Mitochondria are undoubtedly changed in Parkinson’s disease (PD), and mitochondrial functions are disrupted in genetic and pharmacologic models of PD. However, many of these changes might not truly drive neurodegeneration. PD is defined by the particular susceptibility of nigrostriatal dopamine (DA) neurons, but little is understood about the mitochondria in these cells. Here, we critically review the evidence that mitochondrial stressors cause PD. We then consider how changes in the intrinsic function of mitochondria and in their mass, distribution, and dynamics might synergize with an increased need for mitochondria and produce PD, and the importance of understanding how mitochondria contribute to its pathogenesis.
Since their discovery, midbrain dopamine (DA) neurons have been researched extensively, in part because of their diverse functions and involvement in various neuropsychiatric disorders. Over the last few decades, reports have emerged that midbrain DA neurons were not a homogeneous group, but that DA neurons located in distinct anatomical locations within the midbrain had distinctive properties in terms of physiology, function, and vulnerability. Accordingly, several studies focused on identifying heterogeneous gene expression across DA neuron clusters. Here we review the importance of understanding DA neuron heterogeneity at the molecular level, previous studies detailing heterogeneous gene expression in DA neurons, and finally recent work which brings together previous heterogeneous gene expression profiles in a coordinated manner, at single cell resolution.
Protein inclusions made up primarily of misfolded α-synuclein (α-Syn) are the hallmark of a set of disorders known as synucleionopathies, most notably Parkinson’s disease (PD). It is becoming increasingly appreciated that α-Syn misfolding can spread to anatomically connected regions in a prion-like manner. The protein aggregates that ensue are correlated with neurodegeneration in the various yet select neuronal populations that are affected. Recent advances have begun to shed light on the spreading and toxicity mechanisms that may be occurring in PD. Several key emerging themes are arising from this work suggesting that α-Syn mediated neurodegeneration is due to a combination of relative α-Syn expression level, connectivity to affected brain regions, and intrinsic vulnerability to pathology.
alpha-synuclein; Parkinson’s disease; protein misfolding; neurodegeneration; selective vulnerability
The Bacillus anthracis lethal factor (LF) is one component of a tripartite exotoxin partly responsible for persistent anthrax cytotoxicity after initial bacterial infection. Inhibitors of the zinc metalloproteinase have been investigated as potential therapeutic agents, but LF is a challenging target because inhibitors lack sufficient selectivity or possess poor pharmaceutical properties. These structural studies reveal an alternate conformation of the enzyme, induced upon binding of specific inhibitors, that opens a previously unobserved deep pocket termed S1′* which might afford new opportunities to design selective inhibitors that target this subsite.
Lethal factor; Anthrax; Ligand-induced; Conformational change; Structure-based drug design; Zinc hydrolase
Translocation of cell-penetrating peptides is often promoted by increased content of arginine or other guanidinum groups. However, relatively little research has considered the role of these functional groups on antimicrobial peptide activity. This study compared the activity of three histone-derived antimicrobial peptides—buforin II, DesHDAP1, and parasin— with variants that contain only lysine or arginine cationic residues. These peptides operate via different mechanisms as parasin causes membrane permeabilization while buforin II and DesHDAP1 translocate into bacteria. For all peptides, antibacterial activity increased with increased arginine content. Higher arginine content increased permeabilization for parasin while it improved translocation for buforin II and DesHDAP1. These observations provide insight into the relative importance of arginine and lysine in these antimicrobial peptides.
histone-derived antimicrobial peptide; buforin; parasin; membrane translocation; membrane permeabilization; arginine
Morphological disintegration of neurons is coupled invariably to neural death. In particular, disruption of outer segments of photoreceptor neurons triggers photoreceptor death regardless of the pathological stressors. We show that Ranbp2−/−::Tg-Ranbp2CLDm mice with mutations in SUMO-binding motif (SBM) of cyclophilin-like domain (CLD) of Ranbp2 expressed in a null Ranbp2 background lack untoward effects in photoreceptors in the absence of light-stress. However, compared to wild type photoreceptors, light-stress elicits profound disintegration of outer segments of Ranbp2−/−::Tg-Ranbp2CLDm with paradoxical age-dependent resistance of photoreceptors to death and genotype-independent caspase activation. Ranbp2−/−::Tg-Ranbp2CLDm exhibit photoreceptor death-independent changes in ubiquitin-proteasome system (UPS), but death-dependent increase of ubc9 levels. Hence, insidious functional impairment of SBM of Ranbp2’s CLD promotes neuroprotection and uncoupling of photoreceptor degeneration and death against phototoxicity.
