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 3'-UTR and microRNA-24 regulate circadian rhythms by repressing PERIOD2 protein accumulation.
3'-UTR and microRNA-24 regulate circadian rhythms by repressing PERIOD2 protein accumulation.
We previously created two PER2::LUCIFERASE (PER2::LUC) circadian reporter knockin mice that differ only in the 3'-UTR region: , which retains the endogenous 3'-UTR and , where the endogenous 3'-UTR was replaced by an SV40 late poly(A) signal. To delineate the in vivo functions of 3'-UTR, we analyzed circadian rhythms of mice. Interestingly, mice displayed more than threefold stronger amplitude in bioluminescence rhythms than mice, and also exhibited lengthened free-running periods (∼24.0 h), greater phase delays following light pulse, and enhanced temperature compensation relative to Analysis of the 3'-UTR sequence revealed that miR-24, and to a lesser degree miR-30, suppressed PER2 protein translation, and the reversal of this inhibition in augmented PER2::LUC protein level and oscillatory amplitude. Interestingly, mRNA and protein oscillatory amplitude as well as CRY1 protein oscillation were increased in mice, suggesting rhythmic overexpression of PER2 enhances expression of and other core clock genes. Together, these studies provide important mechanistic insights into the regulatory roles of 3'-UTR, miR-24, and PER2 in expression and core clock function., Keywords: 3′-UTR regulation, Per2 gene, Circadian, MiR-24, MicroRNA, Grant Number: R01 GM114424, R01 GM112991, R01 GM111387, , P50 MH074924, R01 AG045828, Publication Note: This NIH-funded author manuscript originally appeared in PubMed Central at https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5651750.
14-3-3 Proteins in Glutamatergic Synapses.
14-3-3 Proteins in Glutamatergic Synapses.
The 14-3-3 proteins are a family of proteins that are highly expressed in the brain and particularly enriched at synapses. Evidence accumulated in the last two decades has implicated 14-3-3 proteins as an important regulator of synaptic transmission and plasticity. Here, we will review previous and more recent research that has helped us understand the roles of 14-3-3 proteins at glutamatergic synapses. A key challenge for the future is to delineate the 14-3-3-dependent molecular pathways involved in regulating synaptic functions., Publication Note: This NIH-funded author manuscript originally appeared in PubMed Central at https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5937437.
14-3-3 and aggresome formation
14-3-3 and aggresome formation
Protein misfolding and aggregation underlie the pathogenesis of many neurodegenerative diseases. In addition to chaperone-mediated refolding and proteasomal degradation, the aggresome-macroautophagy pathway has emerged as another defense mechanism for sequestration and clearance of toxic protein aggregates in cells. Previously, the 14-3-3 proteins were shown to be indispensable for the formation of aggresomes induced by mutant huntingtin proteins. In a recent study, we have determined that 14-3-3 functions as a molecular adaptor to recruit chaperone-associated misfolded proteins to dynein motors for transport to aggresomes. This molecular complex involves a dimeric binding of 14-3-3 to both the dynein-intermediate chain (DIC) and an Hsp70 co-chaperone Bcl-2-associated athanogene 3 (BAG3). As 14-3-3 has been implicated in various neurodegenerative diseases, our findings may provide mechanistic insights into its role in managing misfolded protein stress during the process of neurodegeneration., Keywords: 14-3-3, Aggresomes, Chaperones, Inclusion bodies, Neurodegeneration, Protein aggregation, Protein misfolding, Grant Number: R01 NS050355, NS50355, Publication Note: This NIH-funded author manuscript originally appeared in PubMed Central at https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4189886.
14-3-3 protein targets misfolded chaperone-associated proteins to aggresomes.
14-3-3 protein targets misfolded chaperone-associated proteins to aggresomes.
