Cytoplasmic Protein Research Articles (Page 1)

Proper neural circuit organization requires individual neurons to project to their targets with high specificity. While several guidance molecules have been shown to mediate axonal fasciculation and pathfinding, less is understood about how neurons intracellularly interpret and integrate these cues. Here we provide genetic evidence that the Crk-Associated Substrate (Cas) family of intracellular adaptor proteins is required for proper fasciculation and guidance of two cortical white matter tracts: the Anterior Commissure (AC) and thalamocortical axons (TCAs). Using a Cas Triple Conditional Knock Out (Cas TcKO) mouse model, we show that Cas proteins are required for proper TCA projection by a non-neuronal cortical cell population. We also demonstrate a requirement of the β1-integrin receptor for TCA projection, similarly in a population of non-neuronal cortical cells. Additional analysis of Cas TcKO mutants reveals a role for Cas proteins in AC fasciculation, here within the neurons themselves. This AC fasciculation requirement is not phenocopied in β1-integrin deficient mutants, suggesting that Cas proteins might signal downstream of a different receptor during this axon pathfinding event. These findings implicate Cas proteins as key mediators of cortical axon tract fasciculation and guidance.

Post-transcriptional regulation is particularly prominent during the maternal-to-zygotic transition (MZT), a developmental phase during which a large proportion of maternally provided mRNAs is repressed and cleared from metazoan embryos. RNA-binding proteins (RBPs) are key components of the post-transcriptional regulatory machinery. We show that the ORB2 RBP, the Drosophila ortholog of human Cytoplasmic Polyadenylation Element Binding Protein (hCPEB) 2-4 protein subfamily, binds to hundreds of maternally provided, rare-codon-enriched mRNAs in early embryos; that ORB2 targets are translationally repressed and unstable during the MZT; identify a U-rich motif enriched in ORB2 targets' 3'UTRs; and show that this motif confers ORB2 binding and repression to a luciferase reporter mRNA in S2 tissue culture cells. When tethered to a luciferase reporter, ORB2 and hCPEB2 (but not ORB and hCPEB1) repress translation; the C-terminal Zinc-binding ('ZZ') domain of ORB2 is necessary and sufficient for repression. ORB2 interacts with a suite of post-transcriptional regulators in early embryos; a subset of these interactions is lost upon deletion of the ZZ domain, notably with the Cup repressive complex. ORB2-targets significantly overlap with those previously identified for the repressive RBP, Smaug (SMG). Analysis of the early embryo's translatome in the presence or absence of the endogenous ZZ domain shows that mRNAs bound by ORB2 but not by SMG move onto polysomes upon ZZ domain deletion whereas co-bound transcripts do not, consistent with co-regulation of the latter set of transcripts by both RBPs. Our results assign a function to the ZZ domain and position ORB2 in the post-transcriptional network that regulates maternal transcripts during the Drosophila MZT.

A STAT1-GBP3-STING positive feedback loop governs inflammation, oxidative stress, and DNA damage to trigger acute aortic dissection.

Functional Impact of CYFIP2 RNA Editing on Actin Regulation, Axon Growth, and Spinogenesis.

Background/AimLysine lactylation (Kla) is a recently identified post-translational modification derived from lactate that regulates diverse biological processes. Although Kla has been studied in several cancers, its role in prostate cancer (PC) remains unclear. The objective of this study is to profile Kla in PC in order to explore the mechanisms involved in PC progression.Materials and MethodsWe performed global Kla profiling in PC-3M prostate cancer cells using affinity enrichment with anti-Kla antibodies, followed by LC-MS/MS. Bioinformatics analyses were conducted to explore the functional roles of Kla-modified proteins.ResultsWe identified 681 Kla sites across 379 proteins, with modifications predominantly located in nuclear and cytoplasmic proteins. Enrichment analysis indicated Kla involvement in mRNA splicing, chromatin organization, and glycolysis/gluconeogenesis. Several multifunctional proteins, including AHNAK and nucleolin (NCL) harbor multiple Kla sites. Motif analysis indicated conserved amino acid patterns surrounding Kla sites. Notably, PC-3M cells showed reduced expression of sirtuin (SIRT)3, SIRT5, and SIRT6, which may underlie elevated Kla levels.ConclusionThis study presents the first comprehensive Kla landscape in PCa, suggesting its potential regulatory role in tumor progression. These findings provide a valuable resource for future studies and support Kla as a possible target for therapeutic intervention in prostate cancer.

