Non-coding RNAs (ncRNAs) are increasingly recognized as central players in diverse biological processes. DNA damage response (DDR) elicits a complex signaling cascade, which includes the induction of several ncRNA species. Recent studies indicate that ncRNAs are major regulators of the DDR. DNA-damage induced ncRNAs contribute to regulation of cell cycle, apoptosis and DNA repair, and thus play a key role in maintaining genome stability. This review summarizes the role of ncRNAs in DNA damage and repair.
noncoding RNAs; DNA damage; Repair; miRNAs; Long noncoding RNAs; Genome Integrity
Dominant mutations in the human insulin gene can lead to pancreatic β-cell dysfunction and diabetes mellitus due to toxic folding of a mutant proinsulin. Analogous to a classical mouse model (the Akita mouse), this monogenic syndrome highlights the susceptibility of human β-cells to endoreticular stress due to protein misfolding and aberrant aggregation. The clinical mutations directly or indirectly perturb native disulfide pairing. Whereas the majority of mutations introduce or remove a cysteine (leading in either case to an unpaired residue), non-cysteine-related mutations identify key determinants of folding efficiency. Studies of such mutations suggest that the evolution of insulin has been constrained not only by its structure and function, but also by the susceptibility of its single-chain precursor to impaired foldability.
hormone; human genetics; protein folding; folding efficiency; metabolism
•The interaction between Arabidopsis PEX5 and PEX14N is independent of cargo binding.•The affinity of a PTS1 peptide for PEX5 is unaffected by PEX14N binding.•Arabidopsis PEX5 complexes PTS1 and PTS2 cargoes.•PEX5 and 7 co-isolate with PEX14N, but the PTS2 cargo thiolase does not.•PEX14N does not unload canonical PTS1 cargo peptide in vitro but may play a role in PTS2 release.
PEX5 acts as a cycling receptor for import of PTS1 proteins into peroxisomes and as a co-receptor for PEX7, the PTS2 receptor, but the mechanism of cargo unloading has remained obscure. Using recombinant protein domains we show PEX5 binding to the PEX14N-terminal domain (PEX14N) has no effect on the affinity of PEX5 for a PTS1 containing peptide. PEX5 can form a complex containing both recombinant PTS1 cargo and endogenous PEX7-thiolase simultaneously but isolation of the complex via the PEX14 construct resulted in an absence of thiolase, suggesting a possible role for PEX14 in the unloading of PTS2 cargos.
Structured summary of protein interactions
pMDH1physically interacts with PEX5 by pull down (View interaction)
PEX5Cbinds to PEX14N by filter binding (View interaction)
PEX14Nbinds to PEX5C by pull down (View interaction)
PEX14Nphysically interacts with PEX7 by pull down (View interaction)
PEX5physically interacts with PEX7 by pull down (View interaction)
DCI1physically interacts with PEX5 by pull down (View interaction)
PEX5physically interacts with thiolase PTS2-cargo by pull down (View interaction)
pMDH1physically interacts with PEX7 by pull down (View interaction)
DCI1physically interacts with thiolase PTS2-cargo by pull down (View interaction)
DCI1physically interacts with PEX7 by pull down (View interaction)
PEX14Nphysically interacts with PEX5 by pull down (View interaction)
PEX, peroxisome biogenesis; PMP, peroxisomal membrane protein; PTS, peroxisomal targeting signal; FA, fluorescence anisotropy; FI, fluorescence intensity; HRP, horseradish peroxidase; Peroxisome; PEX5; PEX7; PEX14; PTS1; PTS2; Cargo unloading; Arabidopsis thaliana
The Protein Data Bank (PDB) was established in 1971 as a repository for the three dimensional structures of biological macromolecules. Since then, more than 85,000 biological macromolecule structures have been determined and made available in the PDB archive. Through analysis of the corpus of data, it is possible to identify trends that can be used to inform us about the future of structural biology and to plan the best ways to improve the management of the ever-growing amount of PDB data.