The aggresome is a key cytoplasmic organelle for sequestration and clearance of toxic protein aggregates. Although loading misfolded proteins cargos to dynein motors has been recognized as an important step in the aggresome formation process, the molecular machinery that mediates the association of cargos with the dynein motor is poorly understood. Here, we report a new aggresome-targeting pathway that involves isoforms of 14-3-3, a family of conserved regulatory proteins. 14-3-3 interacts with both the dynein-intermediate chain (DIC) and an Hsp70 co-chaperone Bcl-2-associated athanogene 3 (BAG3), thereby recruiting chaperone-associated protein cargos to dynein motors for their transport to aggresomes. This molecular cascade entails functional dimerization of 14-3-3, which we show to be crucial for the formation of aggresomes in both yeast and mammalian cells. These results suggest that 14-3-3 functions as a molecular adaptor to promote aggresomal targeting of misfolded protein aggregates and may link such complexes to inclusion bodies observed in various neurodegenerative diseases., Keywords: 14-3-3, Adaptor, Aggresome, BAG3, Dynein, Grant Number: R01 NS050355, R15 GM097326, NS50355, Publication Note: This NIH-funded author manuscript originally appeared in PubMed Central at https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3772389.
14-3-3 proteins are required for hippocampal long-term potentiation and associative learning and memory.
14-3-3 proteins are required for hippocampal long-term potentiation and associative learning and memory.
14-3-3 is a family of regulatory proteins highly expressed in the brain. Previous invertebrate studies have demonstrated the importance of 14-3-3 in the regulation of synaptic functions and learning and memory. However, the in vivo role of 14-3-3 in these processes has not been determined using mammalian animal models. Here, we report the behavioral and electrophysiological characterization of a new animal model of 14-3-3 proteins. These transgenic mice, considered to be a 14-3-3 functional knock-out, express a known 14-3-3 inhibitor in various brain regions of different founder lines. We identify a founder-specific impairment in hippocampal-dependent learning and memory tasks, as well as a correlated suppression in long-term synaptic plasticity of the hippocampal synapses. Moreover, hippocampal synaptic NMDA receptor levels are selectively reduced in the transgenic founder line that exhibits both behavioral and synaptic plasticity deficits. Collectively, our findings provide evidence that 14-3-3 is a positive regulator of associative learning and memory at both the behavioral and cellular level., Keywords: 14-3-3 proteins, NMDA receptors, Fear conditioning, Long-term potentiation, Passive avoidance, Grant Number: R01 NS050355, NS50355, Publication Note: This NIH-funded author manuscript originally appeared in PubMed Central at https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3972712.
14-3-3 proteins in neurological disorders.
14-3-3 proteins in neurological disorders.
14-3-3 proteins were originally discovered as a family of proteins that are highly expressed in the brain. Through interactions with a multitude of binding partners, 14-3-3 proteins impact many aspects of brain function including neural signaling, neuronal development and neuroprotection. Although much remains to be learned and understood, 14-3-3 proteins have been implicated in a variety of neurological disorders based on evidence from both clinical and laboratory studies. Here we will review previous and more recent research that has helped us understand the roles of 14-3-3 proteins in both neurodegenerative and neuropsychiatric diseases., Keywords: 14-3-3, Neurodegenerative diseases, Neurodevelopment, Neuropsychiatric diseases, Grant Number: R01 NS050355, Publication Note: This NIH-funded author manuscript originally appeared in PubMed Central at https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3388734.
14-3-3τ promotes surface expression of Cav2.2 (α1B) Ca2+ channels.
14-3-3τ promotes surface expression of Cav2.2 (α1B) Ca2+ channels.
