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Cellular and Molecular Life Sciences :... Apr 2024In our prior investigation, we discerned loss-of-function variants within the gene encoding glutamine-rich protein 2 (QRICH2) in two consanguineous families, leading to...
In our prior investigation, we discerned loss-of-function variants within the gene encoding glutamine-rich protein 2 (QRICH2) in two consanguineous families, leading to various morphological abnormalities in sperm flagella and male infertility. The Qrich2 knockout (KO) in mice also exhibits multiple morphological abnormalities of the flagella (MMAF) phenotype with a significantly decreased sperm motility. However, how ORICH2 regulates the formation of sperm flagella remains unclear. Abnormal glutamylation levels of tubulin cause dysplastic microtubules and flagella, eventually resulting in the decline of sperm motility and male infertility. In the current study, by further analyzing the Qrich2 KO mouse sperm, we found a reduced glutamylation level and instability of tubulin in Qrich2 KO mouse sperm flagella. In addition, we found that the amino acid metabolism was dysregulated in both testes and sperm, leading to the accumulated glutamine (Gln) and reduced glutamate (Glu) concentrations, and disorderly expressed genes responsible for Gln/Glu metabolism. Interestingly, mice fed with diets devoid of Gln/Glu phenocopied the Qrich2 KO mice. Furthermore, we identified several mitochondrial marker proteins that could not be correctly localized in sperm flagella, which might be responsible for the reduced mitochondrial function contributing to the reduced sperm motility in Qrich2 KO mice. Our study reveals a crucial role of a normal Gln/Glu metabolism in maintaining the structural stability of the microtubules in sperm flagella by regulating the glutamylation levels of the tubulin and identifies Qrich2 as a possible novel Gln sensor that regulates microtubule glutamylation and mitochondrial function in mouse sperm.
Topics: Animals; Humans; Male; Mice; Glutamic Acid; Glutamine; Infertility, Male; Mice, Knockout; Microtubules; Mitochondria; Mitochondrial Proteins; Semen; Sperm Motility; Spermatozoa; Tubulin
PubMed: 38597976
DOI: 10.1007/s00018-024-05177-4 -
Neural Regeneration Research Dec 2024Parkinson's disease is characterized by the selective degeneration of dopamine neurons in the nigrostriatal pathway and dopamine deficiency in the striatum. The precise...
Parkinson's disease is characterized by the selective degeneration of dopamine neurons in the nigrostriatal pathway and dopamine deficiency in the striatum. The precise reasons behind the specific degeneration of these dopamine neurons remain largely elusive. Genetic investigations have identified over 20 causative PARK genes and 90 genomic risk loci associated with both familial and sporadic Parkinson's disease. Notably, several of these genes are linked to the synaptic vesicle recycling process, particularly the clathrin-mediated endocytosis pathway. This suggests that impaired synaptic vesicle recycling might represent an early feature of Parkinson's disease, followed by axonal degeneration and the eventual loss of dopamine cell bodies in the midbrain via a "dying back" mechanism. Recently, several new animal and cellular models with Parkinson's disease-linked mutations affecting the endocytic pathway have been created and extensively characterized. These models faithfully recapitulate certain Parkinson's disease-like features at the animal, circuit, and cellular levels, and exhibit defects in synaptic membrane trafficking, further supporting the findings from human genetics and clinical studies. In this review, we will first summarize the cellular and molecular findings from the models of two Parkinson's disease-linked clathrin uncoating proteins: auxilin (DNAJC6/PARK19) and synaptojanin 1 (SYNJ1/PARK20). The mouse models carrying these two PARK gene mutations phenocopy each other with specific dopamine terminal pathology and display a potent synergistic effect. Subsequently, we will delve into the involvement of several clathrin-mediated endocytosis-related proteins (GAK, endophilin A1, SAC2/INPP5F, synaptotagmin-11), identified as Parkinson's disease risk factors through genome-wide association studies, in Parkinson's disease pathogenesis. We will also explore the direct or indirect roles of some common Parkinson's disease-linked proteins (alpha-synuclein (PARK1/4), Parkin (PARK2), and LRRK2 (PARK8)) in synaptic endocytic trafficking. Additionally, we will discuss the emerging novel functions of these endocytic proteins in downstream membrane traffic pathways, particularly autophagy. Given that synaptic dysfunction is considered as an early event in Parkinson's disease, a deeper understanding of the cellular mechanisms underlying synaptic vesicle endocytic trafficking may unveil novel targets for early diagnosis and the development of interventional therapies for Parkinson's disease. Future research should aim to elucidate why generalized synaptic endocytic dysfunction leads to the selective degeneration of nigrostriatal dopamine neurons in Parkinson's disease.