HEN1-dependent methylation of the 3′-terminal nucleotide is a crucial step in plant microRNA (miRNA) biogenesis. Here we report that several viral RNA silencing suppressors (P1/HC-Pro, p21 and p19) inhibit miRNA methylation. These suppressors have distinct effects on different miRNAs. We also show that miRNA* is methylated in vivo in a suppressor-sensitive manner, suggesting that the viral proteins interfere with miRNA/miRNA* duplexes. p19 and p21 bind both methylated and unmethylated miRNA/miRNA* duplexes in vivo. These findings suggest miRNA/miRNA* as the in vivo substrates for the HEN1 miRNA methyltransferase and raise intriguing possibilities regarding the cellular location of miRNA methylation.
Viral RNA silencing suppressor; Methylation; microRNA/microRNA*; HEN1
The development of the hematopoietic system during early embryonic stages occurs in spatially and temporally distinct waves. Hematopoietic stem cells (HSC), the most potent and self‐renewing cells of this system, are produced in the final ‘definitive’ wave of hematopoietic cell generation. In contrast to HSCs in the adult, which differentiate via intermediate progenitor populations to produce functional blood cells, the generation of hematopoietic cells in the embryo prior to HSC generation occurs in the early waves by producing blood cells without intermediate progenitors (such as the ‘primitive’ hematopoietic cells). The lineage relationship between the early hematopoietic cells and the cells giving rise to HSCs, the genetic networks controlling their emergence, and the precise temporal determination of HSC fate remain topics of intense research and debate. This Review article discusses the current knowledge on the step‐wise embryonic establishment of the adult hematopoietic system, examines the roles of pivotal intrinsic regulators in this process, and raises questions concerning the temporal onset of HSC fate determination.
embryo; endothelial‐to‐hematopoietic cell transition; ES cell differentiation; Gata2; hematopoietic development; hematopoietic stem cells; hematopoietic progenitor cells; HSC fate; Runx1
Caspase-3-mediated p65 cleavage is believed to suppress nuclear factor-kappa B (NF-κB)-mediated anti-apoptotic transactivation in cells undergoing apoptosis. However, only a small percentage of p65 is cleaved during apoptosis, not in proportion to the dramatic reduction in NF-κB transactivation. Here we show that the p651-97 fragment generated by Caspase-3 cleavage interferes with ribosomal protein S3 (RPS3), an NF-κB “specifier” subunit, and selectively retards the nuclear translocation of RPS3, thus dampening the RPS3/NF-κB-dependent anti-apoptotic gene expression. Our findings reveal a novel cell fate determination mechanism to ensure cells undergo programed cell death through interfering with the RPS3/NF-κB-conferred anti-apoptotic transcription by the fragment from partial p65 cleavage by activated Caspase-3.
Fate determination; Apoptosis; NF-κB; RPS3; Gene transcription; Caspase-3 cleavage
Reliably pinpointing which specific amino acid residues form the interface(s) between a protein and its binding partner(s) is critical for understanding the structural and physicochemical determinants of protein recognition and binding affinity, and has wide applications in modeling and validating protein interactions predicted by high-throughput methods, in engineering proteins, and in prioritizing drug targets. Here, we review the basic concepts, principles and recent advances in computational approaches to the analysis and prediction of protein-protein interfaces. We point out caveats for objectively evaluating interface predictors, and discuss various applications of data-driven interface predictors for improving energy model-driven protein-protein docking. Finally, we stress the importance of exploiting binding partner information in reliably predicting interfaces and highlight recent advances in this emerging direction.