Surface expression of voltage-gated Ca(2+) (Cav) channels is important for their function in calcium homeostasis in the physiology of excitable cells, but whether or not and how the α1 pore-forming subunits of Cav channels are trafficked to plasma membrane in the absence of the known Cav auxiliary subunits, β and α2δ, remains mysterious. Here we showed that 14-3-3 proteins promoted functional surface expression of the Cav2.2 α1B channel in transfected tsA-201 cells in the absence of any known Cav auxiliary subunit. Both the surface to total ratio of the expressed α1B protein and the current density of voltage step-evoked Ba(2+) current were markedly suppressed by the coexpression of a 14-3-3 antagonist construct, pSCM138, but not its inactive control, pSCM174, as determined by immunofluorescence assay and whole cell voltage clamp recording, respectively. By contrast, coexpression with 14-3-3τ significantly enhanced the surface expression and current density of the Cav2.2 α1B channel. Importantly, we found that between the two previously identified 14-3-3 binding regions at the α1B C terminus, only the proximal region (amino acids 1706-1940), closer to the end of the last transmembrane domain, was retained by the endoplasmic reticulum and facilitated by 14-3-3 to traffic to plasma membrane. Additionally, we showed that the 14-3-3/Cav β subunit coregulated the surface expression of Cav2.2 channels in transfected tsA-201 cells and neurons. Altogether, our findings reveal a previously unidentified regulatory function of 14-3-3 proteins in promoting the surface expression of Cav2.2 α1B channels., Keywords: 14-3-3tau, Calcium Channel, Cav 2.2 Channel, Cav Auxiliary Subunits, Electrophysiology, Endoplasmic Reticulum Retention, Membrane Trafficking, Protein-Protein Interaction, Trafficking, Publication Note: This NIH-funded author manuscript originally appeared in PubMed Central at https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4317001.
Absence Of Specific Yeast Heat-shock Proteins Leads To Abnormal Aggregation And Compromised Autophagic Clearance Of Mutant Huntingtin Proteins
Absence Of Specific Yeast Heat-shock Proteins Leads To Abnormal Aggregation And Compromised Autophagic Clearance Of Mutant Huntingtin Proteins
The functionality of a protein depends on its correct folding, but newly synthesized proteins are susceptible to aberrant folding and aggregation. Heat shock proteins (HSPs) function as molecular chaperones that aid in protein folding and the degradation of misfolded proteins. Trinucleotide (CAG) repeat expansion in the Huntingtin gene (HTT) results in the expression of misfolded Huntingtin protein (Htt), which contributes to the development of Huntington's disease. We previously found that the degradation of mutated Htt with polyQ expansion (Htt103QP) depends on both ubiquitin proteasome system and autophagy. However, the role of heat shock proteins in the clearance of mutated Htt remains poorly understood. Here, we report that cytosolic Hsp70 (Ssa family), its nucleotide exchange factors (Sse1 and Fes1), and a Hsp40 co-chaperone (Ydj1) are required for inclusion body formation of Htt103QP proteins and their clearance via autophagy. Extended induction of Htt103QPGFP leads to the formation of a single inclusion body in wild-type yeast cells, but mutant cells lacking these HSPs exhibit increased number of Htt103QP aggregates. Most notably, we detected more aggregated forms of Htt103QP in sse1 Delta. mutant cells using an agarose gel assay. Increased protein aggregates are also observed in these HSP mutants even in the absence Htt103QP overexpression. Importantly, these HSPs are required for autophagy- mediated Htt103QP clearance, but are less critical for proteasome-dependent degradation. These findings suggest a chaperone network that facilitates inclusion body formation of misfolded proteins and the subsequent autophagic clearance., Keywords: misfolded proteins, degradation, cytosolic proteins, factor fes1, hsp70 chaperones, inclusion-body formation, molecular chaperones, nucleotide exchange factor, quality-control, ubiquitin-proteasome system, Publication Note: The publisher's version of record is available at https://doi.org/10.1371/journal.pone.0191490
Absence of Myocardial Thyroid Hormone Inactivating Deiodinase Results in Restrictive          Cardiomyopathy in Mice
