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PloS One 2024SlyD is a widely-occurring prokaryotic FKBP-family prolyl isomerase with an additional chaperone domain. Often, such as in Escherichia coli, a third domain is found at...
SlyD is a widely-occurring prokaryotic FKBP-family prolyl isomerase with an additional chaperone domain. Often, such as in Escherichia coli, a third domain is found at its C-terminus that binds nickel and provides it for nickel-enzyme biogenesis. SlyD has been found to bind signal peptides of proteins that are translocated by the Tat pathway, a system for the transport of folded proteins across membranes. Using peptide arrays to analyze these signal peptide interactions, we found that SlyD interacted only with positively charged peptides, with a preference for arginines over lysines, and large hydrophobic residues enhanced binding. Especially a twin-arginine motif was recognized, a pair of highly conserved arginines adjacent to a stretch of hydrophobic residues. Using isothermal titration calorimetry (ITC) with purified SlyD and a signal peptide-containing model Tat substrate, we could show that the wild type twin-arginine signal peptide was bound with higher affinity than an RR>KK mutated variant, confirming that positive charges are recognized by SlyD, with a preference of arginines over lysines. The specific role of negative charges of the chaperone domain surface and of hydrophobic residues in the chaperone active site was further analyzed by ITC of mutated SlyD variants. Our data show that the supposed key hydrophobic residues of the active site are indeed crucial for binding, and that binding is influenced by negative charges on the chaperone domain. Recognition of positive charges is likely achieved by a large negatively charged surface region of the chaperone domain, which is highly conserved although individual positions are variable.
Topics: Escherichia coli Proteins; Peptidylprolyl Isomerase; Escherichia coli; Protein Binding; Molecular Chaperones; Protein Sorting Signals; Hydrophobic and Hydrophilic Interactions; Calorimetry; Arginine; Amino Acid Sequence
PubMed: 38917203
DOI: 10.1371/journal.pone.0305823 -
ELife Jun 2024The emergence of new protein functions is crucial for the evolution of organisms. This process has been extensively researched for soluble enzymes, but it is largely...
The emergence of new protein functions is crucial for the evolution of organisms. This process has been extensively researched for soluble enzymes, but it is largely unexplored for membrane transporters, even though the ability to acquire new nutrients from a changing environment requires evolvability of transport functions. Here, we demonstrate the importance of environmental pressure in obtaining a new activity or altering a promiscuous activity in members of the amino acid-polyamine-organocation (APC)-type yeast amino acid transporters family. We identify APC members that have broader substrate spectra than previously described. Using in vivo experimental evolution, we evolve two of these transporter genes, and , toward new substrate specificities. Single mutations on these transporters are found to be sufficient for expanding the substrate range of the proteins, while retaining the capacity to transport all original substrates. Nonetheless, each adaptive mutation comes with a distinct effect on the fitness for each of the original substrates, illustrating a trade-off between the ancestral and evolved functions. Collectively, our findings reveal how substrate-adaptive mutations in membrane transporters contribute to fitness and provide insights into how organisms can use transporter evolution to explore new ecological niches.
Topics: Mutation; Saccharomyces cerevisiae; Amino Acid Transport Systems; Substrate Specificity; Evolution, Molecular; Polyamines; Saccharomyces cerevisiae Proteins; Genetic Fitness; Amino Acids
PubMed: 38916596
DOI: 10.7554/eLife.93971 -
International Journal of... Apr 2024The current meta-analysis aims to explore the potential correlation between natural resistance-associated macrophage protein 1 (NRAMP1) (3'-Untranslated region [3'-UTR])... (Meta-Analysis)
Meta-Analysis Review
A Systemic Review and Meta-analysis on Natural Resistance-associated Macrophage Protein 1 (3'-Untranslated Region) and Nucleotide-binding Oligomerization Domain-2 (rs8057341) Polymorphisms and Leprosy Susceptibility in Asian and Caucasian Populations.
