Ubiquitin-mediated downregulation of the proteasome subunit ADRM1 mediates in vitro capacitation of bovine spermatozoa.

Sophoraflavanone G disrupts human sperm function via impairing CatSper-mediated Ca2+ signaling.

CT10 regulator of kinase (CRK) and CRK-Like (CRKL) are important Src homology 2 (SH2)- and SH3-domain containing signaling adaptors, which drive cell adhesion, motility, differentiation, and proliferation. Their genetically defined and overlapping roles include facilitating proper development of the vertebrate central nervous system. All four members of the SH2-domain containing (SH) protein family are enriched in YXXP motifs, which when the initial tyrosine is phosphorylated form the preferred binding motif of CRK family SH2 domains. We show that ABL kinase drives tyrosine phosphorylation of SH protein YXXP motifs and induces their direct binding to the SH2 domains of CRK family adaptors. The implications of the interactions between CRK/CRKL and SH proteins include signal attenuation and relocation of CRK adaptor signaling.

Protein tyrosine kinases govern essential cellular processes, including proliferation, survival, and migration. Dysregulation of this enzyme family has been implicated in multiple hallmarks of cancer. These kinases catalyze tyrosine phosphorylation in a tightly controlled manner, with substrate recognition dictated by the local chemical environment of surrounding residues. Although several strategies have been developed to define protein tyrosine kinase substrate specificity, these approaches have notable limitations in scale and resolution. To address these challenges, we present a modernized phage display platform integrated with next-generation sequencing, enabling comprehensive and high-throughput profiling of tyrosine kinase substrates. This method enabled the simultaneous assessment of the relative phosphorylation of billions of potential substrates for Hck kinase, followed by analysis of positional and motif enrichment to guide iterative substrate design. The novel substrates developed through this process exhibited robust kinetic behavior (Km < 30 μM) when evaluated by HPLC/MS assays, yielding insights into Hck substrate preferences within multipositional sequence contexts.

Glycoprotein 130 (GP130) is an archetypal class I cytokine receptor that serves as a shared signalling subunit for a group of cytokines known as interleukin 6 (IL-6)-type cytokines. GP130 does not have an intrinsic kinase activity and relies on Janus kinases (JAK) 1, 2, or Tyrosine kinase 2 (TYK2) for phosphorylation of tyrosine residues on its cytoplasmic domain for signal transduction. In this study we investigated the molecular mechanism of GP130 activation. We utilized GP130 constructs that were held in varying states of activation by replacing the entire extracellular region of the receptor with a leucine zipper dimerisation domain and inserted alanine residues into the extracellular juxtamembrane linker sequence to induce rotation of the transmembrane domains. These chimeras differentially activated the associated JAK2 molecules which also correlated with receptor tyrosine phosphorylation status. Förster resonance energy transfer (FRET) microscopy in live cells was used to show that GP130 cytoplasmic domains were in close proximity in active chimeras compared to the inactive receptors, presumably to facilitate JAK protein activation. Coarse-grained molecular dynamics simulations provided a structural rationale for this register-dependent mechanism, showing that active chimeras preferentially maintain compact transmembrane packing and reduced membrane-proximal cytosolic spacing, whereas signalling-deficient constructs sample more heterogeneous transmembrane geometries and increased cytosolic separation. Finally, mutational analysis identified the negatively charged juxtamembrane residues E617 and E619 as critical for signal transduction; charge reversal at these positions in the full-length receptor generated a dominant-negative GP130 variant devoid of signalling capacity.

Temporal characterization of conditions that promote functional capacitation of stallion sperm.