Over activation of c-MYC oncogene (gene locus: 8q24.21) - predominantly due to the amplification of the gene - is a critical genetic imbalance involved in malignant transformation of normal epithelia. Our aim was to explore the differences in c-MYC protein expression in a series of laryngeal (LSCC) and hypopharyngeal squamous cell carcinomas (HPSCCs). We retrospectively analyzed a set of sixty (n=60) paraffin embedded laryngeal-hypopharyngeal squamous cell carcinomas (L-PHSCC) tissue sections by immunocytochemistry (IHC) for the detection of c-MYC protein expression. A digital image analysis (DIA) algorithm was also performed for measuring objectively the corresponding immunostaining intensity levels of the examined protein. The correlation of c-MYC protein expression levels with various clinicopathological characteristics was assessed. High and moderate staining intensity levels of c-MYC protein expression were identified in 46/60 (76.6%) of the examined cases (21/60 and 25/60, respectively), whereas the rest of them demonstrated low expression values (14/60, 23.4%). c-MYC expression was significantly correlated with the anatomic location of the malignancies (p=0.03), with the stage of the disease (p=0.004), as well as with the grade of differentiation (p=0.049). Interestingly, c-MYC diffuse nuclear/cytoplasmic expression was observed mainly in advanced-stage carcinomas. c-MYC overexpression is a common feature in L-HPSCCs, mainly due to c-MYC oncogene overactivation. Increased levels of nuclear and diffuse cytoplasmic c-MYC protein are associated with aggressive phenotypes in patients with L-HPSCCs. Given the emerging interest in oncogene-targeted therapies in modern oncology, identifying patients with distinct c-MYC genetic, epigenetic, and proteomic signatures represents a promising strategy for personalized treatment approaches.

Background/AimClear cell renal cell carcinoma (ccRCC), the most prevalent form of kidney cancer, often presents or recurs as an advanced, aggressive, and lethal disease. Thus, biomarkers are needed to identify patients at risk of developing advanced-stage or treatment-resistant ccRCC. SYNJ2BP, a cytoplasmic scaffolding protein, regulates ACVR2 activity, a key mediator of signaling pathways involved in tumor progression and metastasis. This study aimed to ascertain if SYNJ2BP, a gene highly expressed in normal kidney tissue, may serve as a predictive biomarker for ccRCC.Materials and MethodsBioinformatic analysis and immunohistochemistry were applied to investigate the relationship between SYNJ2BP expression, the immune landscape, and survival outcomes in ccRCC. We utilized data from publicly available databases, including The Cancer Genome Atlas, Gene Set Cancer Analysis (TCGA), and various other databases.ResultsIn-silico analyses revealed that SYNJ2BP expression was significantly down-regulated in ccRCC (Log2FC=0.40, p=2.65E-36; FDR=9.73E-34), compared to normal tissue. Moreover, SYNJ2BP expression was significantly reduced in advanced stages and grades (III and IV; p<0.001) compared to lower stages and grades (I and II). Decreased expression was associated with nodal invasion and metastasis (p<0.0001), unresponsive to treatment (p=0.0052), post-treatment recurrence (p=0.002), lower median overall survival (HR=0.39, 95% CI=0.28-0.54, p<0.0001), disease-specific survival (HR=0.16, 95% CI=0.09-0.27, p<0.0001) and shorter progression-free survival (HR=0.24, 95% CI=0.17-0.35, p<0.0001). Survival trends remained consistent in multivariate Cox regression, where expression remained independently associated with outcome. Consistent with transcript-level findings, immunohistochemistry demonstrated reduced protein expression of SYNJ2BP in ccRCC patients (p<0.05).ConclusionSYNJ2BP is a novel prognostic biomarker for ccRCC and the down-regulation of SYNJ2BP expression is associated with poor survival outcomes and reduced treatment response.

