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Epilepsy is a common neurological disorder, and mutations in genes encoding ion channels or neurotransmitter receptors are frequent causes of monogenic forms of epilepsy. Here we show that abnormal expansions of TTTCA and TTTTA repeats in intron 4 of SAMD12 cause benign adult familial myoclonic epilepsy (BAFME). Single-molecule, real-time sequencing of BAC clones and nanopore sequencing of genomic DNA identified two repeat configurations in SAMD12 . Intriguingly, in two families with a clinical diagnosis of BAFME in which no repeat expansions in SAMD12 were observed, we identified similar expansions of TTTCA and TTTTA repeats in introns of TNRC6A and RAPGEF2 , indicating that expansions of the same repeat motifs are involved in the pathogenesis of BAFME regardless of the genes in which the expanded repeats are located. This discovery that expansions of noncoding repeats lead to neuronal dysfunction responsible for myoclonic tremor and epilepsy extends the understanding of diseases with such repeat expansion. This study identifies TTTCA- and TTTTA-repeat expansions in benign adult familial myoclonic epilepsy. Cortical neurons from affected people exhibit RNA foci containing these expanded repeats, suggesting RNA toxicity as the mechanism underlying disease pathogenesis.

Polycomb Group (PcG) proteins organize chromatin at multiple scales to regulate gene expression. A conserved Sterile Alpha Motif (SAM) in the Polycomb Repressive Complex 1 (PRC1) subunit Polyhomeotic (Ph) has been shown to play an important role in chromatin compaction and large-scale chromatin organization. Ph SAM forms helical head to tail polymers, and SAM-SAM interactions between chromatin-bound Ph/PRC1 are believed to compact chromatin and mediate long-range interactions. To understand the underlying mechanism, here we analyze the effects of Ph SAM on chromatin in vitro. We find that incubation of chromatin or DNA with a truncated Ph protein containing the SAM results in formation of concentrated, phase-separated condensates. Ph SAM-dependent condensates can recruit PRC1 from extracts and enhance PRC1 ubiquitin ligase activity towards histone H2A. We show that overexpression of Ph with an intact SAM increases ubiquitylated H2A in cells. Thus, SAM-induced phase separation, in the context of Ph, can mediate large-scale compaction of chromatin into biochemical compartments that facilitate histone modification. The conserved SAM motif of Polycomb Repressive Complex 1 subunit Ph has been shown to play an important role in chromatin organization. Here, the authors study the effect of Ph SAM on chromatin in vitro, showing that it induces the formation of concentrated, phase-separated condensates, which enhance the ubiquitin ligase activity of PRC1.

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.

Recurrence is a challenge to survival after the initial treatment of esophageal squamous cell carcinoma (ESCC). But, its mechanism remains elusive and there are currently no biomarkers to predict postoperative recurrence. Here, the possibility of sterile alpha motif domain‐containing protein 9 (SAMD9) as a predictor of postoperative recurrence of ESCC is evaluated and the molecular mechanisms by which SAMD9 promotes ESCC recurrence are elucidated. The authors found that the high level of SAMD9 is correlated with postoperative recurrence and poor prognosis of ESCC. Overexpression of SAMD9 promotes tumor stemness, angiogenesis, and EMT, while downregulation of SAMD9 reduced these phenotypes. Mechanistically, it is found that SAMD9 stimulated ubiquitination‐mediated glycogen synthase kinase‐3 beta (GSK‐3β) degradation by interaction with myosin‐9 (MYH9) and TNF receptor‐associated factor 6 (TRAF6), which in turn activated Wnt/β‐catenin pathway. Further, the authors demonstrated that silencing SAMD9 inhibited lung metastasis and tumor formation in vivo. Finally, the authors found that silencing MYH9 or β‐catenin, or overexpressing GSK‐3β inhibited SAMD9‐stimulated ESCC cell stemness, EMT, angiogenesis, metastasis, and tumorigenicity. Together, the findings indicate that the SAMD9/MYH9/GSK3β/β‐catenin axis promotes ESCC postoperative recurrence and that SAMD9 is a crucial target for ESCC therapy. Additionally, SAMD9 has the potential as a predictor of postoperative recurrence in ESCC. This work demonstrates the clinical and functional role of SAMD9 in esophageal squamous cell carcinoma (ESCC). SAMD9 promotes MYH9/TRAF6‐regulated ubiquitination and degradation of GSK3β by binding to MYH9, leading to activation of the β‐catenin signaling pathway, which stimulates EMT, angiogenesis, and stemness of ESCC cells, and ultimately promotes ESCC metastasis and recurrence.

The root growth angle defines how roots grow toward the gravity vector and is among the most important determinants of root system architecture. It controls water uptake capacity, nutrient use efficiency, stress resilience, and, as a consequence, yield of crop plants. We demonstrated that the egt2 (enhanced gravitropism 2) mutant of barley exhibits steeper root growth of seminal and lateral roots and an auxin-independent higher responsiveness to gravity compared to wild-type plants. We cloned the EGT2 gene by a combination of bulked-segregant analysis and whole genome sequencing. Subsequent validation experiments by an independent CRISPR/Cas9 mutant allele demonstrated that egt2 encodes a STERILE ALPHA MOTIF domain–containing protein. In situ hybridization experiments illustrated that EGT2 is expressed from the root cap to the elongation zone. We demonstrated the evolutionary conserved role of EGT2 in root growth angle control between barley and wheat by knocking out the EGT2 orthologs in the A and B genomes of tetraploid durum wheat. By combining laser capture microdissection with RNA sequencing, we observed that seven expansin genes were transcriptionally down-regulated in the elongation zone. This is consistent with a role of EGT2 in this region of the root where the effect of gravity sensing is executed by differential cell elongation. Our findings suggest that EGT2 is an evolutionary conserved regulator of root growth angle in barley and wheat that could be a valuable target for root-based crop improvement strategies in cereals.

