Journal of Biological Chemistry最新文献

{"title":"PARP14 is a writer, reader, and eraser of mono-ADP-ribosylation.","authors":"Archimede Torretta, Constantinos Chatzicharalampous, Carmen Ebenwaldner, Herwig Schüler","doi":"10.1016/j.jbc.2024.107904","DOIUrl":"https://doi.org/10.1016/j.jbc.2024.107904","url":null,"abstract":"","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":"300 12","pages":"107904"},"PeriodicalIF":4.0,"publicationDate":"2024-11-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142647866","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Proteomic study identifies Aurora-A mediated regulation of alternative splicing through multiple splicing factors.","authors":"Arun Prasath Damodaran, Olivia Gavard, Jean-Philippe Gagné, Malgorzata Ewa Rogalska, Amit K Behera, Estefania Mancini, Giulia Bertolin, Thibault Courtheoux, Bandana Kumari, Justine Cailloce, Agnès Mereau, Guy G Poirier, Juan Valcárcel, Thomas Gonatopoulos-Pournatzis, Erwan Watrin, Claude Prigent","doi":"10.1016/j.jbc.2024.108000","DOIUrl":"https://doi.org/10.1016/j.jbc.2024.108000","url":null,"abstract":"<p><p>The cell cycle regulator Aurora-A kinase presents an attractive target for cancer therapies, though its inhibition is also associated with toxic side effects. To gain a more nuanced understanding of Aurora-A function, we applied shotgun proteomics to identify 407 specific protein partners, including several splicing factors. Supporting a role in alternative splicing, we found that Aurora-A localizes to nuclear speckles, the storehouse of splicing proteins. Aurora-A interacts with and phosphorylates splicing factors both in vitro and in vivo, suggesting that it regulates alternative splicing by modulating the activity of these splicing factors. Consistently, Aurora-A inhibition significantly impacts the alternative splicing of 505 genes, with RNA motif analysis revealing an enrichment for Aurora-A interacting splicing factors. Additionally, we observed a significant positive correlation between the splicing events regulated by Aurora-A and those modulated by its interacting splicing factors. An interesting example is represented by CLK1 exon 4, which appears to be regulated by Aurora-A through SRSF3. Collectively, our findings highlight a broad role of Aurora-A in the regulation of alternative splicing.</p>","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":" ","pages":"108000"},"PeriodicalIF":4.0,"publicationDate":"2024-11-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142647937","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"The Hsc70 system maintains the synaptic SNARE protein SNAP-25 in an assembly-competent state and delays its aggregation.","authors":"Karishma Bhasne, Antonia Bogoian-Mullen, Eugenia M Clerico, Lila M Gierasch","doi":"10.1016/j.jbc.2024.108001","DOIUrl":"https://doi.org/10.1016/j.jbc.2024.108001","url":null,"abstract":"<p><p>The complex mechanism of synaptic vesicle fusion with the plasma membrane for neurotransmitter release is initiated by the formation of the SNARE complex at the presynaptic terminal of the neuron. The SNARE complex is composed of four helices contributed by three proteins: one from syntaxin (localized at the plasma membrane), one from synaptobrevin (localized at the synaptic vesicle), and two from the intrinsically disordered and aggregation-prone SNAP-25, which is localized to the plasma membrane by virtue of palmitoylation of cysteine residues. The fusion process is tightly regulated and requires the constitutively expressed Hsp70 chaperone (Hsc70) and its J-protein co-chaperone CSPα. We hypothesize that Hsc70 and CSPα cooperate to chaperone SNAP-25, disfavoring its aggregation and keeping it in a folding state competent for SNARE complex formation. To test this hypothesis, we employed a bottom-up approach and studied the interaction between Hsc70 and CSPα with SNAP-25 in vitro. We showed that the aggregation of SNAP-25 is delayed in the presence of Hsc70 and CSPα. Using a peptide array that spans the sequence of SNAP-25, we identified three potential Hsc70-interacting sequences and designed peptides containing these sequences to test binding in solution. We characterized the interaction of SNAP-25-derived peptides with Hsc70 and CSPα using a combination of biochemical and biophysical techniques, including native-PAGE, binding affinity by fluorescence anisotropy, ATPase-activity of Hsc70, and NMR. We have identified an Hsc70 binding site within SNAP-25 that is likely to represent the site used in the cell to facilitate SNARE complex formation.