Abstract Background and Aims The incidence of acute kidney injury (AKI) in patients is associated with diabetic kidney. Nevertheless, the precise underlying mechanism remains unclear. We previously revealed that methyltransferase 3 (METTL3) mediated N6-methyladenosine (m6A) RNA modifications play critical roles in kidney injuries. This study aimed to explore whether METTL3 promotes AKI sensitivity in diabetic kidney diseases (DKD). Method AKI risk in patients with diabetes was assessed through GWAS. The m6A levels and METTL3 were measured in type 1 and type 2 diabetic mice experiencing AKI. METTL3 knockout or silencing was performed to evaluate its impact on ischemia-reperfusion (I/R)-induced AKI in DKD animals and hypoxia-reoxygenation (HR) and high glucose (HG) treated renal tubular epithelial cells (TECs). Furthermore, downstream targets of METTL3 were identified through m6A sequencing to elucidate its role in the pathogenesis of AKI in DKD. Results The GWAS data from 4111 DKD patients and 5908 AKI patients revealed a 16% higher risk of AKI in DN patients. Our findings confirmed that I/R resulted more severe AKI in both STZ induced type 1 and db/db type 2 diabetes models, leading to renal dysfunction, renal tubular damage, and inflammatory response, suggesting an increased susceptibility to AKI in DKD. Induction of AKI by I/R in diabetic mice and HG+H/R-treated TECs significantly elevated m6A methylation levels and METTL3 expression. Specific knockout of Mettl3 in TECs attenuated I/R-induced AKI in STZ-induced diabetic mice. Meanwhile, silencing METTL3 by tail vein injection of adeno-associated virus 9 Mettl3 shRNA alleviated I/R-induced AKI in db/db mice, improving renal function, tubular injury and inflammatory response. MeRIP-seq analysis indicates that N-myc downstream regulated 1 (NDRG1) as the m6A target of METTL3. Knockdown of Mettl3 significantly decreases the distribution of m6A peaks across Ndrg1 mRNA transcript in HG+HR-treated TECs, with m6A-modified stop codon regions of NDRG1 by METTL3 enhancing stability via the IGF2BP2-dependent pathway. NDRG1, a cytoplasmic protein involved in cellular stress response, interacts with NUR77 to regulate AKI by inhibiting p-P65. Knockdown of METTL3 reduced the expression of NDRG1 and inhibited the activity of NUR77, and suppressed the NF-κB pathway activation, thus alleviating kidney injury. Conclusion METTL3 promotes AKI susceptibility in DKD by the m6A modification of NDRG1 and enhancing its stability via IGF2BP2-dependent mechanisms, thereby exacerbating inflammation by the NDRG1/NUR77/NF-κB signaling pathway. Targeting this pathway (METTL3/NDRG1) may hold therapeutic promise for the treatment of AKI in DKD.

Background: In recent years, indications for liver transplantation have expanded, while the age of transplant recipients has significantly increased due to improvements in perioperative management. As clinical manifestations of posttransplant complications vary and are often nonspecific, the identification of appropriate biomarkers is important for the assessment of graft quality and early recognition of potential complications following liver transplantation. Liver-type FABP (L-FABP) is a small cytoplasmic protein found abundantly in hepatocytes and is involved in the intracellular transport of long-chain fatty acids. Elevated serum levels have been detected in acute and chronic liver failure, kidney failure, and some malignancies. Materials and Methods: We conducted a prospective, single-center study from July 2023 to January 2025, including 29 adult patients who underwent deceased-donor transplantation. Three patients were excluded due to inadequate sample withdrawals. Serum L-FABP was measured preoperatively and on postoperative days 1, 3, 5, 7, and 14. Clinical, surgical, and biochemical data were collected and analyzed using non-parametric statistical tests. Results: L-FABP levels were significantly higher on POD 7 in recipients of grafts from donors ≥ 65 years (p = 0.035), with no corresponding changes in standard liver function markers. While no significant differences in L-FABP levels were found between patients with and without infectious biliary or vascular complications (all p > 0.05), we proved a strong negative correlation between intraoperative blood transfusion volume and L-FABP levels on POD 5 (ρ = −0.677, p < 0.001) and POD 7 (ρ = −0.455, p = 0.025). Conclusions: Our findings suggest that L-FABP holds promise as a biomarker for the early detection of subclinical hepatic graft cellular injury, which is not detected by means of conventional biomarkers for liver function.

Cytoplasmic male sterility (CMS) is caused by mitochondrial genes that are constitutively expressed in plant tissues, although the encoded proteins preferentially accumulate in anthers. The mechanisms regulating CMS protein accumulation remain unclear. Here, we explored this process using wild-abortive CMS (CMS-WA) rice (Oryza sativa). We show that WA352, the causal protein of CMS-WA, is degraded by the ubiquitin-proteasome system (UPS). Structural analysis and protein truncation assays revealed that the N terminus of WA352 is critical for its anchoring to the inner mitochondrial membrane and its UPS-mediated degradation. Functional complementation confirmed that WA352151-352, lacking the N-terminal domain, accumulates constitutively in vegetative tissues, causing a reactive oxygen species burst and retarding rice growth. We further identified three mitochondrion-localized F-box proteins that participate in WA352 ubiquitination and degradation. Our findings demonstrate that UPS-mediated regulation restricts WA352 accumulation to anthers, allowing it to specifically disrupt anther development, thus helping to explain the male-specific effects of CMS genes in plants.