PubMed: 38595283
DOI: 10.4103/NRR.NRR-D-23-01624 -
Nature Cell Biology Apr 2024The localization of transcriptional activity in specialized transcription bodies is a hallmark of gene expression in eukaryotic cells. It remains unclear, however, if...
The localization of transcriptional activity in specialized transcription bodies is a hallmark of gene expression in eukaryotic cells. It remains unclear, however, if and how transcription bodies affect gene expression. Here we disrupted the formation of two prominent endogenous transcription bodies that mark the onset of zygotic transcription in zebrafish embryos and analysed the effect on gene expression using enriched SLAM-seq and live-cell imaging. We find that the disruption of transcription bodies results in the misregulation of hundreds of genes. Here we focus on genes that are upregulated. These genes have accessible chromatin and are poised to be transcribed in the presence of the two transcription bodies, but they do not go into elongation. Live-cell imaging shows that disruption of the two large transcription bodies enables these poised genes to be transcribed in ectopic transcription bodies, suggesting that the large transcription bodies sequester a pause release factor. Supporting this hypothesis, we find that CDK9-the kinase that releases paused polymerase II-is highly enriched in the two large transcription bodies. Overexpression of CDK9 in wild-type embryos results in the formation of ectopic transcription bodies and thus phenocopies the removal of the two large transcription bodies. Taken together, our results show that transcription bodies regulate transcription by sequestering machinery, thereby preventing genes elsewhere in the nucleus from being transcribed.
Topics: Animals; Chromatin; Gene Expression; Positive Transcriptional Elongation Factor B; RNA Polymerase II; Transcription, Genetic; Zebrafish
PubMed: 38589534
DOI: 10.1038/s41556-024-01389-9 -
The Journal of Biological Chemistry May 2024DNA replication in Escherichia coli starts with loading of the replicative helicase, DnaB, onto DNA. This reaction requires the DnaC loader protein, which forms a 6:6...
DNA replication in Escherichia coli starts with loading of the replicative helicase, DnaB, onto DNA. This reaction requires the DnaC loader protein, which forms a 6:6 complex with DnaB and opens a channel in the DnaB hexamer through which single-stranded DNA is thought to pass. During replication, replisomes frequently encounter DNA damage and nucleoprotein complexes that can lead to replication fork collapse. Such events require DnaB re-loading onto DNA to allow replication to continue. Replication restart proteins mediate this process by recruiting DnaB/DnaC to abandoned DNA replication forks. Several dnaC mutations that bypass the requirement for replication restart proteins or that block replication restart have been identified in E. coli. To better understand how these DnaC variants function, we have purified and characterized the protein products of several such alleles. Unlike wild-type DnaC, three of the variants (DnaC 809, DnaC 809,820, and DnaC 811) can load DnaB onto replication forks bound by single-stranded DNA-binding protein. DnaC 809 can also load DnaB onto double-stranded DNA. These results suggest that structural changes in the variant DnaB/DnaC complexes expand the range of DNA substrates that can be used for DnaB loading, obviating the need for the existing replication restart pathways. The protein product of dnaC1331, which phenocopies deletion of the priB replication restart gene, blocks loading through the major restart pathway in vitro. Overall, the results of our study highlight the utility of bacterial DnaC variants as tools for probing the regulatory mechanisms that govern replicative helicase loading.
