic prostate cancer cells. We now report that cav-1 gene transduction (Adcav-1) or recombinant cav-1 (rcav-1) protein treatment of cav-1-negative prostate cancer cell line LP-LNCaP or cav-1-/- endothelial cells potentiated VEGF-stimulated angiogenic signaling.
Downregulation of cav-1 in prostate cancer cell line PC-3 or human umbilical vein endothelial cells (HUVECs) through cav-1 siRNA significantly reduced basal and VEGF-stimulated phosphorylation of VEGFR2 (Y951), PLCγ1 (Y783) and/or Akt (S473 & T308) relative to those in control siRNA treated cells. Additionally rcav-1 stimulation of cav-1 siRNA treated HUVECs restored this signaling pathway. Confocal microscopy and immunoprecipitation analysis revealed association and colocalization of VEGFR2 and PLCγ1 with cav-1 following VEGF stimulation in HUVECs. Interestingly, treatment of HUVECs with cav-1 scaffolding domain (CSD) caused significant reduction in the VEGF-stimulated phosphorylation of VEGFR2, PLCγ1 and Akt suggesting that CSD inhibits cav-1-mediated angiogenic signaling. VEGF stimulation of HUVECs significantly increased tubule length and cell migration, but this stimulatory effect was significantly reduced by cav-1 siRNA and/or CSD treatment.
The present study demonstrates that cav-1 regulates VEGF-stimulated VEGFR2 autophosphorylation and activation of downstream angiogenic signaling, possibly through compartmentalization of specific signaling molecules. Our results provide mechanistic insight into the role of cav-1 in prostate cancer and suggest the use of CSD as a therapeutic tool to suppress angiogenic signaling in prostate cancer.
PMCID: PMC2887683  PMID: 19923922
caveolin-1; VEGFR2; PLCγ1; caveolin-1 scaffolding domain; angiogenesis
25.  Structural and functional protein network analyses predict novel signaling functions for rhodopsin 
Proteomic analyses, literature mining, and structural data were combined to generate an extensive signaling network linked to the visual G protein-coupled receptor rhodopsin. Network analysis suggests novel signaling routes to cytoskeleton dynamics and vesicular trafficking.
Using a shotgun proteomic approach, we identified the protein inventory of the light sensing outer segment of the mammalian photoreceptor.These data, combined with literature mining, structural modeling, and computational analysis, offer a comprehensive view of signal transduction downstream of the visual G protein-coupled receptor rhodopsin.The network suggests novel signaling branches downstream of rhodopsin to cytoskeleton dynamics and vesicular trafficking.The network serves as a basis for elucidating physiological principles of photoreceptor function and suggests potential disease-associated proteins.
Photoreceptor cells are neurons capable of converting light into electrical signals. The rod outer segment (ROS) region of the photoreceptor cells is a cellular structure made of a stack of around 800 closed membrane disks loaded with rhodopsin (Liang et al, 2003; Nickell et al, 2007). In disc membranes, rhodopsin arranges itself into paracrystalline dimer arrays, enabling optimal association with the heterotrimeric G protein transducin as well as additional regulatory components (Ciarkowski et al, 2005). Disruption of these highly regulated structures and processes by germline mutations is the cause of severe blinding diseases such as retinitis pigmentosa, macular degeneration, or congenital stationary night blindness (Berger et al, 2010).
Traditionally, signal transduction networks have been studied by combining biochemical and genetic experiments addressing the relations among a small number of components. More recently, large throughput experiments using different techniques like two hybrid or co-immunoprecipitation coupled to mass spectrometry have added a new level of complexity (Ito et al, 2001; Gavin et al, 2002, 2006; Ho et al, 2002; Rual et al, 2005; Stelzl et al, 2005). However, in these studies, space, time, and the fact that many interactions detected for a particular protein are not compatible, are not taken into consideration. Structural information can help discriminate between direct and indirect interactions and more importantly it can determine if two or more predicted partners of any given protein or complex can simultaneously bind a target or rather compete for the same interaction surface (Kim et al, 2006).
In this work, we build a functional and dynamic interaction network centered on rhodopsin on a systems level, using six steps: In step 1, we experimentally identified the proteomic inventory of the porcine ROS, and we compared our data set with a recent proteomic study from bovine ROS (Kwok et al, 2008). The union of the two data sets was defined as the ‘initial experimental ROS proteome'. After removal of contaminants and applying filtering methods, a ‘core ROS proteome', consisting of 355 proteins, was defined.
In step 2, proteins of the core ROS proteome were assigned to six functional modules: (1) vision, signaling, transporters, and channels; (2) outer segment structure and morphogenesis; (3) housekeeping; (4) cytoskeleton and polarity; (5) vesicles formation and trafficking, and (6) metabolism.