The Wnt/β-catenin signalling pathway regulates ovarian development in the swimming crab Portunus trituberculatus.

RBM20 is one of the genes predisposing to dilated cardiomyopathy (DCM). Several dozen variants associated with DCM have been reported so far. Variants in the arginine/serine-rich domain and the RNA recognition motif domain have been well studied, but the pathogenicity of variants outside of these areas remains unknown. A patient with the Q373fs-RBM20 variant without a typical DCM phenotype was identified in a sudden death cohort. The Q374fs-Rbm20 mouse model was generated to determine the significance of this variant. In mouse experiments, cardiac dysfunction, such as reduced fractional shortening and an extended duration of QRS and the corrected QT interval, were observed in Q374fs-Rbm20 mice by ultrasound echocardiography and electrocardiography. RNA sequencing analysis showed that Q374fs-Rbm20 mice had different splicing patterns, such as Ttn, Ldb3, Camk2d, Obscn, and Ryr2. Casq1, Mybpc2, and Myot expression was also upregulated in Q374fs-Rbm20 mice. A pathway analysis indicated the involvement of some of the 1770 differentially expressed genes in cytoplasmic ribosomal proteins, calcium regulation in cardiac cells, and striated muscle contraction. Our findings suggest that the Q374fs-Rbm20 variant changes gene splicing, affects genes involved in sarcomere structure and calcium handling genes, and presents with cardiac dysfunction.

Background: EHEC O157:H7 causes severe gastrointestinal illness by first colonizing the large intestine. It intimately attaches to the epithelial lining, orchestrating distinctive “attaching and effacing” lesions that disrupt the host’s cellular landscape. While much is known about the well-established virulence factors, there are much to learn about the surface proteins’ roles in a living host. Methods: This study presents the first in vivo characterisation of the surface proteome, i.e., proteosurfaceome, of Escherichia coli O157:H7 EDL933 during intestinal infection, revealing spatial and temporal adaptations critical for colonisation and survival. Using a murine ileal loop model, surface proteomic profiles were analysed at early (3 h) and late (10 h) infection stages across the ileum and colon. Results: In total, 272 proteins were identified, with only 13 shared across all conditions, reflecting substantial niche-specific adaptations. Gene ontology enrichment analyses highlighted dominant roles in metabolic, cellular, and binding functions, while subcellular localisation prediction uncovered cytoplasmic moonlighting proteins with surface activity. Comparative analyses revealed dynamic changes in protein abundance. Conclusions: These findings indicate a coordinated shift from stress adaptation and virulence to nutrient acquisition and persistence and provide a comprehensive view of EHEC O157:H7 surface proteome dynamics during infection, highlighting key adaptive proteins that may serve as targets for future therapeutic and vaccine strategies.

Desmosomes are essential cell-cell adhesion organelles that enable tension-prone tissues, like the skin and heart, to withstand mechanical stress. Desmosomal anomalies are associated with numerous epidermal disorders, cardiomyopathies, and cancer. Despite their critical importance, how desmosomes sense and respond to mechanical stimuli is not understood. Here, we combine super-resolution imaging in epithelial cells and primary cardiomyocytes, FRET-based tension sensors, atomistic computer simulations, and biochemical assays to demonstrate that actomyosin forces induce a conformational change in desmoplakin, a key cytoplasmic desmosomal protein. We show that in human breast cancer MCF7 cells, keratin-19 couples F-actin filaments to desmosomes and regulates the level of actomyosin forces integrated into the desmosomal complex. We demonstrate that actomyosin contractility reorients keratin intermediate filaments and directs force to desmoplakin along the keratin network, plausibly converting the N-terminal plakin domain from a folded to an extended conformation. We also show that desmoplakin undergoes a similar actomyosin force-dependent conformational change in primary cardiomyocytes, with the extent of the change affected by myofibril orientation. Our findings establish that desmoplakin is mechanosensitive and its structural states reflect the level of forces transmitted through the actin network across cell types.

A stable connection between the sperm head (nucleus) and tail (flagellum) is crucial for proper fertility. This linkage is mediated by centrioles, or their remnants, at a structure known as the head-tail coupling apparatus (HTCA). Although many proteins have been implicated at the HTCA, the precise molecular linkage that connects the head and tail is poorly understood. This Review proposes three molecular models for the HTCA based on the presence of three key components: nuclear envelope proteins, cytoplasmic proteins and centriole proteins. As it relates to these models, we discuss the current literature that describes the linkage from nuclear envelope proteins to cytosolic and centriole proteins, including a LINC-complex-based linkage, a nuclear pore complex linkage and a direct linkage that bypasses the outer nuclear membrane. Finally, we discuss outstanding questions in the field and how future studies might delineate the complex molecular machinery at the HTCA.