Protein-protein interactions; machine learning; docking; partner-specific interface prediction; cross validation on protein level; cross validation on instance level; evaluation caveats
Disruption of SMIM1, encoding Small Integral Membrane Protein 1, is responsible for the Vel-negative blood type, a rare but clinically-important blood type. However, the exact nature of the Vel antigen and how it is presented by SMIM1 are poorly understood. Using mass spectrometry we found several sites of phosphorylation in the N-terminal region of SMIM1 and we found the initiating methionine of SMIM1 to be acetylated. Flow cytometry analyses of human erythroleukemia cells expressing N- or C-terminally Flag-tagged SMIM1, several point mutants of SMIM1, and a chimeric molecule between Kell and SMIM1 demonstrated that SMIM1 carries the Vel antigen as a type II membrane protein with a predicted C-terminal extracellular domain of only 3 to 12 amino acids.
type II membrane protein; SMIM; blood group; mass spectrometry; phosphorylation
Embryonic stem cell (ES cell)‐based rat knockout technology, although successfully developed in 2010, has seen very limited usage to date due to low targeting efficiency and a lack of optimized procedures. In this study, we performed gene targeting in ES cells from the Sprague–Dawley (SD) and the Fischer 344 (F344) rat strains using an optimized procedure and the self‐excising neomycin (neo)‐positive selection cassette ACN to successfully generate Leptin and Trp53 knockout rats that did not carry the selection gene. These results demonstrate that our simplified targeting strategy using ACN provides an efficient approach to knock out many other rat genes.
ES cells; gene targeting; homologous recombination; rat; selection‐gene‐free
C2cd4c, encoded by a gene belonging to the C2cd4 family, contains a C2 domain conserved across species and is localized to the cytoplasm. To examine the role of C2cd4c in the pancreas, we studied its localization and generated C2cd4c knockout (KO) mice. C2cd4c was expressed in pancreatic endocrine progenitors at early embryonic stages. When endocrine cells arise from their precursors, C2cd4c is gradually confined to the insulin‐ and pancreatic polypeptide‐expressing cells of the endocrine. In the adult pancreas, C2cd4c is restricted to the beta cells. C2cd4c KO mice showed normal embryonic pancreatic development and adult pancreatic function. Thus, our results suggest that C2cd4c is dispensable for pancreatic development.
C2cd4; insulin; pancreas; pancreatic beta cell
A microRNA (miRNA) is a 21–24 nucleotide RNA product of a non-protein-coding gene. Plants, like animals, have a large number of miRNA-encoding genes in their genomes. The biogenesis of miRNAs in Arabidopsis is similar to that in animals in that miRNAs are processed from primary precursors by at least two steps mediated by RNAse III-like enzymes and that the miRNAs are incorporated into a protein complex named RISC. However, the biogenesis of plant miRNAs consists of an additional step, i.e., the miRNAs are methylated on the ribose of the last nucleotide by the miRNA methyltransferase HEN1. The high degree of sequence complementarity between plant miRNAs and their target mRNAs has facilitated the bioinformatic prediction of miRNA targets, many of which have been subsequently validated. Plant miRNAs have been predicted or confirmed to regulate a variety of processes, such as development, metabolism, and stress responses. A large category of miRNA targets consists of genes encoding transcription factors that play important roles in patterning the plant form.
microRNA; Small interfering RNA; DCL1; HEN1; Auxin; Flower development; Leaf development; Developmental transitions
Production of cellular reactive oxygen species (ROS) is typically associated with protein and DNA damage, toxicity, and death. However, ROS are also essential regulators of signaling and work in concert with redox-sensitive proteins to regulate cell homeostasis during stress. In this review, we focus on the redox regulation of mitophagy, a process that contributes to energetic tone as well as mitochondrial form and function. Mitophagy has been increasingly implicated in diseases including Parkinson’s, Amyotrophic Lateral Sclerosis, and cancer. Although these disease states employ different genetic mutations, they share the common factors of redox dysregulation and autophagic signaling. This review highlights key redox sensitive signaling molecules which can enhance neuronal survival by promoting temporally and spatially controlled autophagic signaling and mitophagy.
Mitophagy; Reactive oxygen species; Energetics; FOXO; HIF; Sirtuin; Atg; p66shc; Neurodegeneration; Amyotrophic lateral sclerosis; Isocitrate dehydrogenase; Mitochondria; Parkinson’s disease
Induced pluripotent stem cells (iPSCs) were first generated 10 years ago. Their ability to differentiate into any somatic cell type of the body including cardiomyocytes has already made them a valuable resource for modelling cardiac disease and drug screening. Initially human iPSCs were used mostly to model known disease phenotypes; more recently, and despite a number of recognised shortcomings, they have proven valuable in providing fundamental insights into the mechanisms of inherited heart disease with unknown genetic cause using surprisingly small cohorts. In this review, we summarise the progress made with human iPSCs as cardiac disease models with special focus on the latest mechanistic insights and related challenges. Furthermore, we suggest emerging solutions that will likely move the field forward.