Topics: Escherichia coli Proteins; Escherichia coli; DnaB Helicases; DNA Replication; DNA, Bacterial; DNA, Single-Stranded; Mutation
PubMed: 38588814
DOI: 10.1016/j.jbc.2024.107275 -
BioRxiv : the Preprint Server For... May 2024Prostate cancer is an exemplar of an enhancer-binding transcription factor-driven disease. The androgen receptor (AR) enhanceosome complex comprised of chromatin and...
Prostate cancer is an exemplar of an enhancer-binding transcription factor-driven disease. The androgen receptor (AR) enhanceosome complex comprised of chromatin and epigenetic coregulators assembles at enhancer elements to drive disease progression. The paralog lysine acetyltransferases p300 and CBP deposit histone marks that are associated with enhancer activation. Here, we demonstrate that p300/CBP are determinant cofactors of the active AR enhanceosome in prostate cancer. Histone H2B N-terminus multisite lysine acetylation (H2BNTac), which is exclusively reliant on p300/CBP catalytic function, marked active enhancers and was notably elevated in prostate cancer lesions relative to the adjacent benign epithelia. Degradation of p300/CBP rapidly depleted acetylation marks associated with the active AR enhanceosome, which was only partially phenocopied by inhibition of their reader bromodomains. Notably, H2BNTac was effectively abrogated only upon p300/CBP degradation, which led to a stronger suppression of p300/CBP-dependent oncogenic gene programs relative to bromodomain inhibition or the inhibition of its catalytic domain. experiments using an orally active p300/CBP proteolysis targeting chimera (PROTAC) degrader (CBPD-409) showed that p300/CBP degradation potently inhibited tumor growth in preclinical models of castration-resistant prostate cancer and synergized with AR antagonists. While mouse p300/CBP orthologs were effectively degraded in host tissues, prolonged treatment with the PROTAC degrader was well tolerated with no significant signs of toxicity. Taken together, our study highlights the pivotal role of p300/CBP in maintaining the active AR enhanceosome and demonstrates how target degradation may have functionally distinct effects relative to target inhibition, thus supporting the development of p300/CBP degraders for the treatment of advanced prostate cancer.
PubMed: 38586029
DOI: 10.1101/2024.03.29.587346 -
MedRxiv : the Preprint Server For... Mar 2024To identify genetic etiologies and genotype/phenotype associations for unsolved ocular congenital cranial dysinnervation disorders (oCCDDs).
PURPOSE
To identify genetic etiologies and genotype/phenotype associations for unsolved ocular congenital cranial dysinnervation disorders (oCCDDs).
METHODS
We coupled phenotyping with exome or genome sequencing of 467 pedigrees with genetically unsolved oCCDDs, integrating analyses of pedigrees, human and animal model phenotypes, and variants to identify rare candidate single nucleotide variants, insertion/deletions, and structural variants disrupting protein-coding regions. Prioritized variants were classified for pathogenicity and evaluated for genotype/phenotype correlations.
RESULTS
Analyses elucidated phenotypic subgroups, identified pathogenic/likely pathogenic variant(s) in 43/467 probands (9.2%), and prioritized variants of uncertain significance in 70/467 additional probands (15.0%). These included known and novel variants in established oCCDD genes, genes associated with syndromes that sometimes include oCCDDs (e.g., genes that fit the syndromic component of the phenotype but had no prior oCCDD association (e.g., ), genes with no reported association with oCCDDs or the syndromic phenotypes (e.g., ), and genes associated with oCCDD phenocopies that had resulted in misdiagnoses.
CONCLUSION
This study suggests that unsolved oCCDDs are clinically and genetically heterogeneous disorders often overlapping other Mendelian conditions and nominates many candidates for future replication and functional studies.
