Search Results (9)

Recently, SASH1 has emerged as a novel protein interactor of a few Eph tyrosine kinase receptors like EphA2. These interactions involve the first N-terminal Sam (sterile alpha motif) domain of SASH1 (SASH1-Sam1) and the Sam domain of Eph receptors. Currently, the functional meaning of the SASH1-Sam1/EphA2-Sam complex is unknown, but EphA2 is a well-established and crucial player in cancer onset and progression. Thus, herein, to investigate a possible correlation between the formation of the SASH1-Sam1/EphA2-Sam complex and EphA2 activity in cancer, cancer-linked mutations in SASH1-Sam1 were deeply analyzed. Our research plan relied first on searching the COSMIC database for cancer-related SASH1 variants carrying missense mutations in the Sam1 domain and then, through a variety of bioinformatic tools and molecular dynamic simulations, studying how these mutations could affect the stability of SASH1-Sam1 alone, leading eventually to a defective fold. Next, through docking studies, with the support of AlphaFold2 structure predictions, we investigated if/how mutations in SASH1-Sam1 could affect binding to EphA2-Sam. Our study, apart from presenting a solid multistep research protocol to analyze structural consequences related to cancer-associated protein variants with the support of cutting-edge artificial intelligence tools, suggests a few mutations that could more likely modulate the interaction between SASH1-Sam1 and EphA2-Sam. Full article

The Sam (Sterile alpha motif) domain of the lipid phosphatase Ship2 (Ship2-Sam) is engaged by the Sam domain of the receptor tyrosine kinase EphA2 (EphA2-Sam) and, this interaction is principally linked to procancer effects. Peptides able to hinder the formation of the EphA2-Sam/Ship2-Sam complex could possess therapeutic potential. Herein, by employing the FoldX software suite, we set up an in silico approach to improve the peptide targeting of the so-called Mid Loop interface of Ship2-Sam, representing the EphA2-Sam binding site. Starting from a formerly identified peptide antagonist of the EphA2-Sam/Ship2-Sam association, first, the most stabilizing mutations that could be inserted in each peptide position were predicted. Then, they were combined, producing a list of potentially enhanced Ship2-Sam ligands. A few of the in silico generated peptides were experimentally evaluated. Interaction assays with Ship2-Sam were performed using NMR and BLI (BioLayer Interferometry). In vitro assays were conducted as well to check for cytotoxic effects against both cancerous and healthy cells, and also to assess the capacity to regulate EphA2 degradation. This study undoubtedly enlarges our knowledge on how to properly target EphA2-Sam/Ship2-Sam associations with peptide-based tools and provides a promising strategy that can be used to target any protein–protein interaction. Full article

The lipid phosphatase Ship2 interacts with the EphA2 receptor by forming a heterotypic Sam (sterile alpha motif)–Sam complex. Ship2 works as a negative regulator of receptor endocytosis and consequent degradation, and anti-oncogenic effects in cancer cells should be induced by hindering its association with EphA2. Herein, a computational approach is presented to investigate the relationship between Ship2-Sam/EphA2-Sam interaction and cancer onset and further progression. A search was first conducted through the COSMIC (Catalogue of Somatic Mutations in Cancer) database to identify cancer-related missense mutations positioned inside or close to the EphA2–Sam and Ship2–Sam reciprocal binding interfaces. Next, potential differences in the chemical–physical properties of mutant and wild-type Sam domains were evaluated by bioinformatics tools based on analyses of primary sequences. Three-dimensional (3D) structural models of mutated EphA2–Sam and Ship2–Sam domains were built as well and deeply analysed with diverse computational instruments, including molecular dynamics, to classify potentially stabilizing and destabilizing mutations. In the end, the influence of mutations on the EphA2–Sam/Ship2–Sam interaction was studied through docking techniques. This in silico approach contributes to understanding, at the molecular level, the mutation/cancer relationship by predicting if amino acid substitutions could modulate EphA2 receptor endocytosis. Full article