cardiac disease modelling; induced pluripotent stem cell‐derived cardiomyocytes; molecular mechanisms
We identified the d‐galacturonic acid (GA)‐responsive transcriptional activator GaaR of the saprotrophic fungus, Aspergillus niger, which was found to be essential for growth on GA and polygalacturonic acid (PGA). Growth of the ΔgaaR strain was reduced on complex pectins. Genome‐wide expression analysis showed that GaaR is required for the expression of genes necessary to release GA from PGA and more complex pectins, to transport GA into the cell, and to induce the GA catabolic pathway. Residual growth of ΔgaaR on complex pectins is likely due to the expression of pectinases acting on rhamnogalacturonan and subsequent metabolism of the monosaccharides other than GA.
gene regulation; pectinase; polygalacturonic acid; transcriptomics; Zn2Cys6 transcription factor
Metallo-β-lactamases are the latest resistance mechanism of pathogenic and opportunistic bacteria against carbapenems, considered as last resort drugs. The worldwide spread of genes coding for these enzymes, together with the lack of a clinically useful inhibitor, have raised a sign of alarm. Inhibitor design has been mostly impeded by the structural diversity of these enzymes. Here we provide a critical review of mechanistic studies of the three known subclasses of metallo- β-lactamases, analyzed at the light of structural and mutagenesis investigations. We propose that these enzymes present a modular structure in their active sites that can be dissected into two halves: one providing the attacking nucleophile, and the second one stabilizing a negatively charged reaction intermediate. These are common mechanistic elements in all metallo-β-lactamases. Nucleophile activation does not necessarily requires a Zn(II) ion, but a Zn(II) center is essential for stabilization of the anionic intermediate. Design of a common inhibitor could be therefore approached based in these convergent mechanistic features despite the structural differences.
Metallo-β-lactamases; Mechanism; Antibiotic Resistance; Zinc enzymes; Drug Design
The sensory epithelium of the mammalian inner ear contains two types of mechanosensory cells: inner (IHC) and outer hair cells (OHC). They both transduce mechanical force generated by sound waves into electrical signals. In their apical end, these cells possess a set of stereocilia representing the mechanosensing organelles. IHC are responsible for detecting sounds and transmitting the acoustic information to the brain by converting graded depolarization into trains of action potentials in auditory nerve fibers. OHC are responsible for the active mechanical amplification process that leads to the fine tuning and high sensitivity of the mammalian inner ear. This active amplification is the consequence of the ability of OHC to alter their cell length in response to changes in membrane potential, and is controlled by an efferent inhibitory innervation. Medial olivocochlear efferent fibers, originating in the brainstem, synapse directly at the base of OHC and release acetylcholine. A very special type of nicotinic receptor, assembled by α9α10 subunits, participates in this synapse. Here we review recent knowledge and the role of both afferent and efferent synapse in the inner ear.
The Bacova_02091 gene in the β‐mannan utilization locus of Bacteroides ovatus encodes a family GH36 α‐galactosidase (BoGal36A), transcriptionally upregulated during growth on galactomannan. Characterization of recombinant BoGal36A reveals unique properties compared to other GH36 α‐galactosidases, which preferentially hydrolyse terminal α‐galactose in raffinose family oligosaccharides. BoGal36A prefers hydrolysing internal galactose substitutions from intact and depolymerized galactomannan. BoGal36A efficiently releases (> 90%) galactose from guar and locust bean galactomannans, resulting in precipitation of the polysaccharides. As compared to other GH36 structures, the BoGal36A 3D model displays a loop deletion, resulting in a wider active site cleft which likely can accommodate a galactose‐substituted polymannose backbone.
