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Membrane fusion in sperm cells is crucial for acrosomal exocytosis and must be preserved to ensure fertilizing capacity. Evolutionarily conserved protein machinery regulates acrosomal exocytosis. Molecular chaperones play a vital role in spermatogenesis and post-testicular maturation. Cysteine string protein (CSP) is a member of the Hsp40 co-chaperones, and the participation of molecular chaperones in acrosomal exocytosis is poorly understood. In particular, the role of CSP in acrosomal exocytosis has not been reported so far. Using western blot and indirect immunofluorescence, we show that CSP is present in human sperm, is palmitoylated, and predominantly bound to membranes. Moreover, using functional assays and transmission electron microscopy, we report that blocking the function of CSP avoided the assembly of transcomplexes and inhibited exocytosis. In summary, here, we describe the presence of CSP in human sperm and show that this protein has an essential role in membrane fusion during acrosomal exocytosis mediating the trans-SNARE complex assembly between the outer acrosomal and plasma membranes. In general, understanding CSP's role is critical in identifying new biomarkers and generating new rational-based approaches to treat male infertility. Summary Sentence: Cysteine string protein is necessary and mediates the trans-SNARE complexes assembly between the outer acrosomal and the plasma membranes in human sperm acrosomal exocytosis mechanism. Graphical Abstract

During the first postnatal week in rodents, cholinergic retinal waves initiate in starburst amacrine cells (SACs), propagating to retinal ganglion cells (RGCs) and visual centers, essential for visual circuit refinement. By modulating exocytosis in SACs, dynamic changes in the protein kinase A (PKA) activity can regulate the spatiotemporal patterns of cholinergic waves. Previously, cysteine string protein-α (CSPα) is found to interact with the core exocytotic machinery by PKA-mediated phosphorylation at serine 10 (S10). However, whether PKA-mediated CSPα phosphorylation may regulate cholinergic waves via SACs remains unknown. Here, we examined how CSPα phosphorylation in SACs regulates cholinergic waves. First, we identified that CSPα1 is the major isoform in developing rat SACs and the inner plexiform layer during the first postnatal week. Using SAC-specific expression, we found that the CSPα1-PKA-phosphodeficient mutant (CSP-S10A) decreased wave frequency, but did not alter the wave spatial correlation compared to control, wild-type CSPα1 (CSP-WT), or two PKA-phosphomimetic mutants (CSP-S10D and CSP-S10E). These suggest that CSPα-S10 phosphodeficiency in SACs dampens the frequency of cholinergic waves. Moreover, the level of phospho-PKA substrates was significantly reduced in SACs overexpressing CSP-S10A compared to control or CSP-WT, suggesting that the dampened wave frequency is correlated with the decreased PKA activity. Further, compared to control or CSP-WT, CSP-S10A in SACs reduced the periodicity of wave-associated postsynaptic currents (PSCs) in neighboring RGCs, suggesting that these RGCs received the weakened synaptic inputs from SACs overexpressing CSP-S10A. Finally, CSP-S10A in SACs decreased the PSC amplitude and the slope to peak PSC compared to control or CSP-WT, suggesting that CSPα-S10 phosphodeficiency may dampen the speed of the SAC-RGC transmission. Thus, via PKA-mediated phosphorylation, CSPα in SACs may facilitate the SAC-RGC transmission, contributing to the robust frequency of cholinergic waves.

Synaptic transmission relies on precisely regulated and exceedingly fast protein-protein interactions that involve voltage-gated channels, the exocytosis/endocytosis machinery as well as signaling pathways. Although we have gained an ever more detailed picture of synaptic architecture much remains to be learned about how synapses are maintained. Synaptic chaperones are \"folding catalysts\" that preserve proteostasis by regulating protein conformation (and therefore protein function) and prevent unwanted protein-protein interactions. Failure to maintain synapses is an early hallmark of several degenerative diseases. Cysteine string protein (CSPα) is a presynaptic vesicle protein and molecular chaperone that has a central role in preventing synaptic loss and neurodegeneration. Over the past few years, a number of different \"client proteins\" have been implicated as CSPα substrates including voltage-dependent ion channels, signaling proteins and proteins critical to the synaptic vesicle cycle. Here we review the ion channels and synaptic protein complexes under the influence of CSPα and discuss gaps in our current knowledge.

