Structural basis of Nrd1–Nab3 heterodimerization

Heterodimerization of RNA binding proteins Nrd1 and Nab3 is essential to communicate the RNA recognition in the nascent transcript with the Nrd1 recognition of the Ser5-phosphorylated Rbp1 C-terminal domain in RNA polymerase II. The structure of a Nrd1–Nab3 chimera reveals the basis of heterodimerization, filling a missing gap in knowledge of this system. The free form of the Nrd1 interaction domain of Nab3 (NRID) forms a multi-state three-helix bundle that is clamped in a single conformation upon complex formation with the Nab3 interaction domain of Nrd1 (NAID). The latter domain forms two long helices that wrap around NRID, resulting in an extensive protein–protein interface that would explain the highly favorable free energy of heterodimerization. Mutagenesis of some conserved hydrophobic residues involved in the heterodimerization leads to temperature-sensitive phenotypes, revealing the importance of this interaction in yeast cell fitness. The Nrd1–Nab3 structure resembles the previously reported Rna14/Rna15 heterodimer structure, which is part of the poly(A)-dependent termination pathway, suggesting that both machineries use similar structural solutions despite they share little sequence homology and are potentially evolutionary divergent.

A Comprehensive Review of Receptor-Type Tyrosine-Protein Phosphatase Gamma (PTPRG) Role in Health and Non-Neoplastic Disease

Protein tyrosine phosphatase receptor gamma (PTPRG) is known to interact with and regulate several tyrosine kinases, exerting a tumor suppressor role in several type of cancers. Its wide expression in human tissues compared to the other component of group 5 of receptor phosphatases, PTPRZ expressed as a chondroitin sulfate proteoglycan in the central nervous system, has raised interest in its role as a possible regulatory switch of cell signaling processes. Indeed, a carbonic anhydrase-like domain (CAH) and a fibronectin type III domain are present in the N-terminal portion and were found to be associated with its role as [HCO3−] sensor in vascular and renal tissues and a possible interaction domain for cell adhesion, respectively. Studies on PTPRG ligands revealed the contactins family (CNTN) as possible interactors. Furthermore, the correlation of PTPRG phosphatase with inflammatory processes in different normal tissues, including cancer, and the increasing amount of its soluble form (sPTPRG) in plasma, suggest a possible role as inflammatory marker. PTPRG has important roles in human diseases; for example, neuropsychiatric and behavioral disorders and various types of cancer such as colon, ovary, lung, breast, central nervous system, and inflammatory disorders. In this review, we sum up our knowledge regarding the latest discoveries in order to appreciate PTPRG function in the various tissues and diseases, along with an interactome map of its relationship with a group of validated molecular interactors.

Primary Hepatoid Adenocarcinoma of Gallbladder With MB21D2/GALNT12/ARID2 Mutations: A Case Report

BackgroundPrimary hepatoid adenocarcinoma of the gallbladder is a relatively rare type of extrahepatic adenocarcinoma. The genetic changes involved in this type of adenocarcinoma were unexplained so far. We reported a rare case of primary hepatoid adenocarcinoma of gallbladder with Mab-21 domain containing 2 (MB21D2), polypeptide N-acetylgalactosaminyltransferase 12 (GALNT12), and AT-rich interaction domain 2 (ARID2) mutations, which was confirmed after surgical resection pathologically.Case SummaryA 69-year-old female with distention of hypogastrium and constipation received enema treatment, but ineffectively. No abnormalities were found on relevant physical examination. Then, the CT and MRI demonstrated a 3.3–4-cm soft tissue mass shadow in the neck of the gallbladder. The primary lesions consisted of two components: high-grade intraepithelial neoplasia of glands and hepatoid glands microscopically after laparoscope cholecystectomy. Immunohistochemical staining showed the sameness and difference of the two areas. Furthermore, tumor mutational burden (TMB) shows that the MB21D2, GALNT12, and ARID2 genes were mutated.ConclusionThis is the first report of primary hepatoid adenocarcinoma of the gallbladder with MB21D2, GALNT12, and ARID2 mutations. This will provide a theoretical basis for genetic changes in rare tumors.