</p>","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":" ","pages":"108001"},"PeriodicalIF":4.0,"publicationDate":"2024-11-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142647863","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"The CTR hydrophobic residues of Nem1 catalytic subunit are required to form a protein phosphatase complex with Spo7 to activate yeast Pah1 PA phosphatase.","authors":"Ruta Jog, Gil-Soo Han, George M Carman","doi":"10.1016/j.jbc.2024.108003","DOIUrl":"10.1016/j.jbc.2024.108003","url":null,"abstract":"<p><p>The Nem1-Spo7 phosphatase complex plays a key role in lipid metabolism as an activator of Pah1 phosphatidate phosphatase, which produces diacylglycerol for the synthesis of triacylglycerol and membrane phospholipids. For dephosphorylation of Pah1, the Nem1 catalytic subunit requires Spo7 for the recruitment of the protein substrate and interacts with the regulatory subunit through its conserved region (residues 251-446). In this work, we found that the Nem1 C-terminal region (CTR) (residues 414-436), which flanks the HAD-like catalytic domain (residues 251-413), contains the conserved hydrophobic residues (L414, L415, L417, L418, L421, V430, L434, and L436) that are necessary for the complex formation with Spo7. AlphaFold predicts that some CTR residues of Nem1 interact with Spo7 conserved regions, whereas some residues interact with the HAD-like domain. By site-directed mutagenesis, Nem1 variants were constructed to lack (Δ(414-446)) or substitute alanines (8A) and arginines (8R) for the hydrophobic residues. When coexpressed with Spo7, the CTR variants of Nem1 did not form a complex with Spo7. In addition, the Nem1 variants were incapable of catalyzing the dephosphorylation of Pah1 in the presence of Spo7. Moreover, the Nem1 variants expressed in nem1Δ cells did not complement the phenotypes characteristic of a defect in the Nem1-Spo7/Pah1 phosphatase cascade function (e.g., lipid synthesis, lipid droplet formation, and phospholipid biosynthetic gene expression). These findings support that Nem1 interacts with Spo7 through its CTR hydrophobic residues to form a phosphatase complex for catalytic activity and physiological functions.</p>","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":" ","pages":"108003"},"PeriodicalIF":4.0,"publicationDate":"2024-11-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142647856","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Impaired branched chain amino acid (BCAA) catabolism during adipocyte differentiation decreases glycolytic flux.","authors":"Courtney R Green, Lynn M Alaeddine, Karl A Wessendorf-Rodriguez, Rory Turner, Merve Elmastas, Justin D Hover, Anne N Murphy, Mikael Ryden, Niklas Mejhert, Christian M Metallo, Martina Wallace","doi":"10.1016/j.jbc.2024.108004","DOIUrl":"https://doi.org/10.1016/j.jbc.2024.108004","url":null,"abstract":"<p><p>Dysregulated branched chain amino acid (BCAA) metabolism has emerged as a key metabolic feature associated with the obese insulin resistant state, and adipose BCAA catabolism is decreased in this context. BCAA catabolism is upregulated early in adipogenesis, but the impact of suppressing this pathway on the broader metabolic functions of the resultant adipocyte remains unclear. Here, we use CRISPR/Cas9 to decrease BCKDHA in 3T3-L1 and human pre-adipocytes, and ACAD8 in 3T3-L1 pre-adipocytes to induce a deficiency in BCAA catabolism through differentiation. We characterize the transcriptional and metabolic phenotype of 3T1-L1 cells using RNAseq and ¹³C metabolic flux analysis within a network spanning glycolysis, tricarboxylic acid (TCA) metabolism, BCAA catabolism, and fatty acid synthesis. While lipid droplet accumulation is maintained in Bckdha-deficient adipocytes, they display a more fibroblast-like transcriptional signature. In contrast, Acad8 deficiency minimally impacts gene expression. Decreased glycolytic flux emerges as the most distinct metabolic feature of 3T3-L1 Bckdha-deficient cells, accompanied by a ∼40% decrease in lactate secretion, yet pyruvate oxidation and utilization for de novo lipogenesis are increased to compensate for loss of BCAA carbon. Deletion of BCKDHA in human adipocyte progenitors also led to a decrease in glucose uptake and lactate secretion, however these cells did not upregulate pyruvate utilisation and lipid droplet accumulation and expression of adipocyte differentiation markers was decreased in BCKDH knockout cells. Overall our data suggest that human adipocyte differentiation may be more sensitive to the impact of decreased BCKDH activity than 3T3-L1 cells, and that both metabolic and regulatory cross-talk exists between BCAA catabolism and glycolysis in adipocytes. Suppression of BCAA catabolism associated with metabolic syndrome may result in a metabolically compromised adipocyte.</p>","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":" ","pages":"108004"},"PeriodicalIF":4.0,"publicationDate":"2024-11-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142647893","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Biophysical characterization of the dystrophin C-terminal domain: Dystrophin interacts differentially with dystrobrevin isoforms.","authors":"Vaibhav Upadhyay, Shashikant Ray, Sudipta Panja, Anthony J Saviola, Nasib Karl Maluf, Krishna M G Mallela","doi":"10.1016/j.jbc.2024.108002","DOIUrl":"https://doi.org/10.1016/j.jbc.2024.108002","url":null,"abstract":"<p><p>Duchenne muscular dystrophy (DMD) gene encodes dystrophin, a large multi-domain protein. Its non-functionality leads to dystrophinopathies like DMD and Becker muscular dystrophy (BMD), for which no cure is yet available. A few therapies targeted towards specific mutations can extend the lifespan of patients, although with limited efficacy and