PubMed: 38585811
DOI: 10.1101/2024.03.22.24304594 -
Neurobiology of Disease Jun 2024Charcot-Marie-Tooth disease (CMT) is a genetic peripheral neuropathy caused by mutations in many functionally diverse genes. The aminoacyl-tRNA synthetase (ARS) enzymes,...
Charcot-Marie-Tooth disease (CMT) is a genetic peripheral neuropathy caused by mutations in many functionally diverse genes. The aminoacyl-tRNA synthetase (ARS) enzymes, which transfer amino acids to partner tRNAs for protein synthesis, represent the largest protein family genetically linked to CMT aetiology, suggesting pathomechanistic commonalities. Dominant intermediate CMT type C (DI-CMTC) is caused by YARS1 mutations driving a toxic gain-of-function in the encoded tyrosyl-tRNA synthetase (TyrRS), which is mediated by exposure of consensus neomorphic surfaces through conformational changes of the mutant protein. In this study, we first showed that human DI-CMTC-causing TyrRS mis-interacts with the extracellular domain of the BDNF receptor TrkB, an aberrant association we have previously characterised for several mutant glycyl-tRNA synthetases linked to CMT type 2D (CMT2D). We then performed temporal neuromuscular assessments of Yars mice modelling DI-CMT. We determined that Yars homozygotes display a selective, age-dependent impairment in in vivo axonal transport of neurotrophin-containing signalling endosomes, phenocopying CMT2D mice. This impairment is replicated by injection of recombinant TyrRS, but not TyrRS, into muscles of wild-type mice. Augmenting BDNF in DI-CMTC muscles, through injection of recombinant protein or muscle-specific gene therapy, resulted in complete axonal transport correction. Therefore, this work identifies a non-cell autonomous pathomechanism common to ARS-related neuropathies, and highlights the potential of boosting BDNF levels in muscles as a therapeutic strategy.
Topics: Animals; Charcot-Marie-Tooth Disease; Brain-Derived Neurotrophic Factor; Mice; Axonal Transport; Disease Models, Animal; Tyrosine-tRNA Ligase; Humans; Mice, Transgenic; Muscle, Skeletal; Receptor, trkB; Mutation
PubMed: 38583640
DOI: 10.1016/j.nbd.2024.106501 -
The Journal of Investigative Dermatology Apr 2024Chronic non-healing wounds negatively impact quality of life and are a significant financial drain on health systems. The risk of infection that exacerbates...
Chronic non-healing wounds negatively impact quality of life and are a significant financial drain on health systems. The risk of infection that exacerbates comorbidities in patients necessitates regular application of wound care. Understanding the mechanisms underlying impaired wound healing are therefore a key priority to inform effective new-generation treatments. In this study, we demonstrate that 14-3-3-mediated suppression of signaling through ROCK is a critical mechanism that inhibits the healing of diabetic wounds. Accordingly, pharmacological inhibition of 14-3-3 by topical application of the sphingo-mimetic drug RB-11 to diabetic wounds on a mouse model of type II diabetes accelerated wound closure more than 2-fold than vehicle control, phenocopying our previous observations in 14-3-3ζ-knockout mice. We also demonstrate that accelerated closure of the wounded epidermis by 14-3-3 inhibition causes enhanced signaling through the Rho-ROCK pathway and that the underlying cellular mechanism involves the efficient recruitment of dermal fibroblasts into the wound and the rapid production of extracellular matrix proteins to re-establish the injured dermis. Our observations that the 14-3-3/ROCK inhibitory axis characterizes impaired wound healing and that its suppression facilitates fibroblast recruitment and accelerated re-epithelialization suggest new possibilities for treating diabetic wounds by pharmacologically targeting this axis.
PubMed: 38582367
DOI: 10.1016/j.jid.2024.03.032 -
European Heart Journal. Cardiovascular... Apr 2024Differentiating cardiac amyloidosis (CA) subtypes is important considering the significantly different therapies for transthyretin (ATTR)-CA and light chain (AL)-CA....