Raman spectroscopy has been widely demonstrated as a powerful tool for analyzing macromolecules characteristic of living organisms. This research focuses on a detailed spectroscopic analysis of lymphocytes affected by infectious mononucleosis caused by Epstein-Barr virus (EBV, HHV-4). As shown, at least 98% of the population is infected with EBV, making it one of the most common viruses. The main aim of this study is to evaluate the capability of Raman spectroscopy to distinguish between healthy lymphocytes from those infected with EBV at an early stage of infection. Blood samples were collected from patients with infectious mononucleosis who were receiving treatment at the University Hospital in Kraków. For comparative purposes, samples from healthy volunteers were also included. To guarantee detailed analysis, Raman spectra were acquired using two excitation wavelengths: 514.5 nm and 785 nm. The two-trace two-dimensional (2T2D) correlation method was performed to recognize the difference between EBV-infected and healthy cells. Initially, mean Raman spectra were calculated for healthy and infected cells and subsequently used to generate synchronous and asynchronous 2T2D correlation maps. Analysis of the asynchronous correlation spectra reveals distinct differences between healthy and diseased lymphocytes for both excitation wavelengths. For example, asynchronous peaks at 514.5 nm excitation indicate the beginning of immunoglobulin formation, while at 785 nm excitation indicate Tyrosine phosphorylation, which corresponds to activation of lymphocytes. The detection of specific biochemical alterations shows the potential of Raman spectroscopy as a non-invasive diagnostic tool in viral infections.

The purpose of this study was to characterize three recurrent, cancer-associated missense mutations in ErbB2, R143Q, R678Q, and V842I, located in the extracellular, juxtamembrane and kinase domains, respectively, to determine how single amino acid substitutions affect receptor organization and dynamics. Using confocal microscopy, Förster resonance energy transfer (FRET) and fluorescence recovery after photobleaching (FRAP), we assessed homo- and heteroassociation, lateral diffusion and tyrosine phosphorylation of ErbB2 cancer-associated variants either alone or co-expressed with EGFR. The only individual biophysical property that differentiated the mutation-activated ErbB2 variants from the wild-type was an accelerated diffusion in the absence of EGFR coexpression. Correlative analysis of the biophysical properties of ErbB2 revealed that ErbB2-activating mutations, including the cancer-associated R143Q, R678Q and V842I mutations, may promote a pre-dimerized receptor state associated with accelerated lateral mobility, but without full-scale activation implied by the lack of growth factor-independent tyrosine phosphorylation. The faster mobility of mutation-activated ErbB2 contrasted with the EGF-induced slowing down of its lateral diffusion. In summary, single amino acid substitutions across ErbB2 domains may modulate receptor dynamics, organization, and responsiveness.

BackgroundPalmitoylacyltransferase DHHC7 (DHHC7) plays a critical role in various biological processes and diseases. However, its expression, localization, and regulatory mechanisms in sperm function remain largely unknown.MethodsImmunofluorescent staining was used to localize DHHC7 in sperm and examine its colocalization with the estrogen receptor (ER), progesterone receptor (PR), and caveolin-1 (CAV1). To explore DHHC7’s role in sperm function, sperm were treated with a specific DHHC7 antibody. Subsequently, sperm motility and motion parameters were analyzed using a computer-assisted sperm analyzer, and the acrosome reaction (an indicator of sperm capacitation) was evaluated using fluorescein isothiocyanate-labeled Pisum sativum agglutinin. To explore the possible regulatory mechanisms of DHHC7 on sperm function, protein palmitoylation was assessed using the acyl-biotin exchange method. Intracellular calcium levels and reactive oxygen species (ROS) production were monitored using fluorescent probes Fluo-4 acetoxymethyl ester (Fluo-4 AM) and 2′,7′-Dichlorodihydrofluorescein diacetate (DCFH-DA), respectively, and protein tyrosine phosphorylation levels and DHHC7 protein in sperm were analyzed using Western blotting.ResultsWe found that DHHC7 was localized in the neck, principal piece, midpiece, and end piece of sperm in both mice and humans. Treatment with DHHC7 antibodies significantly impaired sperm motility, progressive movement, hyperactivation, and the acrosome reaction under capacitated conditions. Furthermore, DHHC7 colocalized with the ER, PR, and CAV1 in mouse sperm. Additionally, DHHC7 antibodies reduced protein palmitoylation and tyrosine phosphorylation levels, and reduced intracellular calcium elevation and ROS generation during mouse sperm capacitation.ConclusionThis study identifies DHHC7 as a regulator of sperm motility and capacitation and suggests that its effects involve palmitoylation-dependent modulation of calcium signaling, tyrosine phosphorylation, and ROS signaling by the spatial association with ER, PR, and CAV1. These findings provide novel insights into the biological functions and regulatory mechanisms of DHHC7 in sperm, highlighting its potential significance in reproductive biology.