Cysteine string protein α (CSPα) is a vesicle protein located in the presynaptic terminal of most synapses. CSPα is an essential molecular co-chaperone that facilitates the correct folding of proteins and the assembly of the exocytic machinery. The absence of this protein leads to altered neurotransmitter release and neurodegeneration in multiple model systems, from flies to mice. In humans, CSPα mutations are associated with the development of neuronal ceroid lipofuscinosis (NCL), a neurodegenerative disease characterized by intracellular accumulation of lysosomal material. Here, we review the physiological role of CSPα and the pathology resulting from the homozygous deletion of the gene or its mutations. In addition, we investigate whether long-term moderate reduction of the protein produces motor dysfunction. We found that 1-year-old CSPα heterozygous mice display a reduced ability to sustain motor unit recruitment during repetitive stimulation, which indicates that physiological levels of CSPα are required for normal neuromuscular responses in mice and, likely, in humans.

Homeostatic synaptic plasticity (HSP) helps neurons and synapses maintain physiologically appropriate levels of output. The fruit fly Drosophila melanogaster larval neuromuscular junction (NMJ) is a valuable model for studying HSP. Here we introduce a genetic tool that allows fruit fly researchers to examine the lifelong maintenance of HSP with a single cross. The tool is a fruit fly stock that combines the GAL4/UAS expression system with RNA interference (RNAi)-based knock down of a glutamate receptor subunit gene. With this stock, we uncover important new information about the maintenance of HSP. We address an open question about the role that presynaptic CaV2-type Ca(2+) channels play in NMJ homeostasis. Published experiments have demonstrated that hypomorphic missense mutations in the CaV2 α1a subunit gene cacophony (cac) can impair homeostatic plasticity at the NMJ. Here we report that reducing cac expression levels by RNAi is not sufficient to impair homeostatic plasticity. The presence of wild-type channels appears to support HSP-even when total CaV2 function is severely reduced. We also conduct an RNAi- and electrophysiology-based screen to identify new factors required for sustained homeostatic signaling throughout development. We uncover novel roles in HSP for Drosophila homologs of Cysteine string protein (CSP) and Phospholipase Cβ (Plc21C). We characterize those roles through follow-up genetic tests. We discuss how CSP, Plc21C, and associated factors could modulate presynaptic CaV2 function, presynaptic Ca(2+) handling, or other signaling processes crucial for sustained homeostatic regulation of NMJ function throughout development. Our findings expand the scope of signaling pathways and processes that contribute to the durable strength of the NMJ.

We have reported previously that myristoylated alanine-rich C kinase substrate (MARCKS) is a key regulatory molecule controlling mucin secretion by airway epithelial cells in vitro and in vivo. The results of those studies supported a mechanism whereby MARCKS, upon phosphorylation by protein kinase C (PKC), translocates from plasma membrane to cytoplasm, where its binding to membranes of intracellular mucin granules is a key component of the secretory pathway. It remains unknown how MARCKS is targeted to and/or preferentially attaches to mucin granule membranes. We hypothesized that the chaperone cysteine string protein (CSP) may play an important role in this process. CSP was shown to associate with membranes of intracellular mucin granules in well-differentiated normal human bronchial epithelial (NHBE) cells in vitro, as determined by ultrastructural immunohistochemistry and Western blotting of isolated granule membranes. CSP in these cells complexed with MARCKS, as shown by co-immunoprecipitation. Given reported associations between CSP and a second chaperone, heat shock protein 70 (HSP70), a role for HSP70 in the MARCKS-dependent secretory mechanism also was investigated. HSP70 appeared to form a trimeric complex with MARCKS and CSP associated with mucin granule membranes within airway epithelial cells. Transfection of the HBE1 human bronchial epithelial cell line with siRNAs targeting sequences of MARCKS, CSP, or HSP70 resulted, in each case, in significant knockdown of expression of these proteins and subsequent attenuation of mucin secretion. The results provide the first evidence that CSP and HSP70, and their interactions with MARCKS, are involved in mucin secretion.

Point mutations in cysteine string protein-α (CSPα) cause dominantly inherited adult-onset neuronal ceroid lipofuscinosis (ANCL), a rapidly progressing and lethal neurodegenerative disease with no treatment. ANCL mutations are proposed to trigger CSPα aggregation/oligomerization, but the mechanism of oligomer formation remains unclear. Here we use purified proteins, mouse primary neurons and patient-derived induced neurons to show that the normally palmitoylated cysteine string region of CSPα loses palmitoylation in ANCL mutants. This allows oligomerization of mutant CSPα via ectopic binding of iron–sulfur (Fe–S) clusters. The resulting oligomerization of mutant CSPα causes its mislocalization and consequent loss of its synaptic SNARE-chaperoning function. We then find that pharmacological iron chelation mitigates the oligomerization of mutant CSPα, accompanied by partial rescue of the downstream SNARE defects and the pathological hallmark of lipofuscin accumulation. Thus, the iron chelators deferiprone (L1) and deferoxamine (Dfx), which are already used to treat iron overload in humans, offer a new approach for treating ANCL.Mutations in cysteine string protein-α (CSPα) cause its aggregation and adult-onset neuronal ceroid lipofuscinosis. Abnormal binding of Fe–S clusters to CSPα mutants is now implicated in driving aggregation, which can be reversed in neurons by clinically approved iron chelators.