It has generally been believed that the diffusion limit set by the nuclear pore for protein is 60kDa. We here studied the cellular localization of several artificial proteins and found that the diffusion limit set by the nuclear pore is not as small as previously thought. The results indicate that the maximal size of protein to diffuse through the nuclear pore complex could be quite larger than 60kDa, thus greatly extending the diffusion limit that the nuclear pore can accommodate.
nuclear pore; protein nuclear translocation; diffusion limit
PGRN and its derived engineered protein, Atsttrin, were reported to antagonize TNFα and protect against inflammatory arthritis (Tang, W., et al, Science, 2011). Here we found that PGRN level was also significantly elevated in skin inflammation. PGRN−/− mice exhibited more severe inflammation following induction of oxazolone. In contrast, recombinant Atsttrin protein effectively attenuated inflammation in mice dermatitis model. In addition, the protective role of PGRN and Atsttrin in dermatitis was probably due to their inhibition on NF-κB signaling. Collectively, PGRN, especially its derived engineered protein, Atsttrin, may represent a potential molecular target for prevention and treatment of inflammatory skin diseases.
Progranulin; Atsttrin; dermatitis; NF-κB signaling
Mutations in with-no-lysine (K) kinase 4 (WNK4) and a ubiquitin E3 ligase complex component kelch-like 3 (KLHL3) both cause pseudohypoaldosteronism II (PHAII), a hereditary form of hypertension. We determined whether WNK4 or its effector is regulated by KLHL3 in Xenopus oocytes. KLHL3 inhibited the positive effect of WNK4 on Na+-Cl− cotransporter (NCC) by decreasing WNK4 protein abundance without decreasing that of NCC and the downstream kinase OSR1 directly. Ubiquitination and degradation of WNK4 were induced by KLHL3. The effect of KLHL3 on WNK4 degradation was blocked by a dominant negative form of cullin 3. All five PHAII mutations of KLHL3 tested disrupted the regulation on WNK4. We conclude that KLHL3 is a substrate adaptor for WNK4 in a ubiquitin E3 ligase complex.
KLHL3; cullin 3; WNK4; ubiquitin E3 ligase; ubiquitination
MauG catalyzes posttranslational modifications of a methylamine dehydrogenase precursor (preMADH) to complete the biosynthesis of its protein-derived tryptophan tryptophylquinone (TTQ) cofactor. Trp199 is present at the site of interaction between MauG and preMADH and is critical to this process as it mediates hole hopping during the inter-protein electron transfer that is required for catalysis. Trp199 was converted to Glu and the structure and reactivity of the W199E/preMADH complex were characterized. The results reveal that the nature of residue 199 is also important for productive complex formation between preMADH and MauG.
Electron transfer; protein-protein interaction; kinetic mechanism; cofactor biosynthesis
Mitochondria process local and global Ca2+ signals. Thereby the spatiotemporal patterns of mitochondrial Ca2+ signals determine whether the metabolism of these organelles is adjusted or cell death is executed. Mitochondrial Ca2+ channels of the inner mitochondrial membrane (IMM) actually implement mitochondrial uptake from cytosolic Ca2+ rises. Despite great efforts in the past, the identity of mitochondrial Ca2+ channels is still elusive. Numerous studies aimed to characterize mitochondrial Ca2+ uniport channels and provided a detailed profile of these great unknowns with important functions. This mini-review revisits previous research on the mechanisms of mitochondrial Ca2+ uptake and aligns them with most recent findings.
Calcium signaling; ER Ca2+ release; grp75; IP3 receptor; Letm1; mCa1; mCa2; MiCa; MCU; Mitochondrial Ca2+ uniporter; Mitofusin; p38MAPK; Uncoupling protein; UCP2/3
Polyglutamine (PolyQ) aggregates are a hallmark of several severe neurodegenerative diseases, expanded CAG-repeat diseases in which inheritance of an expanded polyQ sequence above a pathological threshold is associated with a high risk of disease. Application of vibrational circular dichroism (VCD) reveals that these PolyQ fibril aggregates exhibit a chiral supramolecular organization that is distinct from the supramolecular organization of previously observed amyloid fibrils. PolyQ fibrils grown from monomers with Q repeats 35 and above (Q≥35) exhibit approximately 10-fold enhancement of the same VCD spectrum compared to the already enhanced VCD of fibrils formed from Q repeats 30 and below (Q≤30).