Absence of Myocardial Thyroid Hormone Inactivating Deiodinase Results in Restrictive Cardiomyopathy in Mice
Cardiac injury induces myocardial expression of the thyroid hormone inactivating type 3 deiodinase (D3), which in turn dampens local thyroid hormone signaling. Here, we show that the D3 gene (Dio3) is a tissue-specific imprinted gene in the heart, and thus, heterozygous D3 knockout (HtzD3KO) mice constitute a model of cardiac D3 inactivation in an otherwise systemically euthyroid animal. HtzD3KO newborns have normal hearts but later develop restrictive cardiomyopathy due to cardiac-specific increase in thyroid hormone signaling, including myocardial fibrosis, impaired myocardial contractility, and diastolic dysfunction. In wild-type littermates, treatment with isoproterenol-induced myocardial D3 activity and an increase in the left ventricular volumes, typical of cardiac remodeling and dilatation. Remarkably, isoproterenol-treated HtzD3KO mice experienced a further decrease in left ventricular volumes with worsening of the diastolic dysfunction and the restrictive cardiomyopathy, resulting in congestive heart failure and increased mortality. These findings reveal crucial roles for Dio3 in heart function and remodeling, which may have pathophysiologic implications for human restrictive cardiomyopathy., Keywords: cardiomyopathy, cardiotonic agents, dose-response relationship, gene expression profiling, gene expression regulation, heart, heart failure, infusions, intravenous, iodide peroxidase, isoproterenol, mice, inbred C57BL, muscle proteins, myocardium, RNA messenger, ventricular remodeling, Uncontrolled subjects: Animals, Animals, Newborn, Cardiomyopathy, Restrictive, Cardiotonic Agents, Dose-Response Relationship, Drug, Gene Expression Profiling, Gene Expression Regulation, Heart, Heart Failure, Infusions, Intravenous, Iodide Peroxidase, Isoproterenol, Male, Mice, Mice, Inbred C57BL, Mice, Knockout, Muscle Proteins, Myocardium, RNA, Messenger, Ventricular Remodeling, Note: Originally published in Molecular Endocrinology., Citation: Ueta CB, Oskouei BN, Olivares EL, Pinto JR, Correa MM, Simovic G, Simonides WS, Hare JM, Bianco AC. (2012). Absence of myocardial thyroid hormone inactivating deiodinase results in restrictive cardiomyopathy in mice. Mol Endocrinol, 26(5):809-18. doi: 10.1210/me.2011-1325.
Accelerated healing in NONcNZO10/LtJ type 2 diabetic mice by FGF 1
Accelerated healing in NONcNZO10/LtJ type 2 diabetic mice by FGF 1
The development of novel therapies to treat chronic diabetic ulcers depends upon appropriate animal models for early stage investigation. The NONcNZO10/LtJ mouse is a new polygenic strain developed to more realistically model human metabolic syndrome and obesity-induced Type 2 diabetes; however, detailed wound healing properties have not been reported. In this report we describe a quantitative wound healing study in the NONcNZO10/LtJ mouse using a splinted excisional wound. The rate of wound healing is compared to various controls, and is also quantified in response to topical administration of normal and mutant fibroblast growth factor-1 (FGF-1). Quantitation of re-epithelialization shows that the diabetic condition in the NONcNZO10/LtJ mouse is concomitant with a decreased rate of dermal healing. Furthermore, topical administration of a FGF-1/heparin formulation effectively accelerates re-epithelialization. A similar acceleration can also be achieved by a stabilized mutant form of FGF-1 formulated in the absence of heparin. Such accelerated rates of healing are not associated with any abnormal histology in the healed wounds. The results identify the NONcNZO10/LtJ mouse as a useful model of impaired wound healing in type II diabetes, and further, identify engineered forms of FGF-1 as a potential “second-generation” therapeutic to promote diabetic dermal wound healing., Keywords: Protein engineering, growth factor, diabetic ulcer, protein stability, fibroblast growth factor-1
Activation Profiles of Human Kallikrein-Related Peptidases by Matrix          Metalloproteinases
Activation Profiles of Human Kallikrein-Related Peptidases by Matrix Metalloproteinases
