The SMC5/6 complex: folding chromosomes back into shape when genomes take a break.

High-level folding of chromatin is a key determinant of the shape and functional state of chromosomes. During cell division, structural maintenance of chromosome (SMC) complexes such as condensin and cohesin ensure large-scale folding of chromatin into visible chromosomes. In contrast, the SMC5/6 complex plays more local and context-specific roles in the structural organization of interphase chromosomes with important implications for health and disease. Recent advances in single-molecule biophysics and cryo-electron microscopy revealed key insights into the architecture of the SMC5/6 complex and how interactions connecting the complex to chromatin components give rise to its unique repertoire of interphase functions. In this review, we provide an integrative view of the features that differentiates the SMC5/6 complex from other SMC enzymes and how these enable dramatic reorganization of DNA folding in space during DNA repair reactions and other genome transactions. Finally, we explore the mechanistic basis for the dynamic targeting of the SMC5/6 complex to damaged chromatin and its crucial role in human health.

A peptide array pipeline for the development of Spike-ACE2 interaction inhibitors.

In humans, coronaviruses are the cause of endemic illness and have been the causative agents of more severe epidemics. Most recently, SARS-CoV-2 was the causative agent of the COVID19 pandemic. Thus, there is a high interest in developing therapeutic agents targeting various stages of the coronavirus viral life cycle to disrupt viral propagation. Besides the development of small-molecule therapeutics that target viral proteases, there is also interest molecular tools to inhibit the initial event of viral attachment of the SARS-CoV-2 Spike protein to host ACE2 surface receptor. Here, we leveraged known structural information and peptide arrays to develop an in vitro peptide inhibitor of the Spike-ACE2 interaction. First, from previous co-crystal structures of the Spike-ACE2 complex, we identified an initial 24-residue long region (sequence: STIEEQAKTFLDKFNHEAEDLFYQ) on the ACE2 sequence that encompasses most of the known contact residues. Next, we scanned this 24-mer window along the ACE2 N-terminal helix and found that maximal binding to the SARS-CoV-2 receptor binding domain (CoV2-RBD) was increased when this window was shifted nine residues in the N-terminal direction. Further, by systematic permutation of this shifted ACE2-derived peptide we identified mutations to the wildtype sequence that confer increased binding of the CoV2-RBD. Among these peptides, we identified binding peptide 19 (referred to as BP19; sequence: SLVAVTAAQSTIEEQAKTFLDKFI) as an in vitro inhibitor of the Spike-ACE2 interaction with an IC50 of 2.08 ± 0.38 µM. Overall, BP19 adds to the arsenal of Spike-ACE2 inhibitors, and this study highlights the utility of systematic peptide arrays as a platform for the development of coronavirus protein inhibitors.

Proteome-wide Prediction of Lysine Methylation Leads to Identification of H2BK43 Methylation and Outlines the Potential Methyllysine Proteome.

Protein Lys methylation plays a critical role in numerous cellular processes, but it is challenging to identify Lys methylation in a systematic manner. Here we present an approach combining in silico prediction with targeted mass spectrometry (MS) to identify Lys methylation (Kme) sites at the proteome level. We develop MethylSight, a program that predicts Kme events solely on the physicochemical properties of residues surrounding the putative methylation sites, which then requires validation by targeted MS. Using this approach, we identify 70 new histone Kme marks with a 90% validation rate. H2BK43me2, which undergoes dynamic changes during stem cell differentiation, is found to be a substrate of KDM5b. Furthermore, MethylSight predicts that Lys methylation is a prevalent post-translational modification in the human proteome. Our work provides a useful resource for guiding systematic exploration of the role of Lys methylation in human health and disease.

Insights into The Function and Regulation of Jumonji C Lysine Demethylases as Hypoxic Responsive Enzymes.