The current meta-analysis aims to explore the potential correlation between natural resistance-associated macrophage protein 1 (NRAMP1) (3'-Untranslated region [3'-UTR]) and nucleotide-binding oligomerization domain-2 (NOD2 [rs8057341]) gene polymorphisms and their association with leprosy susceptibility in both Asian and Caucasian populations. Datas were retrieved from case control studies with NOD 2 and NRAMP 1 gene polymorphism associated with leprosy disease. Leprosy emerges as a particularly distinctive ailment among women on a global scale. The NRAMP1 (3'-UTR) and NOD2 (rs8057341) genetic variations play a crucial role in the progression of leprosy. A systematic review of relevant case-control studies was conducted across several databases, including ScienceDirect, PubMed, Google Scholar, and Embase. Utilizing MetaGenyo and Review Manager 5.4 Version, statistical analyses were carried out. Nine case-control studies totaling 3281 controls and 3062 leprosy patients are included in the research, with the objective of examining the potential association between NRAMP1 (3'-UTR) and NOD2 (rs8057341) gene polymorphisms and leprosy risk. The review methodology was registered in PROSPERO (ID520883). The findings reveal a robust association between NRAMP1 (3'-UTR) and NOD2 (rs8057341) gene polymorphisms and leprosy risk across various genetic models. Although the funnel plot analysis did not identify publication bias, bolstering these findings and elucidating potential gene-gene and gene-environment interactions require further comprehensive epidemiological research. This study identified a strong correlation between polymorphisms in the NOD2 (rs8057341) genes and susceptibility to leprosy across two genetic models. Further comprehensive epidemiological investigations are warranted to validate these findings and explore potential interactions between these genes and environmental factors.
Topics: Humans; Leprosy; Genetic Predisposition to Disease; Asian People; White People; Cation Transport Proteins; Nod2 Signaling Adaptor Protein; 3' Untranslated Regions; Polymorphism, Single Nucleotide; Case-Control Studies; Female; Polymorphism, Genetic; Male
PubMed: 38916380
DOI: 10.4103/ijmy.ijmy_43_24 -
BioRxiv : the Preprint Server For... Jun 2024Spermatogenesis is a biological process within the testis that produces haploid spermatozoa for the continuity of species. Sertoli cells are somatic cells in the...
Spermatogenesis is a biological process within the testis that produces haploid spermatozoa for the continuity of species. Sertoli cells are somatic cells in the seminiferous epithelium that orchestrate spermatogenesis. Cyclic reorganization of Sertoli cell actin cytoskeleton is vital for spermatogenesis, but the underlying mechanism remains largely unclear. Here, we report that RNA-binding protein PTBP1 controls Sertoli cell actin cytoskeleton reorganization by programming alternative splicing of actin cytoskeleton regulators. This splicing control enables ectoplasmic specializations, the actin-based adhesion junctions, to maintain the blood-testis barrier and support spermatid transport and transformation. Particularly, we show that PTBP1 promotes actin bundle formation by repressing the inclusion of exon 14 of , a kinase present at the ectoplasmic specialization. Our results thus reveal a novel mechanism wherein Sertoli cell actin cytoskeleton dynamics is controlled post-transcriptionally by utilizing functionally distinct isoforms of actin regulatory proteins, and PTBP1 is a critical regulatory factor in generating such isoforms.
PubMed: 38915624
DOI: 10.1101/2024.06.12.598725 -
BioRxiv : the Preprint Server For... Jun 2024Cdk5 is a highly-conserved, noncanonical cell division kinase important to the terminal differentiation of mammalian cells in multiple organ systems. We previously...