Loneliness Level and Its Associated Factors in Patients With Hemodialysis

Verify the level of loneliness and its relationship with socioeconomic and health conditions, social support, family functionality, and depressive symptoms in patients undergoing hemodialysis. Analysis of secondary data, with a primary cross-sectional investigation, correlational with a quantitative approach, carried out in 2019, with 80 patients with CKD on hemodialysis. Applied instruments: socio-demographic, economic, and health condition characterization, UCLA Loneliness Scale, Medical Outcomes Study Social Support Scale, Patient Health Questionnaire9, and the Family APGAR. Showed female prevalence (55%), white ethnicity (65%), and mean age 59.63 years. In the perception of loneliness, the highest index was (55%), moderately high. Social support scored worst in the Positive Social Interaction domain, mean 74.31. In depression, showed prevalence of severe depression (28.7%). Family functioning showed good score (68.8%). There was a weak negative correlation between loneliness and all domains of Social Support, besides Family Functioning.

Decoupling SARS-CoV-2 ORF6 localization and interferon antagonism

ABSTRACTLike many pathogenic viruses, SARS-CoV-2 must overcome interferon (IFN)-mediated host defenses for infection establishment. To achieve this, SARS-CoV-2 deploys overlapping mechanisms to antagonize IFN production and signaling. The strongest IFN antagonist is the accessory protein ORF6, which localizes to multiple membranous compartments, including the nuclear envelope, where it directly binds the nuclear pore components Nup98-Rae1 to inhibit nuclear translocation of activated STAT1/IRF3 transcription factors. However, a direct cause-and-effect relationship between ORF6 localization and IFN antagonism has yet to be explored experimentally. Here, we use extensive mutagenesis studies to define the structural determinants required for steady-state localization and demonstrate that mis-localized ORF6 variants can still potently inhibit nuclear trafficking and IFN signaling. Additionally, expression of a peptide that mimics the ORF6/Nup98 interaction domain robustly inhibited nuclear trafficking. Furthermore, pharmacologic and mutational approaches combined to suggest that ORF6 is likely a peripheral-membrane protein, opposed to being a transmembrane protein as previously speculated. Thus, ORF6 localization and IFN antagonism are independent activities, which raises the possibility that ORF6 may have additional functions within membrane networks to enhance virus replication.

A distributed residue network permits conformational binding specificity in a conserved family of actin remodelers

Metazoan proteomes contain many paralogous proteins that have evolved distinct functions. The Ena/VASP family of actin regulators consists of three members that share an EVH1 interaction domain with a 100 % conserved binding site. A proteome-wide screen revealed photoreceptor cilium actin regulator (PCARE) as a high-affinity ligand for ENAH EVH1. Here, we report the surprising observation that PCARE is ~100-fold specific for ENAH over paralogs VASP and EVL and can selectively bind ENAH and inhibit ENAH-dependent adhesion in cells. Specificity arises from a mechanism whereby PCARE stabilizes a conformation of the ENAH EVH1 domain that is inaccessible to family members VASP and EVL. Structure-based modeling rapidly identified seven residues distributed throughout EVL that are sufficient to differentiate binding by ENAH vs. EVL. By exploiting the ENAH-specific conformation, we rationally designed the tightest and most selective ENAH binder to date. Our work uncovers a conformational mechanism of interaction specificity that distinguishes highly similar paralogs and establishes tools for dissecting specific Ena/VASP functions in processes including cancer cell invasion.

Phosphorylation-dependent BRD4 dimerization and implications for therapeutic inhibition of BET family proteins

Bromodomain-containing protein 4 (BRD4) is an epigenetic reader and oncology drug target that regulates gene transcription through binding to acetylated chromatin via bromodomains. Phosphorylation by casein kinase II (CK2) regulates BRD4 function, is necessary for active transcription and is involved in resistance to BRD4 drug inhibition in triple-negative breast cancer. Here, we provide the first biophysical analysis of BRD4 phospho-regulation. Using integrative structural biology, we show that phosphorylation by CK2 modulates the dimerization of human BRD4. We identify two conserved regions, a coiled-coil motif and the Basic-residue enriched Interaction Domain (BID), essential for the BRD4 structural rearrangement, which we term the phosphorylation-dependent dimerization domain (PDD). Finally, we demonstrate that bivalent inhibitors induce a conformational change within BRD4 dimers in vitro and in cancer cells. Our results enable the proposal of a model for BRD4 activation critical for the characterization of its protein-protein interaction network and for the development of more specific therapeutics.