Viruses hijack the host cell machinery to promote viral replication; however, the mechanism by which metabolic reprogramming regulates innate antiviral immunity in the host remains elusive. Herein, we explore how the hexosamine biosynthesis pathway (HBP) and O-linked-N-acetylglucosaminylation (O-GlcNAcylation) regulate host antiviral response against hepatitis B virus (HBV) and We conducted a metabolomics assay to evaluate metabolic responses of host cells to HBV infection. We systematically explored the role of HBP and protein O-GlcNAcylation in regulating HBV infection in cell and mouse models. O-linked N-acetylglucosamine (O-GlcNAc) target proteins were identified via liquid chromatography-tandem mass spectrometry (LC-MS) and co-immunoprecipitation assays. Additionally, we also examined uridine diphosphate (UDP)-GlcNAc biosynthesis and O-GlcNAcylation levels in patients with chronic hepatitis B (CHB). HBV infection upregulated GLUT1 expression on the hepatocyte surface and facilitated glucose uptake, which provides substrates to HBP to synthesize UDP-GlcNAc, leading to an increase in protein O-GlcNAcylation. Pharmacological or transcriptional inhibition of HBP and O-GlcNAcylation promoted HBV replication. Mechanistically, O-GlcNAc transferase (OGT)-mediated O-GlcNAcylation of sterile alpha motif and histidine/aspartic acid domain-containing protein 1 (SAMHD1) on Ser93 stabilizes SAMHD1 and enhances its antiviral activity. Analysis of clinical samples revealed that UDP-GlcNAc level was increased, and SAMHD1 was O-GlcNAcylated in patients with CHB. HBP-mediated O-GlcNAcylation positively regulates host antiviral response against HBV and . The findings reveal a link between HBP, O-GlcNAc modification, and innate antiviral immunity by targeting SAMHD1.

No efficient treatment exists for nephrotic syndrome (NS), a frequent cause of chronic kidney disease. Here we show mutations in six different genes ( MAGI2, TNS2, DLC1, CDK20, ITSN1, ITSN2 ) as causing NS in 17 families with partially treatment-sensitive NS (pTSNS). These proteins interact and we delineate their roles in Rho-like small GTPase (RLSG) activity, and demonstrate deficiency for mutants of pTSNS patients. We find that CDK20 regulates DLC1. Knockdown of MAGI2 , DLC1 , or CDK20 in cultured podocytes reduces migration rate. Treatment with dexamethasone abolishes RhoA activation by knockdown of DLC1 or CDK20 indicating that steroid treatment in patients with pTSNS and mutations in these genes is mediated by this RLSG module. Furthermore, we discover ITSN1 and ITSN2 as podocytic guanine nucleotide exchange factors for Cdc42. We generate Itsn2 - L knockout mice that recapitulate the mild NS phenotype. We, thus, define a functional network of RhoA regulation, thereby revealing potential therapeutic targets. Nephrotic syndrome is the second most common chronic kidney disease but there are no targeted treatment strategies available. Here the authors identify mutations of six genes codifying for proteins involved in cytoskeleton remodelling and modulation of small GTPases in 17 families with nephrotic syndrome.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) induces immune-mediated type 1 interferon (IFN-1) production, the pathophysiology of which involves sterile alpha motif and histidine-aspartate domain-containing protein 1 (SAMHD1) tetramerization and the cytosolic DNA sensor cyclic-GMP-AMP synthase (cGAS)–stimulator of interferon genes (STING) signaling pathway. As a result, type I interferonopathies are exacerbated. Aspirin inhibits cGAS-mediated signaling through cGAS acetylation. Acetylation contributes to cGAS activity control and activates IFN-1 production and nuclear factor-κB (NF-κB) signaling via STING. Aspirin and dapsone inhibit the activation of both IFN-1 and NF-κB by targeting cGAS. We define these as anticatalytic mechanisms. It is necessary to alleviate the pathologic course and take the lag time of the odds of achieving viral clearance by day 7 to coordinate innate or adaptive immune cell reactions.

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.

Plant shoots typically grow upward in opposition to the pull of gravity. However, exceptions exist throughout the plant kingdom. Most conspicuous are trees with weeping or pendulous branches. While such trees have long been cultivated and appreciated for their ornamental value, the molecular basis behind the weeping habit is not known. Here, we characterized a weeping tree phenotype in Prunus persica (peach) and identified the underlying genetic mutation using a genomic sequencing approach. Weeping peach tree shoots exhibited a downward elliptical growth pattern and did not exhibit an upward bending in response to 90 degrees reorientation. The causative allele was found to be an uncharacterized gene, Ppa013325, having a 1.8-Kb deletion spanning the 5' end. This gene, dubbed WEEP, was predominantly expressed in phloem tissues and encodes a highly conserved 129-amino acid protein containing a sterile alpha motif (SAM) domain. Silencing WEEP in the related tree species Prunus domestica (plum) resulted in more outward, downward, and wandering shoot orientations compared to standard trees, supporting a role for WEEP in directing lateral shoot growth in trees. This previously unknown regulator of branch orientation, which may also be a regulator of gravity perception or response, provides insights into our understanding of how tree branches grow in opposition to gravity and could serve as a critical target for manipulating tree architecture for improved tree shape in agricultural and horticulture applications.