Abstract The 15 human kallikrein-related peptidases (KLKs) are clinically important biomarkers and therapeutic targets of interest in inflammation, cancer, and neurodegenerative disease. KLKs are secreted as inactive pro-forms (pro-KLKs) that are activated extracellularly by specific proteolytic release of their amino-terminal pro-peptide, and this is a key step in their functional regulation. Physiologically relevant KLK regulatory cascades of activation have been described in skin desquamation and semen liquefaction, and work by a large number of investigators has elucidated pairwise and autolytic activation relationships among the KLKs with the potential for more extensive activation cascades. More recent work has asked whether functional intersection of KLKs with other types of regulatory proteases exists. Such studies show a capacity for members of the thrombostasis axis to act as broad activators of pro-KLKs. In the present report, we ask whether such functional intersection is possible between the KLKs and the members of the matrix metalloproteinase (MMP) family by evaluating the ability of the MMPs to activate pro-KLKs. The results identify MMP-20 as a broad activator of pro-KLKs, suggesting the potential for intersection of the KLK and MMP axes under pathological dysregulation of MMP-20 expression., Keywords: activation cascade, enamelysin, kallikrein-related peptidase, KLK, matrix metalloproteinase 20 (MMP-20), Note: Originally published in Biological Chemistry. The final publication is available at www.degruyter.com., Citation: Yoon, H., Blaber, S., Li, W., Scarisbrick, I. and Blaber, M. (2013). Activation Profiles of Human Kallikrein-related Peptidases by Matrix Metalloproteinases. Biol Chem. 394 (1): 137-47. doi: 10.1515/hsz-2012-0249.
Activation Profiles of Human Kallikrein-Related Peptidases by Proteases of the          Thrombostasis Axis
Activation Profiles of Human Kallikrein-Related Peptidases by Proteases of the Thrombostasis Axis
The human kallikrein-related peptidases (KLKs) comprise 15 members (KLK1-15) and are the single largest family of serine proteases. The KLKs are utilized, or proposed, as clinically important biomarkers and therapeutic targets of interest in cancer and neurodegenerative disease. All KLKs appear to be secreted as inactive pro-forms (pro-KLKs) that are activated extracellularly by specific proteolytic release of their N-terminal pro-peptide. This processing is a key step in the regulation of KLK function. Much recent work has been devoted to elucidating the potential for activation cascades between members of the KLK family, with physiologically relevant KLK regulatory cascades now described in skin desquamation and semen liquefaction. Despite this expanding knowledge of KLK regulation, details regarding the potential for functional intersection of KLKs with other regulatory proteases are essentially unknown. To elucidate such interaction potential, we have characterized the ability of proteases associated with thrombostasis to hydrolyze the pro-peptide sequences of the KLK family using a previously described pro-KLK fusion protein system. A subset of positive hydrolysis results were subsequently quantified with proteolytic assays using intact recombinant pro-KLK proteins. Pro-KLK6 and 14 can be activated by both plasmin and uPA, with plasmin being the best activator of pro-KLK6 identified to date. Pro-KLK11 and 12 can be activated by a broad-spectrum of thrombostasis proteases, with thrombin exhibiting a high degree of selectivity for pro-KLK12. The results show that proteases of the thrombostasis family can efficiently activate specific pro-KLKs, demonstrating the potential for important regulatory interactions between these two major protease families., Keywords: kallikrein-related peptidases, KLK, activation cascade, thrombostasis, plasmin, thrombin, inflammation, Uncontrolled subjects: Enzyme Activation, Factor Xa, Fibrinolysin, Fibroblast Growth Factor 1, Humans, Hydrolysis, Kallikreins, Peptide Hydrolases, Plasma Kallikrein, Recombinant Fusion Proteins, Thrombosis, Note: Originally published in Protein Science, Citation: Yoon, H., Blaber, S.I., Evans, D.M., Trim, J., Juliano, M.A., Scarisbrick, I.A. and Blaber, M. (2008). Activation profiles of human kallikrein-related peptidases by proteases of the thrombostasis axis. Prot. Sci. 17, 1998-2007.
Acupoint Sensitization, Acupuncture Analgesia, Acupuncture on Visceral Functional Disorders, and Its Mechanism.
Acupoint Sensitization, Acupuncture Analgesia, Acupuncture on Visceral Functional Disorders, and Its Mechanism.