Polyglutamine; PolyQ; fibril aggregates; chirality; vibrational circular dichroism; VCD; deep ultraviolet resonance Raman spectroscopy; DUVRR; hydrogen-deuterium exchange; CAG-repeat; neurodegenerative diseases
Fibroblast growth factors (FGFs) and their receptors are expressed in a variety of mammalian tissues, playing a role in development and cell proliferation. While analyzing human sperm motility, we found that sperm treated with endo-β-galactosidase (EBG), which specifically hydrolyzes poly-N-acetyllactosamine type glycans (polyLacs), enhanced motility. Mass spectrometry analysis revealed that sperm-associated polyLacs are heavily fucosylated, consistent with Lewis Y antigen. Immunohistochemistry of epididymis using an anti-Lewis Y antibody before and after EBG treatment suggested that polyLacs carrying the Lewis Y epitope are synthesized in epididymal epithelia and secreted to seminal fluid. EBG-treated sperm elevated cAMP levels and calcium influx, indicating activation of fibroblast growth factor signaling. Seminal fluid polyLacs bound to FGFs in vitro, and impaired FGF-mediated signaling in HEK293T cells.
Calorie restriction is reported to enhance survival and delay the onset of age-related decline in many different species. Several proteins have been proposed to play a role in mediating the response to calorie restriction, including the target of rapamycin kinase, sirtuins, and AMP kinase. An enhanced mechanistic understanding of calorie restriction has popularized the concept of “calorie restriction mimetics”, drugs that mimic the beneficial effects of caloire restriction without requiring a reduction in nutrient intake. In theory, such drugs should delay the onset and progression of multiple age-related diseases, similar to calorie restriction in mammals. Despite the potential benefits of such calorie restriction mimetics, however, relatively little is known about the interaction between genetic variation and individual response to calorie restriction. Limited evidence from model systems indicates that genotype plays a large role in determining both the magnitude and direction of effect that calorie restriction has on longevity. Here we present an overview of these data from the perspective of using yeast as a model to study aging and describe an approach we are taking to further characterize the molecular mechanisms underlying genotype-dependent responses to calorie restriction.
replicative lifespan; longevity; yeast; caloric restriction; calorie restriction; genotype by environment interaction
Semaphorins are known modulators of axonal sprouting and angiogenesis. In the retina, we identified a distinct and almost exclusive expression of Semaphorin 3F in the outer layers. Interestingly, these outer retinal layers are physiologically avascular. Using functional in vitro models, we report potent anti-angiogenic effects of Semaphorin 3F on both retinal and choroidal vessels. In addition, human retinal pigment epithelium isolates from patients with pathologic neovascularization of the outer retina displayed reduced Semaphorin 3F expression in 10 out of 15 patients. Combined, these results elucidate a functional role for Semaphorin 3F in the outer retina where it acts as a vasorepulsive cue to maintain physiologic avascularity.
Sema3F; Retina; AMD; RPE; Choroid
Cluster 1 streptokinases (SK1) from Streptococcus pyogenes (GAS) show substantially higher human plasminogen (hPg) activation activities and tighter hPg binding affinities than cluster 2b streptokinases (SK2b) in solution. The extent to which the different domains of SK are responsible for these differences is unknown. We exchanged each of the three known SK domains (α, β, and γ) between SK1 and SK2b and assessed the function of the resulting variants. Our results show that primary structural differences in the β-domains dictate these functional differences. This first report on the primary structure–functional relationship between naturally occurring SK1 and SK2b sheds new light on the mechanism of hPg activation by SK, a critical virulence determinant in this species of human pathogenic bacteria.
Streptokinase; Fibrinolysis; Protein domains; Virulence; Streptococcus pyogenes
•EPAC1 promotes AP1-dependent transcription in HUVECs.•Delineation of the minimal, EPAC1-responsive SOCS3 promoter in human cells.•Phosphorylated c-Jun (Ser 63) is constitutively associated with the SOCS3 promoter.•Phosphorylation of c-Jun on Ser63 requires activation of PKA, but not EPAC1.•c-Jun is required for SOCS3 induction by cyclic AMP in MEFs.
The cyclic AMP sensor, EPAC1, activates AP1-mediated transcription in HUVECs. Correspondingly, induction of the SOCS3 minimal promoter by EPAC1 requires a single AP1 site that constitutively binds phosphorylated (Ser63) c-Jun in DNA-pull-down assays. c-Jun (Ser63) becomes further phosphorylated following cyclic AMP stimulation and specific activation of protein kinase A (PKA), but not through selective activation of EPAC1. Moreover, despite a requirement for c-Jun for SOCS3 induction in fibroblasts, phospho-null c-Jun (Ser63/73Ala) had little effect on SOCS3 induction by cyclic AMP in HUVECs. AP1 activation and SOCS3 induction by EPAC1 in HUVECs therefore occur independently of c-Jun phosphorylation on Ser63.