Cdk5 is a highly-conserved, noncanonical cell division kinase important to the terminal differentiation of mammalian cells in multiple organ systems. We previously identified Pef1, the ortholog of cdk5, as regulator of chronological lifespan. To reveal the processes impacted by Pef1, we developed APEX2-biotin phenol-mediated proximity labeling in Efficient labeling required a short period of cell wall digestion and eliminating glucose and nitrogen sources from the medium. We identified 255 high-confidence Pef1 neighbors in growing cells and a novel Pef1-interacting partner, the DNA damage response protein Rad24. The Pef1-Rad24 interaction was validated by reciprocal proximity labeling and co-immunoprecipitation. Eliminating Pef1 partially rescued the DNA damage sensitivity of cells lacking Rad24. To monitor how Pef1 neighbors change under different conditions, cells induced for autophagy were labeled and 177 high-confidence Pef1 neighbors were identified. Gene ontology (GO) analysis of the Pef1 neighbors identified proteins participating in processes required for autophagosome expansion including regulation of actin dynamics and vesicle-mediated transport. Some of these proteins were identified in both exponentially growing and autophagic cells. Pef1-APEX2 proximity labeling therefore identified a new Pef1 function in modulating the DNA damage response and candidate processes that Pef1 and other cdk5 orthologs may regulate.
PubMed: 38915521
DOI: 10.1101/2024.06.12.598664 -
BioRxiv : the Preprint Server For... Jun 2024Idiopathic pulmonary fibrosis is a fatal disease characterized by the TGF-β-dependent activation of lung fibroblasts, leading to excessive deposition of collagen...
Idiopathic pulmonary fibrosis is a fatal disease characterized by the TGF-β-dependent activation of lung fibroblasts, leading to excessive deposition of collagen proteins and progressive replacement of healthy lung with scar tissue. We and others have shown that fibroblast activation is supported by metabolic reprogramming, including the upregulation of the synthesis of glycine, the most abundant amino acid found in collagen protein. How fibroblast metabolic reprogramming is regulated downstream of TGF-β is incompletely understood. We and others have shown that TGF-β-mediated activation of the Mechanistic Target of Rapamycin Complex 1 (mTORC1) and downstream upregulation of Activating Transcription Factor 4 (ATF4) promote increased expression of the enzymes required for glycine synthesis; however, whether mTOR and ATF4 regulate other metabolic pathways in lung fibroblasts has not been explored. Here, we used RNA sequencing to determine how both ATF4 and mTOR regulate gene expression in human lung fibroblasts following TGF-β. We found that ATF4 primarily regulates enzymes and transporters involved in amino acid homeostasis as well as aminoacyl-tRNA synthetases. mTOR inhibition resulted not only in the loss of ATF4 target gene expression, but also in the reduced expression of glycolytic enzymes and mitochondrial electron transport chain subunits. Analysis of TGF-β-induced changes in cellular metabolite levels confirmed that ATF4 regulates amino acid homeostasis in lung fibroblasts while mTOR also regulates glycolytic and TCA cycle metabolites. We further analyzed publicly available single cell RNAseq data sets and found increased expression of ATF4 and mTOR metabolic targets in pathologic fibroblast populations from the lungs of IPF patients. Our results provide insight into the mechanisms of metabolic reprogramming in lung fibroblasts and highlight novel ATF4 and mTOR-dependent pathways that may be targeted to inhibit fibrotic processes.
PubMed: 38915485
DOI: 10.1101/2024.06.12.598694 -
Journal of Inflammation Research 2024Alzheimer's disease (AD) is the most common neurodegenerative illness, characterized by memory loss and cognitive decline, accounting for 60-80% of dementia cases. AD is... (Review)
Review
Alzheimer's disease (AD) is the most common neurodegenerative illness, characterized by memory loss and cognitive decline, accounting for 60-80% of dementia cases. AD is characterized by senile plaques made up of amyloid β (Aβ) protein, intracellular neurofibrillary tangles caused by hyperphosphorylation of tau protein linked with microtubules, and neuronal loss. Currently, therapeutic treatments and nanotechnological developments are effective in treating the symptoms of AD, but a cure for the illness has not yet been found. Recently, the increased study of extracellular vesicles (EVs) has led to a growing awareness of their significant involvement in neurodegenerative disorders, including AD. Exosomes are small extracellular vesicles that transport various components including messenger RNAs, non-coding RNAs, proteins, lipids, DNA, and other bioactive compounds from one cell to another, facilitating information transmission and material movement. There is growing evidence indicating that exosomes have complex functions in AD. Exosomes may have a dual role in Alzheimer's disease by contributing to neuronal death and also helping to alleviate the pathological progression of the disease. Therefore, the primary aim of this review is to outline the updated understandings on exosomes biogenesis and many functions of exosomes in the generation, conveyance, distribution, and elimination of hazardous proteins related to Alzheimer's disease. This review is intended to provide novel insights for understanding the development, specific treatment, and early detection of Alzheimer's disease.