Publication Note: This NIH-funded author manuscript originally appeared in PubMed Central at https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4537726.
Acute BDNF treatment upregulates GluR1-SAP97 and GluR2-GRIP1 interactions
Acute BDNF treatment upregulates GluR1-SAP97 and GluR2-GRIP1 interactions
Brain-derived neurotrophic factor (BDNF) plays several prominent roles in synaptic plasticity and in learning and memory formation. Reduced BDNF levels and altered BDNF signaling have been reported in several brain diseases and behavioral disorders, which also exhibit reduced levels of AMPAr subunits. BDNF treatment acutely regulates AMPA receptor expression and function, including synaptic AMPAr subunit trafficking, and implicates several well defined signaling molecules that are required to elicit long term potentiation and depression (LTP and LTD, respectively). Long term encoding of synaptic events, as in long term memory formation, requires AMPAr stabilization and maintenance. However, factors regulating AMPAr stabilization in neuronal cell membranes and synaptic sites are not well characterized. In this study, we examine the effects of acute BDNF treatment on levels of AMPAr-associated scaffolding proteins and on AMPAr subunit-scaffolding protein interactions. We also examine the effects of BDNF-dependent enhanced interactions between AMPAr subunits with their specific scaffolding proteins on the accumulation of both types of proteins. Our results show that acute BDNF treatment upregulates the interactions between AMPAr subunits (GluR1 and GluR2) with their scaffold proteins SAP97 and GRIP1, respectively, leading to prolonged increased accumulation of both categories of proteins, albeit with distinct mechanisms for GluR1 and GluR2. Our findings reveal a new role for BDNF in the long term maintenance of AMPA receptor subunits and associated scaffolding proteins at synapses and further support the role of BDNF as a key regulator of synaptic consolidation. These results have potential implications for recent findings implicating BDNF and AMPAr subunits in various brain diseases and behavioral disorders., Publication Note: This NIH-funded author manuscript originally appeared in PubMed Central at https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3584105.
Akt mediated phosphorylation of LARP6; critical step in biosynthesis of type I collagen
Akt mediated phosphorylation of LARP6; critical step in biosynthesis of type I collagen
La ribonucleoprotein domain family, member 6 (LARP6) is the RNA binding protein, which regulates translation of collagen mRNAs and synthesis of type I collagen. Posttranslational modifications of LARP6 and how they affect type I collagen synthesis have not been studied. We show that in lung fibroblasts LARP6 is phosphorylated at 8 serines, 6 of which are located within C-terminal domain. Phosphorylation of LARP6 follows a hierarchical order; S451 phosphorylation being a prerequisite for phosphorylations of other serines. Inhibition of PI3K/Akt pathway reduced the phosphorylation of LARP6, but had no effect on the S451A mutant, suggesting that PI3K/Akt pathway targets S451 and we have identified Akt as the responsible kinase. Overexpression of S451A mutant had dominant negative effect on collagen biosynthesis; drastically reduced secretion of collagen and induced hyper-modifications of collagen alpha 2 (I) polypeptides. This indicates that LARP6 phosphorylation at S451 is critical for regulating translation and folding of collagen polypeptides. Akt inhibitor, GSK-2141795, which is in clinical trials for treatment of solid tumors, reduced collagen production by human lung fibroblasts with EC50 of 150 nM. This effect can be explained by inhibition of LARP6 phosphorylation and suggests that Akt inhibitors may be effective in treatment of various forms of fibrosis., Keywords: 2-dimensional electrophoresis, 3'-untranslated region, alpha-1(i) messenger-rna, cells, human lung fibroblasts, idiopathic pulmonary-fibrosis, inhibition, osteogenesis imperfecta, protein-kinase kinase, translation, Publication Note: The publisher’s version of record is available at http://www.dx.doi.org/10.1038/srep22597
Akt mediated phosphorylation of LARP6; critical step in biosynthesis of type I collagen.
Akt mediated phosphorylation of LARP6; critical step in biosynthesis of type I collagen.