Novel echocardiographic pixel intensity quantification method for differentiating transthyretin cardiac amyloidosis from light chain cardiac amyloidosis and other phenocopies.
AIMS
Differentiating cardiac amyloidosis (CA) subtypes is important considering the significantly different therapies for transthyretin (ATTR)-CA and light chain (AL)-CA. Therefore, an echocardiographic method to distinguish ATTR-CA from AL-CA would provide significant value. We assessed a novel echocardiographic pixel intensity method to quantify myocardial calcification to differentiate ATTR-CA from phenocopies of CA and from AL-CA, specifically.
METHODS AND RESULTS
167 patients with ATTR-CA (n=53), AL-CA (n=32), hypertrophic cardiomyopathy (n=37), and advanced chronic kidney disease (n=45) were retrospectively evaluated. The septal reflectivity ratio (SRR) was measured as the average pixel intensity of the visible anterior septal wall divided by the average pixel intensity of the visible posterior lateral wall. SRR and other myocardial strain-based echocardiographic measures were evaluated with receiver operator characteristic analysis to evaluate accuracy in distinguishing ATTR-CA from AL-CA and other forms of left ventricular hypertrophy. Mean septal reflectivity ratio (SRR) was significantly higher in the ATTR-CA cohort compared to the other cohorts (p <0.001). SRR demonstrated the largest AUC (0.91, p<0.0001) for distinguishing ATTR from all other cohorts and specifically for distinguishing ATTR-CA from AL-CA (AUC=0.90, p<0.0001, specificity 96%, sensitivity 63%). There was excellent inter- and intra-operator reproducibility with an ICC of 0.91 (p <0.001) and 0.89 (p <0.001), respectively.
CONCLUSION
The SRR is a reproducible and robust parameter for differentiating ATTR-CA from other phenocopies of CA and specifically ATTR-CA from AL-CA.
PubMed: 38581424
DOI: 10.1093/ehjci/jeae095 -
Journal of Cell Science May 2024Endosomal-lysosomal trafficking is accompanied by the acidification of endosomal compartments by the H+-V-ATPase to reach low lysosomal pH. Disruption of the correct pH...
Endosomal-lysosomal trafficking is accompanied by the acidification of endosomal compartments by the H+-V-ATPase to reach low lysosomal pH. Disruption of the correct pH impairs lysosomal function and the balance of protein synthesis and degradation (proteostasis). Here, we treated mammalian cells with the small dipeptide LLOMe, which is known to permeabilize lysosomal membranes, and find that LLOMe also impacts late endosomes (LEs) by neutralizing their pH without causing membrane permeabilization. We show that LLOMe leads to hyperactivation of Rab7 (herein referring to Rab7a), and disruption of tubulation and mannose-6-phosphate receptor (CI-M6PR; also known as IGF2R) recycling on pH-neutralized LEs. pH neutralization (NH4Cl) and expression of Rab7 hyperactive mutants alone can both phenocopy the alterations in tubulation and CI-M6PR trafficking. Mechanistically, pH neutralization increases the assembly of the V1G1 subunit (encoded by ATP6V1G1) of the V-ATPase on endosomal membranes, which stabilizes GTP-bound Rab7 via RILP, a known interactor of Rab7 and V1G1. We propose a novel pathway by which V-ATPase and RILP modulate LE pH and Rab7 activation in concert. This pathway might broadly contribute to pH control during physiologic endosomal maturation or starvation and during pathologic pH neutralization, which occurs via lysosomotropic compounds and in disease states.
Topics: Animals; Humans; Adaptor Proteins, Signal Transducing; Endosomes; HeLa Cells; Hydrogen-Ion Concentration; Lysosomes; Protein Transport; rab7 GTP-Binding Proteins; Receptor, IGF Type 2; Vacuolar Proton-Translocating ATPases
PubMed: 38578235
DOI: 10.1242/jcs.261765