high costs, emphasizing the need for more general treatments. Dystrophin's complex structure with poorly understood domains and the presence of multiple isoforms with varied expression patterns in different tissues pose challenges in therapeutic development. The C-terminal (CT) domain of dystrophin is less understood in terms of its structure-function, although it has been shown to perform important functional roles by interacting with another cytosolic protein, dystrobrevin. Dystrophin and dystrobrevin stabilize the sarcolemma membrane by forming a multi-protein complex called dystrophin-associated glycoprotein complex (DAGC) that is destabilized in DMD. Dystrobrevin has two major isoforms, alpha and beta, with tissue-specific expression patterns. Here, we characterize the CT domain of dystrophin and its interactions with the two dystrobrevin isoforms. We show that the CT domain is non-globular and shows reversible urea denaturation as well as thermal denaturation. It interacts with dystrobrevin isoforms differentially, with differences in binding affinity and the mode of interaction. We further show that the amino acid differences in the C-terminal region of dystrobrevin isoforms contribute to these differences. These results have implications for the stability of DAGC in different tissues and explain the differing symptoms associated with DMD patients affecting organs beyond the skeletal muscles.</p>","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":" ","pages":"108002"},"PeriodicalIF":4.0,"publicationDate":"2024-11-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142647761","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Structural determinants of M2R involved in inhibition by Sigma-1R.","authors":"Chang Liu, I-Shan Chen, Muruj Barri, Ruth Murrell-Lagnado, Yoshihiro Kubo","doi":"10.1016/j.jbc.2024.108006","DOIUrl":"https://doi.org/10.1016/j.jbc.2024.108006","url":null,"abstract":"<p><p>Sigma-1 receptor (S1R) is a multimodal chaperone protein which is implicated in various pathophysiological conditions including drug addiction, Alzheimer's disease and amyotrophic lateral sclerosis (ALS). S1R interacts with various ion channels and receptors on endoplasmic reticulum or plasma membrane (PM). It has been reported that S1R colocalizes with the M2-muscarinic acetylcholine receptor (M2R) on the soma of motoneurons, although a functional interaction between these two proteins hasn't been established. Here, we investigated the regulation of M2R signalling by S1R using electrophysiological recordings of GIRK currents in HEK293T cells. We observed that S1R strongly inhibited M2R-mediated activation of GIRK1/2, but the disease mutant linked to ALS, S1R E102Q, did not. The inhibitory effect of S1R was selective for M2R and wasn't seen when S1R was co-expressed with other G_i/o coupled receptors including M4R. Chimeric and mutant receptors of M2R and M4R were generated and analysed, and this highlighted Ala401 in the transmembrane 6 domain (TM6) of M2R and Glu172 as well as Glu175 in the extracellular loop 2 region of M2R, as essential for the inhibition by S1R. Co-immunoprecipitation confirmed the physical interaction between M2R and S1R. Immunocytochemical labelling of M2R and S1R expressed in HeLa cells, HEK293T cells and cultured hippocampal neurons, showed clear PM expression of M2R throughout the cell which was decreased by coexpression with S1R but was still apparent. Taken together, our results show that S1R interacts with M2R to reduce both its PM expression and function, and this involves TM6 and the extracellular loop 2.</p>","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":" ","pages":"108006"},"PeriodicalIF":4.0,"publicationDate":"2024-11-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142647944","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"On the specificity of the recognition of m6A-RNA by YTH reader domains.","authors":"Julian Widmer, Andreas Vitalis, Amedeo Caflisch","doi":"10.1016/j.jbc.2024.107998","DOIUrl":"https://doi.org/10.1016/j.jbc.2024.107998","url":null,"abstract":"<p><p>Most processes of life are the result of polyvalent interactions between macromolecules, often of heterogeneous types and sizes. Frequently, the times associated with these interactions are prohibitively long for interrogation using atomistic simulations. Here, we study the recognition of N6-methylated adenine (m⁶A) in RNA by the reader domain YTHDC1, a prototypical, cognate pair that challenges simulations through its composition and required timescales. Simulations of RNA pentanucleotides in water reveal that the unbound state can impact (un)binding kinetics in a manner that is both model- and sequence-dependent. This is important because there are two contributions to the specificity of the recognition of the Gm⁶AC motif: from the sequence adjacent to the central adenine and from its methylation. Next, we establish a reductionist model consisting of an RNA trinucleotide binding to the isolated reader domain in high salt. An adaptive sampling protocol allows us to quantitatively study the dissociation of this complex. Through joint analysis of a data set including both the cognate and control sequences (GAC, Am⁶AA, and AAA), we derive that both contributions to specificity, sequence and methylation, are significant and in good agreement with experimental numbers. Analysis of the kinetics suggests that flexibility in both the RNA and the YTHDC1 recognition loop leads to many low-populated unbinding pathways. This multiple-pathway mechanism might be dominant for the binding of unstructured polymers, including RNA and peptides, to proteins when their association is driven by polyvalent, electrostatic interactions.