This study aimed to identify diagnostic gene biomarkers for colorectal cancer (CRC) by analyzing differentially expressed genes (DEGs) in tumor and adjacent normal samples across five colon cancer gene-expression profiles (GSE10950, GSE25070, GSE41328, GSE74602, GSE142279) from the Gene Expression Omnibus (GEO) database. Intersecting identified DEGs with the module with the highest correlation to gene expression patterns of tumor samples in the gene co-expression network analysis revealed 283 overlapped genes. Centrality measures were calculated for these genes in the reconstructed STRING protein–protein interaction network. Applying LASSO logistic regression, eleven genes were ultimately recognized as candidate diagnostic genes. Among these genes, the area under the receiver operating characteristic curve (AUROC) values for nine genes (CDC25B, CDK4, IQGAP3, MMP1, MMP7, SLC7A5, TEAD4, TRIB3, and UHRF1) surpassed the threshold of 0.92 in both the training and validation sets. We evaluated the diagnostic performance of these genes with four machine learning algorithms: random forest (RF), support vector machines (SVM), artificial neural network (ANN), and gradient boosting machine (GBM). In the testing dataset (GSE21815 and GSE106582), the AUROC scores were greater than 0.95 for all of the machine learning algorithms, indicating the high diagnostic performance of the nine genes. Besides, these nine genes are also significantly correlated to twelve immune cells, namely Mast cells activated, Macrophages M0, M1, and M2, Neutrophils, T cells CD4 memory activated, T cells follicular helper, T cells CD8, T cells CD4 memory resting, B cells memory, Plasma cells, and Mast cells resting (P < 0.05). These results strongly suggest that all of the nine genes have the potential to serve as reliable diagnostic biomarkers for CRC.

DnaJ heat shock protein family member C5 beta (DNAJC5B), also known as cysteine-string protein beta, exhibits a prominent expression in testicular tissue and plays an important role in acrosomal exocytosis in vitro. Nevertheless, the precise role and underlying mechanism of DNAJC5B in spermatogenesis and male fertility remain poorly understood. The meta-analysis of RNA-sequencing datasets from porcine and murine testes reveals that Dnajc5b could be a pivotal factor in spermatogenesis. This study illustrates that male fertility declines with an increased ratio of abnormal spermatozoa in germ-cell knockout Dnajc5b mice. DNAJC5B has been identified as a mitochondrial protein with high expression in spermatids. The absence of DNAJC5B induces a cascade of mitochondrial damages, including oxidative stress, mitochondrial stress in the testes, and lower mitochondrial membrane potential of spermatozoa. In vivo and in vitro evidence demonstrates that DNAJC5B mitigates excessive cellular autophagy and mitophagy via DNAJ domain under environmental stress conditions, such as starvation or exposure to mitochondrial uncouplers FCCP and CCCP. This study highlights the important role of DNAJC5B in safeguarding male fertility by preserving mitochondrial function and regulating autophagy during spermiogenesis.

Neuronal ceroid lipofuscinoses (NCL) are a group of inherited neurodegenerative disorders with lysosomal pathology ( CLN1 - 14 ). Recently, mutations in the DNAJC5/CLN4 gene, which encodes the presynaptic co-chaperone CSPα were shown to cause autosomal-dominant NCL. Although 14 NCL genes have been identified, it is unknown if they act in common disease pathways. Here we show that two disease-associated proteins, CSPα and the depalmitoylating enzyme palmitoyl-protein thioesterase 1 ( PPT1/CLN1 ) are biochemically linked. We find that in DNAJC5/CLN4 patient brains, PPT1 is massively increased and mis-localized. Surprisingly, the specific enzymatic activity of PPT1 is dramatically reduced. Notably, we demonstrate that CSPα is depalmitoylated by PPT1 and hence its substrate. To determine the consequences of PPT1 accumulation, we compared the palmitomes from control and DNAJC5/CLN4 patient brains by quantitative proteomics. We discovered global changes in protein palmitoylation, mainly involving lysosomal and synaptic proteins. Our findings establish a functional link between two forms of NCL and serve as a springboard for investigations of NCL disease pathways.