The Drosophila PRC1 complex regulates gene expression by modifying histone proteins and chromatin architecture. Two PRC1 subunits, PSC and Ph, are most implicated in chromatin architecture. In vitro, PRC1 compacts chromatin and inhibits transcription and nucleosome remodeling. The long disordered C-terminal region of PSC (PSC-CTR) is important for these activities, while Ph has little effect. In cells, Ph is important for condensate formation, long-range chromatin interactions, and gene regulation, and its polymerizing sterile alpha motif (SAM) is implicated in these activities. In vitro, truncated Ph containing the SAM and two other conserved domains (mini-Ph) undergoes phase separation with chromatin, suggesting a mechanism for SAM-dependent condensate formation in vivo. How the distinct activities of PSC and Ph on chromatin function together in PRC1 is not known. To address this question, we analyzed structures formed with large chromatin templates and PRC1 in vitro. PRC1 bridges chromatin into extensive fibrillar networks. Ph, its SAM, and SAM polymerization activity have little effect on these structures. Instead, the PSC-CTR controls their growth, and is sufficient for their formation. To understand how phase separation driven by Ph SAM intersects with the chromatin bridging activity of the PSC-CTR, we used mini-Ph to form condensates with chromatin and then challenged them with PRC1 lacking Ph (PRC1ΔPh). PRC1ΔPh converts mini-Ph chromatin condensates into clusters of small non-fusing condensates and bridged fibers. These condensates retain a high level of chromatin compaction and do not intermix. Thus, phase separation of chromatin by mini-Ph, followed by the action of the PSC-CTR, creates a unique chromatin organization with regions of high nucleosome density and extraordinary stability. We discuss how this coordinated sequential activity of two proteins found in the same complex may occur and the possible implications of stable chromatin architectures in maintaining transcription states. Full article

Among the diverse protein binding modules, Sam (Sterile alpha motif) domains attract attention due to their versatility. They are present in different organisms and play many functions in physiological and pathological processes by binding multiple partners. The EphA2 receptor contains a Sam domain at the C-terminus (EphA2-Sam) that is able to engage protein regulators of receptor stability (including the lipid phosphatase Ship2 and the adaptor Odin). Ship2 and Odin are recruited by EphA2-Sam through heterotypic Sam-Sam interactions. Ship2 decreases EphA2 endocytosis and consequent degradation, producing chiefly pro-oncogenic outcomes in a cellular milieu. Odin, through its Sam domains, contributes to receptor stability by possibly interfering with ubiquitination. As EphA2 is upregulated in many types of tumors, peptide inhibitors of Sam-Sam interactions by hindering receptor stability could function as anticancer therapeutics. This review describes EphA2-Sam and its interactome from a structural and functional perspective. The diverse design strategies that have thus far been employed to obtain peptides targeting EphA2-mediated Sam-Sam interactions are summarized as well. The generated peptides represent good initial lead compounds, but surely many efforts need to be devoted in the close future to improve interaction affinities towards Sam domains and consequently validate their anticancer properties. Full article

The Connector Enhancer of Kinase Suppressor of Ras-2 (CNKSR2), also known as CNK2 or MAGUIN, is a scaffolding molecule that contains functional protein binding domains: Sterile Alpha Motif (SAM) domain, Conserved Region in CNK (CRIC) domain, PSD-95/Dlg-A/ZO-1 (PDZ) domain, Pleckstrin Homology (PH) domain, and C-terminal PDZ binding motif. CNKSR2 interacts with different molecules, including RAF1, ARHGAP39, and CYTH2, and regulates the Mitogen-Activated Protein Kinase (MAPK) cascade and small GTPase signaling. CNKSR2 has been reported to control the development of dendrite and dendritic spines in primary neurons. CNKSR2 is encoded by the CNKSR2 gene located in the X chromosome. CNKSR2 is now considered as a causative gene of the Houge type of X-linked syndromic mental retardation (MRXHG), an X-linked Intellectual Disability (XLID) that exhibits delayed development, intellectual disability, early-onset seizures, language delay, attention deficit, and hyperactivity. In this review, we summarized molecular features, neuronal function, and neurodevelopmental disorder-related variations of CNKSR2. Full article