In step 3, a protein-protein interaction network was constructed based on the literature mining. Since for most of the interactions experimental evidence was co-immunoprecipitation, or pull-down experiments, and in addition many of the edges in the network are supported by single experimental evidence, often derived from high-throughput approaches, we refer to this network, as ‘fuzzy ROS interactome'. Structural information was used to predict binary interactions, based on the finding that similar domain pairs are likely to interact in a similar way (‘nature repeats itself') (Aloy and Russell, 2002). To increase the confidence in the resulting network, edges supported by a single evidence not coming from yeast two-hybrid experiments were removed, exception being interactions where the evidence was the existence of a three-dimensional structure of the complex itself, or of a highly homologous complex. This curated static network (‘high-confidence ROS interactome') comprises 660 edges linking the majority of the nodes. By considering only edges supported by at least one evidence of direct binary interaction, we end up with a ‘high-confidence binary ROS interactome'. We next extended the published core pathway (Dell'Orco et al, 2009) using evidence from our high-confidence network. We find several new direct binary links to different cellular functional processes (Figure 4): the active rhodopsin interacts with Rac1 and the GTP form of Rho. There is also a connection between active rhodopsin and Arf4, as well as PDEδ with Rab13 and the GTP-bound form of Arl3 that links the vision cycle to vesicle trafficking and structure. We see a connection between PDEδ with prenyl-modified proteins, such as several small GTPases, as well as with rhodopsin kinase. Further, our network reveals several direct binary connections between Ca2+-regulated proteins and cytoskeleton proteins; these are CaMK2A with actinin, calmodulin with GAP43 and S1008, and PKC with 14-3-3 family members.
In step 4, part of the network was experimentally validated using three different approaches to identify physical protein associations that would occur under physiological conditions: (i) Co-segregation/co-sedimentation experiments, (ii) immunoprecipitations combined with mass spectrometry and/or subsequent immunoblotting, and (iii) utilizing the glycosylated N-terminus of rhodopsin to isolate its associated protein partners by Concanavalin A affinity purification. In total, 60 co-purification and co-elution experiments supported interactions that were already in our literature network, and new evidence from 175 co-IP experiments in this work was added. Next, we aimed to provide additional independent experimental confirmation for two of the novel networks and functional links proposed based on the network analysis: (i) the proposed complex between Rac1/RhoA/CRMP-2/tubulin/and ROCK II in ROS was investigated by culturing retinal explants in the presence of an ROCK II-specific inhibitor (Figure 6). While morphology of the retinas treated with ROCK II inhibitor appeared normal, immunohistochemistry analyses revealed several alterations on the protein level. (ii) We supported the hypothesis that PDEδ could function as a GDI for Rac1 in ROS, by demonstrating that PDEδ and Rac1 co localize in ROS and that PDEδ could dissociate Rac1 from ROS membranes in vitro.
In step 5, we use structural information to distinguish between mutually compatible (‘AND') or excluded (‘XOR') interactions. This enables breaking a network of nodes and edges into functional machines or sub-networks/modules. In the vision branch, both ‘AND' and ‘XOR' gates synergize. This may allow dynamic tuning of light and dark states. However, all connections from the vision module to other modules are ‘XOR' connections suggesting that competition, in connection with local protein concentration changes, could be important for transmitting signals from the core vision module.
In the last step, we map and functionally characterize the known mutations that produce blindness.
In summary, this represents the first comprehensive, dynamic, and integrative rhodopsin signaling network, which can be the basis for integrating and mapping newly discovered disease mutants, to guide protein or signaling branch-specific therapies.
Orchestration of signaling, photoreceptor structural integrity, and maintenance needed for mammalian vision remain enigmatic. By integrating three proteomic data sets, literature mining, computational analyses, and structural information, we have generated a multiscale signal transduction network linked to the visual G protein-coupled receptor (GPCR) rhodopsin, the major protein component of rod outer segments. This network was complemented by domain decomposition of protein–protein interactions and then qualified for mutually exclusive or mutually compatible interactions and ternary complex formation using structural data. The resulting information not only offers a comprehensive view of signal transduction induced by this GPCR but also suggests novel signaling routes to cytoskeleton dynamics and vesicular trafficking, predicting an important level of regulation through small GTPases. Further, it demonstrates a specific disease susceptibility of the core visual pathway due to the uniqueness of its components present mainly in the eye. As a comprehensive multiscale network, it can serve as a basis to elucidate the physiological principles of photoreceptor function, identify potential disease-associated genes and proteins, and guide the development of therapies that target specific branches of the signaling pathway.
doi:10.1038/msb.2011.83
PMCID: PMC3261702  PMID: 22108793
protein interaction network; rhodopsin signaling; structural modeling

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