La ribonucleoprotein domain family, member 6 (LARP6) is the RNA binding protein, which regulates translation of collagen mRNAs and synthesis of type I collagen. Posttranslational modifications of LARP6 and how they affect type I collagen synthesis have not been studied. We show that in lung fibroblasts LARP6 is phosphorylated at 8 serines, 6 of which are located within C-terminal domain. Phosphorylation of LARP6 follows a hierarchical order; S451 phosphorylation being a prerequisite for phosphorylations of other serines. Inhibition of PI3K/Akt pathway reduced the phosphorylation of LARP6, but had no effect on the S451A mutant, suggesting that PI3K/Akt pathway targets S451 and we have identified Akt as the responsible kinase. Overexpression of S451A mutant had dominant negative effect on collagen biosynthesis; drastically reduced secretion of collagen and induced hyper-modifications of collagen α2 (I) polypeptides. This indicates that LARP6 phosphorylation at S451 is critical for regulating translation and folding of collagen polypeptides. Akt inhibitor, GSK-2141795, which is in clinical trials for treatment of solid tumors, reduced collagen production by human lung fibroblasts with EC50 of 150 nM. This effect can be explained by inhibition of LARP6 phosphorylation and suggests that Akt inhibitors may be effective in treatment of various forms of fibrosis., Grant Number: R01 DK059466, 5R01DK059466-11, Publication Note: This NIH-funded author manuscript originally appeared in PubMed Central at https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4773855.
Allosteric Transmission along a Loosely Structured Backbone Allows a Cardiac Troponin C Mutant to Function with Only One Ca Ion.
Allosteric Transmission along a Loosely Structured Backbone Allows a Cardiac Troponin C Mutant to Function with Only One Ca Ion.
Hypertrophic cardiomyopathy (HCM) is one of the most common cardiomyopathies and a major cause of sudden death in young athletes. The Ca sensor of the sarcomere, cardiac troponin C (cTnC), plays an important role in regulating muscle contraction. Although several cardiomyopathy-causing mutations have been identified in cTnC, the limited information about their structural defects has been mapped to the HCM phenotype. Here, we used high-resolution electron-spray ionization mass spectrometry (ESI-MS), Carr-Purcell-Meiboom-Gill relaxation dispersion (CPMG-RD), and affinity measurements of cTnC for the thin filament in reconstituted papillary muscles to provide evidence of an allosteric mechanism in mutant cTnC that may play a role to the HCM phenotype. We showed that the D145E mutation leads to altered dynamics on a μs-ms time scale and deactivates both of the divalent cation-binding sites of the cTnC C-domain. CPMG-RD captured a low populated protein-folding conformation triggered by the Glu-145 replacement of Asp. Paradoxically, although D145E C-domain was unable to bind Ca, these changes along its backbone allowed it to attach more firmly to thin filaments than the wild-type isoform, providing evidence for an allosteric response of the Ca-binding site II in the N-domain. Our findings explain how the effects of an HCM mutation in the C-domain reflect up into the N-domain to cause an increase of Ca affinity in site II, thus opening up new insights into the HCM phenotype., Keywords: Calcium-binding protein, Cardiomyopathy, Nuclear magnetic resonance (NMR), Protein structure, Small-angle X-ray scattering (SAXS), Troponin, Grant Number: R01 HL128683, Publication Note: This NIH-funded author manuscript originally appeared in PubMed Central at https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5313108.