Deoxynucleoside triphosphate (dNTP) molecules are essential for the replication and maintenance of genomic information in both cells and a variety of viral pathogens. While the process of dNTP biosynthesis by cellular enzymes, such as ribonucleotide reductase (RNR) and thymidine kinase (TK), has been extensively investigated, a negative regulatory mechanism of dNTP pools was recently found to involve sterile alpha motif (SAM) domain and histidine-aspartate (HD) domain-containing protein 1, SAMHD1. When active, dNTP triphosphohydrolase activity of SAMHD1 degrades dNTPs into their 2′-deoxynucleoside (dN) and triphosphate subparts, steadily depleting intercellular dNTP pools. The differential expression levels and activation states of SAMHD1 in various cell types contributes to unique dNTP pools that either aid (i.e., dividing T cells) or restrict (i.e., nondividing macrophages) viral replication that consumes cellular dNTPs. Genetic mutations in SAMHD1 induce a rare inflammatory encephalopathy called Aicardi–Goutières syndrome (AGS), which phenotypically resembles viral infection. Recent publications have identified diverse roles for SAMHD1 in double-stranded break repair, genome stability, and the replication stress response through interferon signaling. Finally, a series of SAMHD1 mutations were also reported in various cancer cell types while why SAMHD1 is mutated in these cancer cells remains to investigated. Here, we reviewed a series of studies that have begun illuminating the highly diverse roles of SAMHD1 in virology, immunology, and cancer biology. Full article

Trefoil factors (TFFs) are regulatory peptides playing critical roles in mucosal repair and protection against a variety of insults within the gastrointestinal tract. This work aimed to explore the effects of deoxynivalenol (DON) on intestinal TFFs expression using in vivo and in vitro models. In an animal trial, twenty-four 28-d-old barrows (Duroc × Landrace × Large White; initial body weight = 7.6 ± 0.7 kg) were randomly divided into three treatments for 28 days, including a control diet (0.61 mg DON/kg feed), and two levels of DON-contaminated diets containing 1.28 and 2.89 mg DON/kg feed, respectively. Piglets exposed to DON had lower mRNA expression of TFF1, TFF2, TFF3, as well as Claudin-4 in the intestine (P < 0.05). Dietary DON exposure decreased the protein levels of TFF2 and TFF3 in the jejunum as demonstrated by western blot and immunohistochemistry. In intestinal porcine epithelial cells (IPEC-J2), DON depressed the mRNA expression of TFF2, TFF3, and Claudin-4. Overexpression of sterile alpha motif (SAM) pointed domain E26 transformation-specific (ETS) factor (SPDEF) was found to attenuate DON-induced suppression of TFFs in IPEC-J2 cells. Altogether, our work shows, for the first time, that dietary DON exposure depresses the expression of intestinal TFFs in piglets. Given the fundamental role of TFFs in intestinal mucosal homeostasis, our observations indicate that the DON content in animal feed should be strictly controlled based on the existing regulation for DON. Full article

Macrophages are natural target cells of human immunodeficiency virus type 1 (HIV-1). Viral replication appears to be delayed in these cells compared to lymphocytes; however, little is known about the kinetics of early post-entry events. Time-of-addition experiments using several HIV-1 inhibitors and the detection of reverse transcriptase (RT) products with droplet digital PCR (ddPCR) revealed that early replication was delayed in primary human monocyte-derived macrophages of several donors and peaked late after infection. Direct imaging of reverse-transcription and pre-integration complexes (RTC/PIC) by click-labeling of newly synthesized DNA further confirmed our findings and showed a concomitant shift to the nuclear stage over time. Altering the entry pathway enhanced infectivity but did not affect kinetics of viral replication. The addition of viral protein X (Vpx) enhanced productive infection and accelerated completion of reverse transcription and nuclear entry. We propose that sterile alpha motif (SAM) and histidine/aspartate (HD) domain-containing protein 1 (SAMHD1) activity lowering deoxyribonucleotide triphosphate (dNTP) pools is the principal factor delaying early HIV-1 replication in macrophages. Full article