PubMed: 38911990
DOI: 10.2147/JIR.S466821 -
Brain Communications 2024While voltage-gated potassium channels have critical roles in controlling neuronal excitability, they also have non-ion-conducting functions. Kv8.1, encoded by the KCNV1...
While voltage-gated potassium channels have critical roles in controlling neuronal excitability, they also have non-ion-conducting functions. Kv8.1, encoded by the KCNV1 gene, is a 'silent' ion channel subunit whose biological role is complex since Kv8.1 subunits do not form functional homotetramers but assemble with Kv2 to modify its ion channel properties. We profiled changes in ion channel expression in amyotrophic lateral sclerosis patient-derived motor neurons carrying a superoxide dismutase 1(A4V) mutation to identify what drives their hyperexcitability. A major change identified was a substantial reduction of KCNV1/Kv8.1 expression, which was also observed in patient-derived neurons with C9orf72 expansion. We then studied the effect of reducing KCNV1/Kv8.1 expression in healthy motor neurons and found it did not change neuronal firing but increased vulnerability to cell death. A transcriptomic analysis revealed dysregulated metabolism and lipid/protein transport pathways in KCNV1/Kv8.1-deficient motor neurons. The increased neuronal vulnerability produced by the loss of KCNV1/Kv8.1 was rescued by knocking down Kv2.2, suggesting a potential Kv2.2-dependent downstream mechanism in cell death. Our study reveals, therefore, unsuspected and distinct roles of Kv8.1 and Kv2.2 in amyotrophic lateral sclerosis-related neurodegeneration.
PubMed: 38911266
DOI: 10.1093/braincomms/fcae202 -
Cureus May 2024Introduction Type 2 diabetes mellitus (T2DM), a prevalent chronic metabolic disorder, necessitates multifaceted treatment approaches. Emerging studies highlight the...
Retrospective Observational Study on Assessing Sitagliptin and Dapagliflozin as a Fixed-Dose Combination in the Indian Population With Type 2 Diabetes Mellitus: The SIDAXA Study.
Introduction Type 2 diabetes mellitus (T2DM), a prevalent chronic metabolic disorder, necessitates multifaceted treatment approaches. Emerging studies highlight the cardiovascular advantages of sodium-glucose transport protein 2 (SGLT2) and dipeptidyl peptidase 4 (DPP-4) inhibitors in T2DM. This investigation delves into the synergistic effects of the fixed-dose combination (FDC) of sitagliptin and dapagliflozin, offering insights into its safety and efficacy for the Indian population. Methods This real-world, retrospective, observational study spanned 328 cases across 111 Indian centres, evaluating the safety, efficacy, and clinical utilization of the sitagliptin and dapagliflozin FDC in T2DM patients after obtaining ethical approval. Assessments at baseline, week four, and week 12 encompassed hemoglobin A1C (HbA1C), fasting plasma glucose (FPG), postprandial blood glucose (PPBG), low-density lipoprotein cholesterol (LDL-C), systolic blood pressure (SBP), diastolic blood pressure (DBP), and weight change. The statistical analysis was done using Statistical Package for Social Sciences (SPSS) version 29.0.1.0(171) (IBM Corp., Armonk, NY, USA) with a significance level p<0.05. Results Study participants [mean age: 51.14±5.55 years, 77.74% (n=255) males, 22.26% (n=73) females] exhibited prevalent risk factors like sedentary lifestyle (n=167, 50.91%) and smoking (n=147, 44.82%). Comorbidities included hypertension (n=235, 71.65%) and dyslipidaemia (n=139, 42.38%). Metformin (n=282, 85.98%) and sulfonylurea (n=134, 40.85%) were commonly prescribed concomitant oral antidiabetic agents (OADs). FDC administration