O-GlcNAc cycling in neuroinflammation: From molecular mechanisms and therapeutic perspectives.

OGT's inner circle: Protein interactions and functional impact.

In eukaryotes, protein secretion plays essential roles in intercellular communications and extracellular niche-building. Protein secretion generally requires a signal sequence that targets cargos to the canonical secretory pathway consisting of the endoplasmic reticulum (ER), the Golgi apparatus, plasma membrane, and vesicles moving between these compartments. However, cytoplasmic proteins lacking signal sequences (e.g., IL1β, Acb1, FGF2) have been detected, and many have defined functions in the extracellular space, suggesting unconventional protein secretion (UcPS) via alternative pathways. In recent years, scientists have uncovered many new UcPS paradigms, reporting a plethora of mechanisms that collectively form a new field. The inaugural Cold Spring Harbor Asia (CSHA) conference on "Molecular Mechanisms and Physiology of Unconventional Secretion" is the first meeting to bring these researchers together, providing a collegial platform for information sharing at this exciting frontier of cell biology research.

Small cell lung cancer (SCLC) is aggressive, with limited treatment progress for decades. Stathmin 1 (STMN1) is a cytoplasmic phosphorylated protein that is specific to cancerous tissues including SCLC and is associated with malignancy. Pyrrole-imidazole polyamide (PIP) compounds decrease gene expression by binding to specific DNA sites and disturbing RNA transcription. In this study, we synthesized a novel chlorambucil-conjugated PIP compound targeting both the STMN1 promoter and STMN1 DNA sequences (Chb-STMN1 PIP) to evaluate its therapeutic efficacy against SCLC. We examined the expression of STMN1 in surgically resected tissues and cell line database. Suppression of STMN1 by Chb-STMN1 PIP was analyzed using RT-PCR, WB, and CAGE-seq in vitro. The anti-tumor effects of Chb-STMN1 PIP were evaluated in vitro and in vivo. STMN1 mRNA expression was higher in cell lines with more STMN1 copy number alterations. In vitro studies showed that Chb-STMN1 PIP treatment resulted in significant STMN1 suppression and cell viability reduction compared to the control groups. Administration of Chb-STMN1 PIP to an SCLC xenograft mouse model also showed a tumor reduction effect and significantly suppressed angiogenesis, proliferation potency, and cell viability of SCLC. CAGE analysis indicated that the expression of STMN1 was suppressed in Chb-STMN1 PIP-treated cells compared to that in control cells. A novel compound, Chb-STMN1 PIP, induced significant anti-tumor effects and suppressed STMN1 expression in SCLC cell lines. Furthermore, SCLC xenograft mouse models showed tumor shrinkage and reduced malignant properties. Targeting STMN1 with our developed PIP compound appears to be a novel strategy and promising in SCLC.

Bulk organelle‑fractionation masks cell‑to‑cell heterogeneity, and existing microfluidic methods cannot reliably reconnect each isolated organelle to its parent cell, an essential capability for multiomics readouts. VacTrap, a high-throughput microfluidic device that isolates and spatially indexes single nuclei from mammalian cells is developed. The VacTrap device consisted of three aligned layers: 1) a Bis-gel microwells layer with a "trapdoor" (BAC-gel) base, fabricated atop a through-hole glass slide; 2) a polydimethylsiloxane (PDMS) microwell layer to receive transferred nuclei; and 3) a vacuum manifold. VacTrap operation begins with cell cytoplasmic lysis using differential detergent fractionation (DDF) to release intact nuclei into the Bis-gel microwells, while cytoplasmic proteins are electrophoresed into the Bis-gel layer. Subsequent addition of dithiothreitol (DTT) and vacuum dissolves the trapdoors within 3-5min, synchronously transferring nuclei into the PDMS microwells, achieving 98% efficiency across 80% of trapdoors. To verify fractionation of the cytoplasmic proteins from each cell nucleus, selectprotein targets are successfully detected by in situ immunoprobing in the archival Bis-gel layer. To verify the fractionation and collection of individual intact nuclei, the morphology analysis confirms preservation of the nuclear features. By introducing spatial indexing of nuclei back to the originating cell, VacTrap provides a robust, automated cell-preparation platform for single-cell multiomics applications.