Combinatorial Analysis of AT-Rich Interaction Domain 1A and CD47 in Gastric Cancer Patients Reveals Markers of Prognosis

Background: The AT-rich interaction domain 1A (ARID1A) is thought to be a tumor suppressive gene, and most of its mutations result in loss of expression of ARID1A protein. Combined with SIRPα on the surface of macrophages, CD47 on the surface of cancer cells can send an antiphagocytic “Don’t eat me” signal to the immune system that helps to avoid immune surveillance. However, the relationship between ARID1A and CD47 expression and their prognostic value in gastric cancer (GC) are still unknown.Methods: In this study, we evaluated ARID1A and CD47 expression in 154 GC patients’ tissues using tissue microarray. Expressions of ARID1A and CD47 in GC cell lines were determined by western blot and quantitative reverse transcriptase–polymerase chain reaction (qRT-PCR) techniques, and cell membranous CD47 expression was quantified by flow cytometry. In addition, chromatin immunoprecipitation (ChIP)–qPCR was used to determine the aspects of regulation of CD47 by ARID1A. The proportions of tumor-infiltrating immune cells were estimated on The Cancer Genome Atlas (TCGA) data set by using quanTIseq and EPIC algorithms. The infiltration of M1-polarized macrophages, M2-polarized macrophages, and regulatory T cells (Tregs) in GC tissues was determined by multispectral immunofluorescence.Results: A significant correlation was found between loss of ARID1A and high expression of CD47 at protein level in GC. By integrating 375 bulk RNA sequencing samples from TCGA data set, we found that mutated ARID1A correlated with high CD47 expression. In GC cell lines, knockdown of ARID1A significantly increased CD47 expression both at protein and mRNA levels as measured by western blot, qRT-PCR, and flow cytometry. Moreover, ChIP-qPCR revealed that CD47 was a direct downstream target gene of ARID1A in GC. Utilizing univariate and multivariate survival analyses, we found that patients with ARID1AlossCD47high expression had a worse prognosis. Estimation of infiltrating immune cells on TCGA data set showed that a higher infiltration proportion of M2 macrophages and Tregs was found in ARID1AmutatedCD47high expression subgroup. Furthermore, application of multispectral immunofluorescence revealed a higher infiltration proportion of M2 macrophages and Tregs in ARID1AlossCD47high GC tissues.Conclusion: Loss of ARID1A is strongly correlated with high CD47 expression in GC, and combination of ARID1A and CD47 is a promising prognosis factor in GC.

An Overview of the Primary Cilium and RPGRIP1L: The Signalling Hub’s Anchor for Organ Development and Homeostasis

Research on the primary cilium has been growing exponentially in the past several decades due to its functions as a cell signalling hub, which defects leads to several disorders and abnormalities collectively known as ciliopathies. Among other parts of the primary cilium structures, the transition zone is the area whose defects lead to the most severe clinical manifestations and high lethality. The ciliary transition zone consists of multiple protein modules that are hypothesized to be anchored by the RPGRIP1L protein. Despite its importance, RPGRIP1L studies remain hidden from the limelight, and our understanding of the protein remains scattered. This review summarizes the clinical manifestations and molecular mechanisms of the RPGRIP1L in the primary cilium. We then take a closer look at each RPGRIP1L’s protein domain to understand how each domain ensures proper functions and localization of RPGRIP1L. The three domains of RPGRIP1L are postulated to be involved in different roles. While the coiled coil domain is vital for scaffolding the protein to the centriolar structure, the ability of the C2 domain to interact with lipid allows the formation of ‘lipid gate’ at the transition zone. The high variability of the RPGR interaction domain enable the RPGRIP1L to interact with multiple different proteins, making it an ideal anchor for other ciliary protein modules in the transition zone.