Dosage imbalance of dual specificity tyrosine phosphorylation regulated kinase 1A (DYRK1A) is a feature of several neurodevelopmental and neurodegenerative diseases, including Down syndrome, DYRK1A syndrome, autism spectrum disorders, Alzheimer's disease and Parkinson's disease. Thus, manipulating DYRK1A activity in the brain has emerged as a potential therapeutic target for neurological disorders. Several DYRK1A inhibitors have shown promise for improving cognition in rodent models of Down syndrome and Alzheimer's disease, for example, but the ability of these inhibitors to affect DYRK1A levels or activity in relevant human cells has not been established. We filled this gap by testing the effects of a new DYRK1A inhibitor on trisomy 21 induced pluripotent stem cell (iPSC)-derived neural progenitor cells and neurons, in which DYRK1A expression and activity are increased. Our results demonstrated that Leucettinib-21, a potent and selective low-molecular-mass pharmacological inhibitor of DYRK1A, decreases DYRK1A activity in human trisomy 21 iPSC-derived neural progenitor cells and cortical neurons. Leucettinib-21 reduces DYRK1A activity in a relevant human disease model, supporting future human trials.

CaMKIIδ splice variants differentially regulate vascular smooth muscle cell motility.

Vertebrate neural tube closure requires planar cell polarity (PCP) signaling to coordinate cell behaviors in the neuroepithelium. In the Xenopus neural plate, PCP is marked by enrichment of the core PCP protein Vangl2 at the anterior edge of every cell, but the distribution of the extracellular factors modulating this asymmetry remains largely unknown. Here, we identify the secreted protein R-spondin2 (Rspo2) as a modulator of neural plate PCP. Rspo2 exhibits predominantly anterior localization in neuroepithelial cells. Morpholino-mediated knockdown of Rspo2 causes neural tube closure defects and disrupts the anterior enrichment of Vangl2. Rspo2 associates with Vangl2 and inhibits FGF receptor-dependent Vangl2 tyrosine phosphorylation in vivo , as shown by both depletion and overexpression experiments. Rspo2 domain analysis shows that the thrombospondin domain contributes to both anterior Rspo2 membrane enrichment and inhibition of Vangl2 phosphorylation. Together, these findings identify a role of Rspo2 in PCP signaling in the Xenopus neural plate and support a model in which anteriorly localized Rspo2 helps maintain Vangl2 asymmetry while limiting Vangl2 tyrosine phosphorylation during neural plate morphogenesis.

Stress can elevate plasma homocysteine (Hcy) levels, a key factor in the development of various diseases, leading to hyperhomocysteinemia (HHcy). The enzyme Cystathionine β-synthase (CBS) functions in the trans-sulfuration pathway, which links the methionine cycle to glutathione synthesis. This pathway is essential for the metabolism of homocysteine. However, the precise mechanism by which stress regulates hepatic CBS expression remains unclear. The present study aimed to elucidate the molecular mechanism by which CBS expression is regulated in the livers of restraint-stressed rats. Our results showed that stress-induced over-secretion of epinephrine and norepinephrine (E/NE) activated the β-adrenergic receptor, leading to elevated serum IL-6 levels. Treatment of rat primary hepatocytes with IL-6 for 1h suppressed both CBS activity and Cbs mRNA expression, concomitant with an enhancement of Sp3-mediated transcriptional repression. Furthermore, we found that IL-6 activates NF-κB via the tyrosine phosphorylation pathway, which in turn potentiates Sp3-mediated repression of Cbs transcription. These findings suggest that E/NE contributes to stress-induced HHcy by inhibiting Cbs transcription through the upregulation of Sp3, and that the IL-6/NF-κB axis plays a critical role in the dysregulation of Hcy metabolism.