Cyclic AMP; Transcription; SOCS3; EPAC1; c-Jun; AP1
•SNP rs1143684 results in either a Phe or Leu at position 47 in human NQO2.•NQO2-L47 has a slightly lower kcat/Km; it also has a lower Ki,app with resveratrol.•NQO2-L47 is more unstable to proteolysis and thermal denaturation.•NQO2-F47 (but not NQO2-L47) shows negative cooperativity towards resveratrol.•NQO2-L47 exists as multiple conformers in solution.
There are two common forms of NRH-quinone oxidoreductase 2 (NQO2) in the human population resulting from SNP rs1143684. One has phenylalanine at position 47 (NQO2-F47) and the other leucine (NQO2-L47). Using recombinant proteins, we show that these variants have similar steady state kinetic parameters, although NQO2-L47 has a slightly lower specificity constant. NQO2-L47 is less stable towards proteolytic digestion and thermal denaturation than NQO2-F47. Both forms are inhibited by resveratrol, but NQO2-F47 shows negative cooperativity with this inhibitor. Thus these data demonstrate, for the first time, clear biochemical differences between the variants which help explain previous biomedical and epidemiological findings.
rs1143684; Resveratrol; Cooperativity; Dihydronicotinamide riboside
Diabetic peripheral neuropathy is a major chronic diabetic complication. We have previously shown that in type 1 diabetic streptozotocin-treated mice, insulin- and TNF-α co-expressing bone marrow-derived cells (BMDCs) induced by hyperglycemia travel to nerve tissues where they fuse with nerve cells, causing premature apoptosis and nerve dysfunction. Here we show that similar BMDCs also occur in type 2 diabetic high-fat diet (HFD) mice. Furthermore, we found that hyperglycemia induces the co-expression of insulin and TNF-α in c-kit+Sca-1+lineage+ (KSL) progenitor cells, which maintain the same expression pattern in the progeny, which in turn participates in the fusion with neurons when transferred to normoglycemic animals.
Neuropathy; Hyperglycemia; Hematopoietic stem cell; Cell–cell fusion; Stem cell abnormalities
Metalloproteins have long been recognized as key determinants of endogenous contrast in magnetic resonance imaging (MRI) of biological subjects. More recently, both natural and engineered metalloproteins have been harnessed as biotechnological tools to probe gene expression, enzyme activity, and analyte concentrations by MRI. Metalloprotein MRI probes are paramagnetic and function by analogous mechanisms to conventional gadolinium or iron oxide-based MRI contrast agents. Compared with synthetic agents, metalloproteins typically offer worse sensitivity, but the possibilities of using protein engineering and targeted gene expression approaches in conjunction with metalloprotein contrast agents are powerful and sometimes definitive strengths. This review summarizes theoretical and practical aspects of metalloprotein-based contrast agents, and discusses progress in the exploitation of these proteins for molecular imaging applications.
metalloprotein; magnetic resonance imaging; protein engineering; contrast agent; sensor; molecular imaging
Recent experiments inside cells and in cytomimetic conditions have demonstrated that the crowded environments found therein can significantly reshape the energy landscapes of individual protein molecules and their oligomers. The resulting shifts in populations of conformational and oligomeric states have numerous biological consequences, including on the efficiency of replication and transcription, the development of aggregation-related diseases, and the efficacy of small-molecule drugs. Some of the effects of crowding can be anticipated from hard-particle theoretical models, but the in vitro and in vivo measurements indicate that these effects are often subtle and complex. These observations, coupled with recent computational studies at the atomistic level, suggest that the latter detailed modeling may be required to yield a quantitative understanding on the influences of the crowded cellular environments.
macromolecular crowding; protein folding; protein binding; protein aggregation; postprocessing
Pancreatic islet amyloid is a characteristic feature of type 2 diabetes. The major protein component of islet amyloid is the polypeptide hormone known as islet amyloid polypeptide (IAPP, or amylin). IAPP is stored with insulin in the β-cell secretory granules and is released in response to the stimuli that lead to insulin secretion. IAPP is normally soluble and is natively unfolded in its monomeric state, but forms islet amyloid in type 2 diabetes. Islet amyloid is not the cause of type 2 diabetes, but it leads to β-cell dysfunction and cell death, and contributes to the failure of islet cell transplantation. The mechanism of IAPP amyloid formation is not understood and the mechanisms of cytotoxicity are not fully defined.