significantly reduced HbA1c by 1.05 ± 0.83% (p < 0.0001) at week 12. FPG and PPBG showed significant reductions of 22.98 ± 22.23 mg/dL (p < 0.0001), 165.50 ± 37.02 mg/dL and 40.94 ± 36.04 mg/dL (p < 0.0001) at four weeks respectively. By week 12, significant reductions were noted in SBP (14.61±13.98mmHg reduction, p-value <0.0001), DBP (7.80±8.45mmHg reduction, p-value <0.0001), and LDL-C levels (18.14±23.95 mg/dL reduction, p-value <0.0001). In patients with established cardiovascular disease, there was reduction in HbA1c levels by 1.02 ± 0.63% after 12 weeks, with FPG decreasing by 54.52 ± 32.67 mg/dL and PPBG decreasing by 88.73 ± 44.90 mg/dL. Treatment-emergent adverse events included headache, changes in micturition, genital mycotic infection, and nausea and diarrhoea which were mild, transient, and necessitated no treatment discontinuation. Conclusion The FDC of sitagliptin and dapagliflozin significantly improved glycaemic control and lipid profiles in T2DM patients, particularly those with coronary artery disease. It demonstrated a favourable safety profile in the Indian population, signifying its potential as an effective and well-tolerated therapeutic option in patients with established cardiovascular disease.
PubMed: 38910691
DOI: 10.7759/cureus.60815 -
Biophysical Journal Jun 2024The ATP-binding cassette (ABC) transporter P-glycoprotein (P-gp) is a multidrug efflux pump that is overexpressed in a variety of cancers and associated with the drug...
The ATP-binding cassette (ABC) transporter P-glycoprotein (P-gp) is a multidrug efflux pump that is overexpressed in a variety of cancers and associated with the drug resistance phenomenon. P-gp structures were previously determined in detergent and in nanodiscs, in which different transmembrane helix conformations were found, "straight" and "kinked", respectively, indicating a possible role of the lipid environment on the P-gp structural ensemble. Here, we investigate the dynamic conformational ensembles and protein-lipid interactions of two human P-gp inward-open conformers, straight and kinked, employing all-atom molecular dynamics simulations in asymmetric multicomponent lipid bilayers that mimic the highly specialized hepatocyte membrane in which P-gp is expressed. The two conformers are found to differ in terms of the accessibility of the substrate cavity. The MD simulations show how cholesterol and different lipid species wedge, snorkel, and partially enter within the cavity of the straight P-gp conformer solved in detergent. However, the access to the cavity of kinked P-gp conformer solved in nanodiscs is restricted. Furthermore, the volume and dynamic fluctuations of the substrate cavity largely differ between the two P-gp structures, and are modulated by the presence (or absence) of cholesterol in the membrane and/or of ATP. From the mechanistic perspective, the findings indicate that the straight conformer likely precedes the kinked conformer in the functional working cycle of P-gp, with the latter conformation representing a post substrate-bound state. The inaccessibility of the main transmembrane cavity in the kinked conformer might be crucial in preventing substrate disengagement and transport withdrawal. Remarkably, in our unbiased MD simulations, one transmembrane helix (TM10) of the straight conformer underwent a spontaneous conformational transition to a kinked conformation, underlining the relevance of both conformations in a native phospholipid environment and revealing structural descriptors defining the transition between two P-gp conformers.
PubMed: 38909280
DOI: 10.1016/j.bpj.2024.06.020