Capacitation is a key maturation process that enables spermatozoa to acquire fertilizing ability and can be induced in vitro using capacitation media. Because capacitation protocols differ markedly among laboratories, we compared three compositionally distinct Hepes-, Tris-, and TALP-based media. This study was performed in boar spermatozoa using 3–6 biological replicates of pooled ejaculates depending on the assay, with 46 ejaculate samples from 12 boars in total. The aim was to determine whether such non-standardized conditions differentially affect signaling pathways leading to capacitation and thereby influence the detection of commonly used capacitation markers. We found clear differences among the tested media. All three induced capacitation-associated events, but their functional and molecular effects were not equivalent. The Hepes-based medium supported sperm motility most effectively, increasing total and progressive motility to 60.0% and 48.7%, respectively, after 1 h of incubation and maintaining the highest motility throughout the incubation period. In contrast, the Tris-based medium maintained lower but relatively stable motility, whereas the TALP-based medium showed a rapid decline in total motility from 53.1% to 15.2% during the first hour. The TALP-based medium induced the highest and most sustained protein kinase A (PKA) activity, reaching 0.047 U/mL at 0 h and 0.040 U/mL after 3 h, whereas the Hepes- and Tris-based media showed lower and less sustained activity ranging from 0.003 to 0.030 U/mL during incubation. In addition, distinct patterns of protein tyrosine phosphorylation were observed depending on the medium used. In particular, the TALP-based medium containing bicarbonate and bovine serum albumin (BSA) and the Hepes-based medium with the highest BSA concentration were associated with the highest levels of total protein tyrosine phosphorylation. Phosphoproteomic analysis further revealed condition-specific phosphorylation events, indicating that sperm maturation is dynamically regulated by the surrounding molecular environment. In contrast, no significant differences were detected in oxidative phosphorylation or in electron transport system complexes among the tested media. These findings show that differences in capacitation media composition, particularly in bicarbonate and BSA content, can markedly alter signaling outcomes and the interpretation of capacitation markers, with important implications for reproductive technologies and experimental standardization.

Intricate phosphorylation-dependent regulatory mechanisms enable viruses to diversify the functions of their proteins, profoundly shaping viral replication and pathogenicity. Although phosphoproteomic analyses have produced an expanding catalog of phosphorylation sites on viral proteins, a considerable proportion of these modifications are likely non-functional. This creates a pressing need for a prioritization strategy to predict functionally relevant phosphorylation sites. To address this, we developed an evolution-guided prioritization strategy that integrates phosphoproteomic data with genus-level conservation. Using this strategy, we prioritized two tyrosine phosphorylation sites for functional analysis. Notably, phosphorylation at one of these sites, UL7 Tyr-89, functions as a tissue-specific regulatory mechanism that fine-tunes UL7 activity and modulates herpes simplex virus 1 replication and pathogenicity in the central nervous system. This strategy provides a practical framework for prioritizing candidate regulatory sites for functional investigation in viral replication and pathogenicity.

Wiskott-Aldrich Syndrome protein (WASp) is an actin nucleation-promoting factor that regulates the dynamic rearrangements of the actin cytoskeleton following T cell receptor (TCR) engagement. Recognition of antigen by T cells leads to TCR signal transduction, which involves the phosphorylation of various proteins in close proximity to the TCR, known as proximal TCR signalling. Proximal TCR signalling is initiated by the phosphorylation of signalling proteins including CD3 subunits, lymphocyte-specific protein tyrosine kinase (Lck) and zeta-chain-associated protein kinase 70 (ZAP70). Activation of these proteins initiates the formation of a signalosome, which is required for actin polymerisation and gene expression. The role of WASp in relation to proximal TCR signalling is largely unknown. In the present study, we knocked out WASp in Jurkat T cells using the CRISPR-Cas9 system to evaluate its effect on proximal TCR signalling. As expected, Jurkat T cells lacking WASp exhibited impaired actin polymerisation. Following TCR triggering, phosphorylation of CD3, Lck and ZAP70 was markedly reduced. There was also a failure in the recruitment of Lck and ZAP70 to the TCR. Impaired proximal TCR signalling (reduced tyrosine phosphorylation of CD3, Lck and ZAP70) was exclusively associated with decreased CD69 and CD25 expression. Therefore, besides its role in actin rearrangement, WASp is also required for proximal TCR signalling.