Islet Amyloid Polypeptide; Amylin; Amyloid; Type 2 Diabetes; IAPP
Native mass spectrometry, or as is sometimes called “native electrospray (ESI)” allows proteins in their native or near-native protein in solution to be introduced into gas phase and interrogated by MS. This approach is now a powerful tool to investigate protein complexes. This article reviews the background of native MS of protein complexes and describes its strengths, taking photosynthetic pigment-protein complexes as examples. Native MS can be utilized in combination with other MS-based approaches to obtain complementary information to that provided by tools such as X-ray crystallography and NMR spectroscopy to understand the structure-function relationships of protein complexes. When additional information beyond that provided by native MS is required, other MS-based strategies can be successfully applied to augment the results of native MS.
ESI-MS; Native ESI; photosynthesis; pigment-protein complex; top-down
Small Heat Shock Proteins (sHSPs) are a diverse family of molecular chaperones that delay protein aggregation through interactions with nonnative and aggregate-prone protein states. This function has been shown to be important to cellular viability and sHSP function/dysfunction is implicated in many diseases, including Alzheimer’s and Alexander disease. Though their gene products are small, many sHSPs assemble into a distribution of large oligomeric states that undergo dynamic subunit exchange. These inherent properties present significant experimental challenges for characterizing sHSP oligomers. Of the ten mammalian sHSPs, the human sHSP 03B1;B crystallin is a paradigm example of sHSP oligomeric properties. Advances in our understanding of sHSP structure, oligomeric distribution, and dynamics have prompted the proposal of several models for the oligomeric states of B. The aim of this review is to highlight characteristics of αB crystallin (αB) that are key to understanding its structure and function. The current state of knowledge, existing models, and outstanding questions that remain to be addressed are presented.
small heat shock protein; αB crystallin; oligomers; pseudoatomic models
Post-translational modifications (PTMs) occur on nearly all proteins. Many domains within proteins are modified on multiple amino acid sidechains by diverse enzymes to create a myriad of possible protein species. How these combinations of PTMs lead to distinct biological outcomes is only beginning to be understood. This manuscript highlights several examples of combinatorial PTMs in proteins, and describes recent technological developments, which are driving our ability to understand how PTM patterns may “code” for biological outcomes.
post-translational modifications; histones; phosphorylation; epigenetics; signaling; methylation
The aging process is characterized by gradual changes to an organism's macromolecules, which negatively impacts biological processes. The complex macromolecular structure of chromatin regulates all nuclear processes requiring access to the DNA sequence. As such, maintenance of chromatin structure is an integral component to deter premature aging. In this review, we describe current research that links aging to chromatin structure. Histone modifications influence chromatin compaction and gene expression and undergo many changes during aging. Histone protein levels also decline during aging, dramatically affecting chromatin structure. Excitingly, lifespan can be extended by manipulations that reverse the age-dependent changes to chromatin structure, indicating the pivotal role chromatin structure plays during aging.
aging; chromatin; histone modifications; epigenetics
The interaction of the membrane traversing stator subunits a and b of the rotary ATP synthase was probed by substitution of a single Cys into each subunit with subsequent Cu+2 catalyzed cross-linking. Extensive interaction between the transmembrane (TM) region of one b subunit and TM2 of subunit a was indicated by cross-linking with 6 Cys pairs introduced into these regions. Additional disulfide cross-linking was observed between the N-terminus of subunit b and the periplasmic loop connecting TM4 and TM5 of subunit a. Finally, benzophenone-4-maleimide derivatized Cys in the 2–3 periplasmic loop of subunit a were shown to cross-link with the periplasmic N-terminal region of subunit b. These experiments help to define the juxtaposition of subunits b and a in the ATP synthase.
ATP synthase; transmembrane helix; disulfide cross-linking; benzophenone-4-maleimide; subunit a; subunit b