Cellular responses to hypoxia (low oxygen) are governed by oxygen sensitive signaling pathways. Such pathways, in part, are controlled by enzymes with oxygen-dependent catalytic activity, of which the role of prolyl 4-hydroxylases has been widely reviewed. These enzymes inhibit hypoxic response by inducing the oxygen-dependent degradation of hypoxia-inducible factor 1α, the master regulator of the transcriptional hypoxic response. Jumonji C domain-containing lysine demethylases are similar enzymes which share the same oxygen-dependent catalytic mechanism as prolyl 4- hydroxylases. Traditionally, the role of lysine demethylases has been studied in relation to demethylation activity against histone substrates, however, within the past decade an increasing number of nonhistone protein targets have been revealed, some of which have a key role in survival in the hypoxic tumor microenvironment. Within this review, we highlight the involvement of methyllysine in the hypoxic response with a focus on the HIF signaling pathway, the regulation of demethylase activity by oxygen, and provide insights into notable areas of future hypoxic demethylase research.

PRMT7 methylates eukaryotic translation initiation factor 2α and regulates its role in stress granule formation.

Protein arginine methyltransferases (PRMTs) are a family of enzymes that modify proteins by methylating the guanidino nitrogen atoms of arginine residues to regulate cellular processes such as chromatin remodeling, pre-mRNA splicing, and signal transduction. PRMT7 is the single type III PRMT solely capable of arginine monomethylation. To date, other than histone proteins, there are very few identified substrates of PRMT7. We therefore performed quantitative mass spectrometry experiments to identify PRMT7's interactome and potential substrates to better characterize the enzyme's biological function(s) in cells. These experiments revealed that PRMT7 interacts with and can methylate eukaryotic translation initiation factor 2 alpha (eIF2α), in vitro and in breast cancer cells. Furthermore, we uncovered a potential regulatory interplay between eIF2α arginine methylation by PRMT7 and stress-induced phosphorylation status of eIF2α at serine 51. Finally, we demonstrated that PRMT7 is required for eIF2α-dependent stress granule formation in the face of various cellular stresses. Altogether, our findings implicate PRMT7 as a novel mediator of eIF2α-dependent cellular stress response pathways.

Association between miRNA-146a rs2910164 (G/C) polymorphism with the susceptibility to chronic HBV infection and spontaneous viral clearance in an Iranian population.

Hepatitis B virus (HBV) infection is one of the clinical dilemmas in chronic liver diseases. MicroRNAs (miRNAs) are small noncoding RNA molecules that play an important role in the pathogenesis of liver diseases and single nucleotide polymorphisms (SNPs) in miRNA genes affect the clinical course of HBV infection. Previous studies have shown that miRNA-146a rs2910164 polymorphism can be associated with the pathogenesis of liver diseases such as hepatocellular carcinoma. The present study investigated the association between miRNA-146a rs2910164 polymorphism and susceptibility to HBV infection in an Iranian population. The study comprised 266 patients with chronic HBV infection, 172 patients with spontaneous viral clearance (SVC) after acute HBV infection, and 266 healthy control adjusted for sex and age. The genotyping of the miRNA-146a rs2910164 polymorphism was performed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Our data revealed that GG genotype and G allele of miRNA-146a rs2910164 SNP is dominated (P < 0.001) in patients with chronic HBV infection (Odds ratio [OR] = 3.92; 95% confidence interval [CI] = 2.1-7.32). miRNA-146a rs2910164 polymorphism showed a statistically significant association (P < 0.001) between CC genotype and allele C with SVC (OR = 2.92; 95% CI = 1.56-546). Our findings suggest miRNA-146a SNP (C/G) in our population may be associated with the susceptibility to HBV infection and CC genotype is associated with SVC. Also, the GG genotype and G allele at miRNA-146a rs2910164 is associated with chronic HBV infection in our population.

HELB Is a Feedback Inhibitor of DNA End Resection.