CLDN18.2 has emerged as a promising therapeutic target in gastric and gastro-oesophageal junction cancers. However, its clinical efficacy in pancreatic ductal adenocarcinoma (PDAC) has been modest, suggesting the presence of regulatory mechanisms impairing its efficacy. We aim to investigate how O-linked N-acetylglucosaminylation (O-GlcNAcylation) affects CLDN18.2 subcellular localisation, tumour progression and therapeutic resistance in PDAC, while exploring strategies to restore treatment sensitivity. This study used samples from patients with PDAC, along with the following models: humanised patient-derived xenograft (PDX), patient-derived organoids (PDOs), orthotopic PDO xenograft, KPC mice (LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx1-Cre) and KPC-Cldn18.2 knockout (KO) mice. KRAS (Kirsten rat sarcoma viral oncogene homolog) mutation and hyperglycaemia cooperatively drive CLDN18.2 O-GlcNAcylation at T204, promoting CLDN18.2 cytoplasmic accumulation. O-GlcNAcylated CLDN18.2 promotes pancreatic cancer migration, invasion and metastases and reduces its sensitivity to anti-CLDN18.2 based targeted therapy. Mechanistically, O-GlcNAcylated CLDN18.2 exhibits reduced binding to PTP1B, leading to enhanced tyrosine phosphorylation. O-GlcNAcylated CLDN18.2 recruits Src via SH2 domain, triggering Src activation. Genetic (T204A) or pharmacological blockade of O-GlcNAcylation restores CLDN18.2 membrane localisation and suppresses tumour progression. Therapeutically, low-dose MRTX1133 (KRASG12D inhibitor) reduces O-GlcNAcylation and synergises with CLDN18.2-targeted therapy in KRAS mutant PDAC models with minimal side effects. KRAS mutations and hyperglycaemia drive O-GlcNAcylation of CLDN18.2 at its C-terminal T204 site. O-GlcNAcylated CLDN18.2 promotes poor prognosis and reduces the effectiveness of CLDN18.2-targeted therapies. Low dose MRTX1133 restores CLDN18.2 membrane localisation by reducing its O-GlcNAcylation and augments CLDN18.2-targeted therapy efficacy, offering a novel combinatorial strategy for KRAS-mutant PDAC.

Infertility is a global health problem, with male factors contributing to nearly half of all cases. Up to 30% of male infertility is classified as idiopathic, in part because routine semen analysis does not assess sperm fertilizing competence. Capacitation is a complex process that endows spermatozoa with the competence to fertilize the oocyte, and it depends on oxidant-driven phosphorylation events. These events include increased PKA substrate and tyrosine phosphorylation, which promote hyperactivated motility and the acrosome reaction. These pathways are normally restrained by decapacitation factors that must be relieved in the female reproductive tract before capacitation can proceed. Protein CoAlation is an antioxidant modification of protein thiols through a disulfide bond with coenzyme A (CoASH). We previously detected protein CoAlation in human spermatozoa and observed that its levels decline during capacitation, but its function was unknown. We hypothesized that protein CoAlation functions as a decapacitation mechanism that prevents redox signalling, enabling oxidative activation of phosphorylation events during capacitation. Using spermatozoa from healthy human donors, we leveraged subcellular fractionation, immunocytochemistry, computer-assisted sperm analysis (CASA), and immunoblotting to determine the sperm protein CoAlation profile, assess CoASH biosynthetic enzymes, and test how pharmacological modulation of CoAlation levels influences capacitation. CoAlated proteins were distributed across intracellular sperm compartments, and spermatozoa possess the CoASH biosynthetic enzymes PANK2 and CoASY, indicating an intrinsic capacity for CoAlation. Inhibition of CoASH biosynthesis reduced CoAlation and enhanced PKA substrate phosphorylation, tyrosine phosphorylation, hyperactivated motility, and the progesterone-induced acrosome reaction under capacitating conditions. Pantothenic acid supplementation increased CoAlation and suppressed these processes without impairing viability or baseline motility. These findings indicate that high levels of protein CoAlation in several protein bands are a pre-existing feature of the non-capacitated state that restrains the redox-regulated events of capacitation and that its decline is required to permit sperm capacitation. CoAlation levels may emerge as a biomarker of sperm capacitation and fertilizing competence.

HIV-1 Tat-induced VAPB disruption initiates a cascade of organellar failures culminating in neuronal lipid accumulation

Age-dependent induction of ER stress in retinal pigment epithelium impairs phagocytosis via ADAM17-dependent MERTK shedding.