</p>","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":" ","pages":"107998"},"PeriodicalIF":4.0,"publicationDate":"2024-11-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142647924","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"The antibacterial activity of a prophage-encoded fitness factor is neutralized by two cognate immunity proteins.","authors":"Andrea G Alexei, Nathan P Bullen, Stephen R Garrett, David Sychantha, John C Whitney","doi":"10.1016/j.jbc.2024.108007","DOIUrl":"https://doi.org/10.1016/j.jbc.2024.108007","url":null,"abstract":"<p><p>The human gastrointestinal tract is a competitive environment inhabited by dense polymicrobial communities. Bacteroides, a genus of Gram-negative anaerobes, are prominent members of this ecological niche. Bacteroides spp. uses a repertoire of mechanisms to compete for resources within this environment such as the delivery of proteinaceous toxins into neighbouring competitor bacteria and the ability to consume unique metabolites available in the gut. In recent work, Bacteroides stercoris gut colonization was linked to the activity of a prophage-encoded ADP-ribosyltransferase, which was found to stimulate the release of the metabolite inosine from host epithelial cells. This fitness factor, termed Bxa, shares a similar genomic arrangement to bacterial toxins encoded within interbacterial antagonism loci. Here, we report that Bxa also possesses antibacterial ADP-ribosyltransferase activity, raising the question of how Bxa-producing bacteria resist intoxication prior to Bxa's release from cells. To this end, we identify two cognate immunity proteins, Bsi and BAH, that neutralize Bxa's antibacterial activity using distinct mechanisms. BAH acts as an enzymatic immunity protein that reverses Bxa ADP-ribosylation whereas Bsi physically interacts with Bxa and blocks its ADP-ribosylation activity. We also find that the N-terminal domain of Bxa is dispensable for toxicity and homologous domains in other bacteria are fused to a diverse array of predicted toxins found throughout the Bacteroidaceae, suggesting that Bxa belongs to a broader prophage encoded polymorphic toxin system. Overall, this work shows that Bxa is a promiscuous ADP-ribosyltransferase and that B. stercoris possesses mechanisms to protect itself from the toxic activity of this prophage encoded fitness factor.</p>","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":" ","pages":"108007"},"PeriodicalIF":4.0,"publicationDate":"2024-11-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142647950","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"O-glycosylation is essential for cell surface expression of the transcobalamin receptor CD320.","authors":"Chunyu Du, Wenjun Guo, Mengting Wang, Zibin Zhou, Tiantian Zhou, Meng Liu, Ningzheng Dong, Qingyu Wu","doi":"10.1016/j.jbc.2024.107997","DOIUrl":"https://doi.org/10.1016/j.jbc.2024.107997","url":null,"abstract":"<p><p>CD320 is a cell surface receptor that mediates vitamin B₁₂ uptake in most tissues. To date, the mechanisms that regulate CD320 expression on the cell surface are not fully understood. In this work, we studied CD320 expression in transfected human embryonic kidney (HEK) 293 and hepatoma HepG2 cells. By glycosidase and trypsin digestion, monensin and brefeldin treatment, western blotting, flow cytometry, and lectin biding, we found that CD320 underwent N- and O-glycosylation and sialylation, resulting in a ∼70-kDa band that formed a high-molecular weight complex on the cell surface. Site-directed mutagenesis altering Asn126, Asn195 and Asn213 residues, individually or together, abolished N-glycosylation in CD320 but did not block its intracellular trafficking and expression on the cell surface in HEK293 and HepG2 cells. In contrast, treatment of the cells with Ben-gal, a structural analog of GalNAc-α-1-O-Ser/Thr, inhibited O-glycosylation and cell surface expression of CD320, and decreased vitamin B₁₂ uptake. Analysis of CD320 deletion mutants indicated that O-glycosylation sites in a Ser/Thr-rich region near the transmembrane domain were important for CD320 expression on the cell surface. These results reveal an important role of O-glycans, but not N-glycans, in the intracellular trafficking and cell surface expression of CD320, providing new insights into the cellular mechanisms in regulating CD320 function and vitamin B₁₂ metabolism.</p>","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":" ","pages":"107997"},"PeriodicalIF":4.0,"publicationDate":"2024-11-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142647913","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}