Bacteroides ovatus; galactomannan modification; GH36 α‐galactosidase; polysaccharide utilization locus
This article describes a rapid UPLC‐MS/MS method to quantitate novel bile acids in biological fluids and the evaluation of their diagnostic potential in Niemann‐Pick C (NPC). Two new compounds, NPCBA1 (3β‐hydroxy,7β‐N‐acetylglucosaminyl‐5‐cholenoic acid) and NPCBA2 (probably 3β,5α,6β‐trihydroxycholanoyl‐glycine), were observed to accumulate preferentially in NPC patients: median plasma concentrations of NPCBA1 and NPCBA2 were 40‐ and 10‐fold higher in patients than in controls. However, NPCBA1 concentrations were normal in some patients because they carried a common mutation inactivating the GlcNAc transferase required for the synthesis of this bile acid. NPCBA2, not containing a GlcNAc moiety, is thus a better NPC biomarker.
bile acids; Biomarkers; GlcNAc transferase; Niemann‐Pick C; screening; UPLC‐MS/MS
The stability of β‐catenin is very important for canonical Wnt signaling. A protein complex including Axin/APC/GSK3β phosphorylates β‐catenin to be degraded by ubiquitination with β‐TrCP. In the recent study, we isolated WDR26, a protein that binds to Axin. Here, we found that WDR26 is a negative regulator of the canonical Wnt signaling pathway, and that WDR26 affected β‐catenin levels. In addition, WDR26/Axin binding is involved in the ubiquitination of β‐catenin. These results suggest that WDR26 plays a negative role in β‐catenin degradation in the Wnt signaling pathway.
Axin1; ubiquitination; WDR26; Wnt; β‐catenin
Chronic obstructive pulmonary disease (COPD) is a common, highly debilitating disease of the airways, primarily caused by smoking. Chronic inflammation and structural remodelling are key pathological features of this disease, in part caused by the aberrant function of airway smooth muscle (ASM) cells under the regulation of transforming growth factor (TGF)‐β. miRNA are short, noncoding gene transcripts involved in the negative regulation of specific target genes, through their interactions with mRNA. Previous studies have proposed that mRNA‐145 (miR‐145) may interact with SMAD3, an important downstream signalling molecule of the TGF‐β pathway. TGF‐β was used to stimulate primary human ASM cells isolated from healthy nonsmokers, healthy smokers and COPD patients. This resulted in a TGF‐β‐dependent increase in CXCL8 and IL‐6 release, most notably in the cells from COPD patients. TGF‐β stimulation increased SMAD3 expression, only in cells from COPD patients, with a concurrent increased miR‐145 expression. Regulation of miR‐145 was found to be negatively controlled by pathways involving the MAP kinases, MEK‐1/2 and p38 MAPK. Subsequent, overexpression of miR‐145 (using synthetic mimics) in ASM cells from patients with COPD suppressed IL‐6 and CXCL8 release, to levels comparable to the nonsmoker controls. Therefore, this study suggests that miR‐145 negatively regulates pro‐inflammatory cytokine release from ASM cells in COPD by targeting SMAD3.
COPD; inflammation; microRNA
Mechanisms underlying the association between fibroblastic growth factor 23 (FGF-23) and inflammation are uncertain. We found that FGF-23 was markedly up-regulated in LPS/INF-γ-induced proinflammatory M1 macrophages and Hyp mouse-derived peritoneal macrophages, but not in IL-4-induced M2 anti-inflammatory macrophages. NF-κB and JAK/STAT1 pathways mediated the increased transcription of FGF-23 in response to M1 polarization. FGF-23 stimulated TNF-α, but not IL-6, expression in M0 macrophages and suppressed Arginase-1 expression in M2 macrophages through FGFR-mediated mechanisms. 1,25(OH)2D stimulated Arginase-1 expression and inhibited FGF-23 stimulation of TNF-α. FGF-23 has proinflammatory paracrine functions and counter-regulatory actions to 1,25(OH)2D on innate immune responses.
1,25(OH)2D; FGF-23; interferon gamma; Klotho; lipopolysaccharide; macrophages
A-Synuclein is found in plaques associated with Parkinson’s and other neurodegenerative diseases. Changes in α-synuclein oligomerization are thought to give rise to nucleation of neurodegenerative plaques. Here, we investigated the effect of hydrostatic pressure on the aggregation of α-synuclein in cultured neuronal cells. We found that hydrostatic pressure is associated with a transition from monomeric to higher order α-synuclein aggregates. We then tested whether this aggregation is associated with the loss of binding partners, such as phospholipase Cβ. We found that increased pressure reduces the level of PLCβ1 and the amount of α-synuclein / PLCβ1 complexes. These studies suggest that pressure promotes release of α-synuclein from protein partners promoting its oligomerization.