DNA double-strand break repair by homologous recombination is initiated by the formation of 3' single-stranded DNA (ssDNA) overhangs by a process termed end resection. Although much focus has been given to the decision to initiate resection, little is known of the mechanisms that regulate the ongoing formation of ssDNA tails. Here we report that DNA helicase B (HELB) underpins a feedback inhibition mechanism that curtails resection. HELB is recruited to ssDNA by interacting with RPA and uses its 5'-3' ssDNA translocase activity to inhibit EXO1 and BLM-DNA2, the nucleases catalyzing resection. HELB acts independently of 53BP1 and is exported from the nucleus as cells approach S phase, concomitant with the upregulation of resection. Consistent with its role as a resection antagonist, loss of HELB results in PARP inhibitor resistance in BRCA1-deficient tumor cells. We conclude that mammalian DNA end resection triggers its own inhibition via the recruitment of HELB.

Artificial citrate operon confers mineral phosphate solubilization ability to diverse fluorescent pseudomonads.

Citric acid is a strong acid with good cation chelating ability and can be very efficient in solubilizing mineral phosphates. Only a few phosphate solubilizing bacteria and fungi are known to secrete citric acids. In this work, we incorporated artificial citrate operon containing NADH insensitive citrate synthase (gltA1) and citrate transporter (citC) genes into the genome of six-plant growth promoting P. fluorescens strains viz., PfO-1, Pf5, CHAO1, P109, ATCC13525 and Fp315 using MiniTn7 transposon gene delivery system. Comprehensive biochemical characterization of the genomic integrants and their comparison with plasmid transformants of the same operon in M9 minimal medium reveals the highest amount of ∼7.6±0.41 mM citric and 29.95±2.8 mM gluconic acid secretion along with ∼43.2±3.24 mM intracellular citrate without affecting the growth of these P. fluorescens strains. All genomic integrants showed enhanced citric and gluconic acid secretion on Tris-Cl rock phosphate (TRP) buffered medium, which was sufficient to release 200-1000 µM Pi in TRP medium. This study demonstrates that MPS ability could be achieved in natural fluorescent pseudomonads by incorporation of artificial citrate operon not only as plasmid but also by genomic integration.

Remodulation of central carbon metabolic pathway in response to arsenite exposure in Rhodococcus sp. strain NAU-1.

Arsenite-tolerant bacteria were isolated from an organic farm of Navsari Agricultural University (NAU), Gujarat, India (Latitude: 20°55'39.04″N; Longitude: 72°54'6.34″E). One of the isolates, NAU-1 (aerobic, Gram-positive, non-motile, coccobacilli), was hyper-tolerant to arsenite (As(III), 23 mM) and arsenate (As(V), 180 mM). 16S rRNA gene of NAU-1 was 99% similar to the 16S rRNA genes of Rhodococcus (Accession No. HQ659188). Assays confirmed the presence of membrane bound arsenite oxidase and cytoplasmic arsenate reductase in NAU-1. Genes for arsenite transporters (arsB and ACR3(1)) and arsenite oxidase gene (aoxB) were confirmed by PCR. Arsenite oxidation and arsenite efflux genes help the bacteria to tolerate arsenite. Specific activities of antioxidant enzymes (catalase, ascorbate peroxidase, superoxide dismutase and glutathione S-transferase) increased in dose-dependent manner with arsenite, whereas glutathione reductase activity decreased with increase in As(III) concentration. Metabolic studies revealed that Rhodococcus NAU-1 produces excess of gluconic and succinic acids, and also activities of glucose dehydrogenase, phosphoenol pyruvate carboxylase and isocitrate lyase were increased, to cope with the inhibited activities of glucose-6-phosphate dehydrogenase, pyruvate dehydrogenase and α-ketoglutarate dehydrogenase enzymes respectively, in the presence of As(III). Enzyme assays revealed the increase in direct oxidative and glyoxylate pathway in Rhodococcus NAU-1 in the presence of As(III).