Alternative Folding Nuclei Definitions Facilitate the Evolution of a Symmetric Protein Fold from a Smaller Peptide Motif
Alternative Folding Nuclei Definitions Facilitate the Evolution of a Symmetric Protein Fold from a Smaller Peptide Motif
Protein 3° structure symmetry is a defining feature of nearly a third of protein folds and is generally thought to result from a combination of gene duplication, fusion, and truncation events. Such events represent major replication errors, involving substantial alteration of protein 3° structure as well as causing regions of exact repeating 1° structure, both of which are generally considered deleterious to protein folding. Thus, the prevalence of symmetric protein folds is counterintuitive and suggests a specific, yet unexplained, robustness. Using a designed β-trefoil protein, we show that purely symmetric 1° structure enables utilization of alternative definitions of the critical folding nucleus in response to gross structural rearrangement. Thus, major replication errors producing 1° structure symmetry can conserve foldability. The results provide an explanation for the prevalence of symmetric protein folds, and highlight a critical role for 1° structure symmetry in protein evolution., Keywords: protein folding, protein evolution, folding nucleus, protein design
Amide Hydrogens Reveal A Temperature-dependent Structural Transition That Enhances Site-ii Ca2+ -binding Affinity In A C-domain Mutant Of Cardiac Troponin C
Amide Hydrogens Reveal A Temperature-dependent Structural Transition That Enhances Site-ii Ca2+ -binding Affinity In A C-domain Mutant Of Cardiac Troponin C
The hypertrophic cardiomyopathy-associated mutant D145E, in cardiac troponin C (cTnC) C-domain, causes generalised instability at multiple sites in the isolated protein. As a result, structure and function of the mutant are more susceptible to higher temperatures. Above 25 degrees C there are large, progressive increases in N-domain Ca2+-binding affinity for D145E but only small changes for the wild-type protein. NMR-derived backbone amide temperature coefficients for many residues show a sharp transition above 30-40 degrees C, indicating a temperature-dependent conformational change that is most prominent around the mutated EF-hand IV, as well as throughout the C-domain. Smaller, isolated changes occur in the N-domain. Cardiac skinned fibres reconstituted with D145E are more sensitive to Ca2+ than fibres reconstituted with wild-type, and this defect is amplified near body-temperature. We speculate that the D145E mutation destabilises the native conformation of EF-hand IV, leading to a transient unfolding and dissociation of helix H that becomes more prominent at higher temperatures. This creates exposed hydrophobic surfaces that may be capable of binding unnaturally to a variety of targets, possibly including the N-domain of cTnC when it is in its open Ca2+-saturated state. This would constitute a potential route for propagating signals from one end of TnC to the other., Keywords: skeletal-muscle, hypertrophic cardiomyopathy, high-pressure, calcium-binding, chemical-shift index, n-domain, nmr-spectroscopy, protein secondary structure, regulatory domain, skinned ventricular-muscle, Publication Note: The publisher's version of record is available at https://doi.org/10.1038/s41598-017-00777-6
Amide hydrogens reveal a temperature-dependent structural transition that enhances site-II Ca(2+)-binding affinity in a C-domain mutant of cardiac troponin C.
Amide hydrogens reveal a temperature-dependent structural transition that enhances site-II Ca(2+)-binding affinity in a C-domain mutant of cardiac troponin C.
The hypertrophic cardiomyopathy-associated mutant D145E, in cardiac troponin C (cTnC) C-domain, causes generalised instability at multiple sites in the isolated protein. As a result, structure and function of the mutant are more susceptible to higher temperatures. Above 25 °C there are large, progressive increases in N-domain Ca(2+)-binding affinity for D145E but only small changes for the wild-type protein. NMR-derived backbone amide temperature coefficients for many residues show a sharp transition above 30-40 °C, indicating a temperature-dependent conformational change that is most prominent around the mutated EF-hand IV, as well as throughout the C-domain. Smaller, isolated changes occur in the N-domain. Cardiac skinned fibres reconstituted with D145E are more sensitive to Ca(2+) than fibres reconstituted with wild-type, and this defect is amplified near body-temperature. We speculate that the D145E mutation destabilises the native conformation of EF-hand IV, leading to a transient unfolding and dissociation of helix H that becomes more prominent at higher temperatures. This creates exposed hydrophobic surfaces that may be capable of binding unnaturally to a variety of targets, possibly including the N-domain of cTnC when it is in its open Ca(2+)-saturated state. This would constitute a potential route for propagating signals from one end of TnC to the other., Grant Number: R01 HL128683, Publication Note: This NIH-funded author manuscript originally appeared in PubMed Central at https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5429600.

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