PKD-dependent PARP12-catalyzed mono-ADP-ribosylation of Golgin-97 is required for E-cadherin transport from Golgi to plasma membrane

Adenosine diphosphate (ADP)-ribosylation is a posttranslational modification involved in key regulatory events catalyzed by ADP-ribosyltransferases (ARTs). Substrate identification and localization of the mono-ADP-ribosyltransferase PARP12 at the trans-Golgi network (TGN) hinted at the involvement of ARTs in intracellular traffic. We find that Golgin-97, a TGN protein required for the formation and transport of a specific class of basolateral cargoes (e.g., E-cadherin and vesicular stomatitis virus G protein [VSVG]), is a PARP12 substrate. PARP12 targets an acidic cluster in the Golgin-97 coiled-coil domain essential for function. Its mutation or PARP12 depletion, delays E-cadherin and VSVG export and leads to a defect in carrier fission, hence in transport, with consequent accumulation of cargoes in a trans-Golgi/Rab11–positive intermediate compartment. In contrast, PARP12 does not control the Golgin-245–dependent traffic of cargoes such as tumor necrosis factor alpha (TNFα). Thus, the transport of different basolateral proteins to the plasma membrane is differentially regulated by Golgin-97 mono-ADP-ribosylation by PARP12. This identifies a selective regulatory mechanism acting on the transport of Golgin-97– vs. Golgin-245–dependent cargoes. Of note, PARP12 enzymatic activity, and consequently Golgin-97 mono-ADP-ribosylation, depends on the activation of protein kinase D (PKD) at the TGN during traffic. PARP12 is directly phosphorylated by PKD, and this is essential to stimulate PARP12 catalytic activity. PARP12 is therefore a component of the PKD-driven regulatory cascade that selectively controls a major branch of the basolateral transport pathway. We propose that through this mechanism, PARP12 contributes to the maintenance of E-cadherin–mediated cell polarity and cell–cell junctions.

Small Endogenous Ligands Modulation of Nerve Growth Factor Bioactivity: A Structural Biology Overview

Experiments with cell cultures and animal models have provided solid support for the assumption that Nerve Growth Factor (NGF) plays a key role in the regulation of neuronal cell survival and death. Recently, endogenous ligands have been proposed as physiological modulators of NGF biological activity as part of this regulatory cascade. However, the structural and mechanistic determinants for NGF bioactivity remain to be elucidated. We recently unveiled, by an integrated structural biology approach, the ATP binding sites of NGF and investigated the effects on TrkA and p75NTR receptors binding. These results pinpoint ATP as a genuine endogenous modulator of NGF signaling, paving the way to the characterization of not-yet-identified chemical diverse endogenous biological active small molecules as novel modulators of NGF. The present review aims at providing an overview of the currently available 3D structures of NGF in complex with different small endogenous ligands, featuring the molecular footprints of the small molecules binding. This knowledge is essential for further understanding the functional role of small endogenous ligands in the modulation of neurotrophins signaling in physiological and pathological conditions and for better exploiting the therapeutic potentialities of NGF.

Identification of downstream effectors of retinoic acid specifying the zebrafish pancreas by integrative genomics

Retinoic acid (RA) is a key signal for the specification of the pancreas. Still, the gene regulatory cascade triggered by RA in the endoderm remains poorly characterized. In this study, we investigated this regulatory network in zebrafish by combining RNA-seq, RAR ChIP-seq and ATAC-seq assays. By analysing the effect of RA and of the RA receptor (RAR) inverse-agonist BMS493 on the transcriptome and on the chromatin accessibility of endodermal cells, we identified a large set of genes and regulatory regions regulated by RA signalling. RAR ChIP-seq further defined the direct RAR target genes in zebrafish, including hox genes as well as several pancreatic regulators like mnx1, insm1b, hnf1ba and gata6. Comparison of zebrafish and murine RAR ChIP-seq data highlighted the conserved direct target genes and revealed that some RAR sites are under strong evolutionary constraints. Among them, a novel highly conserved RAR-induced enhancer was identified downstream of the HoxB locus and driving expression in the nervous system and in the gut in a RA-dependent manner. Finally, ATAC-seq data unveiled the role of the RAR-direct targets Hnf1ba and Gata6 in opening chromatin at many regulatory loci upon RA treatment.

LuxT is a Global Regulator of Low-Cell Density Behaviors Including Type III Secretion, Siderophore Production, and Aerolysin Secretion in Vibrio harveyi

Quorum sensing (QS) is a chemical communication process in which bacteria produce, release, and detect extracellular signaling molecules called autoinducers. Via combined transcriptional and post-transcriptional regulatory mechanisms, QS allows bacteria to collectively alter gene expression on a population-wide scale. Recently, the LuxT transcription factor was shown to control V. harveyiqrr1, encoding the Qrr1 small RNA that functions at the core of the QS regulatory cascade. Here, we use RNA-Sequencing to reveal that, beyond control of qrr1, LuxT is a global regulator of 414 V. harveyi genes including those involved in type III secretion, siderophore production, and aerolysin toxin biosynthesis. Importantly, LuxT directly represses swrZ, encoding a transcription factor, and LuxT control of type III secretion, siderophore, and aerolysin genes occurs by two mechanisms, one that is SwrZ-dependent and one that is SwrZ-independent. All of these target genes specify QS-controlled behaviors that are enacted when V. harveyi is at low cell density. Thus, LuxT and SwrZ function in parallel with QS to drive particular low cell density behaviors. Phylogenetic analyses reveal that luxT is highly conserved among Vibrionaceae, but swrZ is less well conserved. In a test case to examine the relationship between LuxT and SwrZ, we find that in Aliivibrio fischeri, LuxT also functions as a swrZ repressor, and LuxT activates A. fischeri siderophore production via swrZ repression. Our results indicate that LuxT is a major regulator among Vibrionaceae, and, in the species that also possess swrZ, LuxT functions with SwrZ to control gene expression.

miR-31-NUMB Cascade Modulates Monocarboxylate Transporters to Increase Oncogenicity and Lactate Production of Oral Carcinoma Cells

Oral squamous cell carcinoma (OSCC) is among the leading causes of cancer-associated death worldwide. miR-31 is an oncogenic miRNA in OSCC. NUMB is an adaptor protein capable of suppressing malignant transformation. Disruption of the miR-31-NUMB regulatory axis has been demonstrated in malignancies. Mitochondrial dysfunction and adaptation to glycolytic respiration are frequent events in malignancies. Monocarboxylate transporters (MCTs) function to facilitate lactate flux in highly glycolytic cells. Upregulation of MCT1 and MCT4 has been shown to be a prognostic factor of OSCC. Here, we reported that miR-31-NUMB can modulate glycolysis in OSCC. Using the CRISPR/Cas9 gene editing strategy, we identified increases in oncogenic phenotypes, MCT1 and MCT4 expression, lactate production, and glycolytic respiration in NUMB-deleted OSCC subclones. Transfection of the Numb1 or Numb4 isoform reversed the oncogenic induction elicited by NUMB deletion. This study also showed, for the first time, that NUMB4 binds MCT1 and MCT4 and that this binding increases their ubiquitination, which may decrease their abundance in cell lysates. The disruptions in oncogenicity and metabolism associated with miR-31 deletion and NUMB deletion were partially rescued by MCT1/MCT4 expression or knockdown. This study demonstrated that NUMB is a novel binding partner of MCT1 and MCT4 and that the miR-31-NUMB-MCT1/MCT4 regulatory cascade is present in oral carcinoma.

Rational pharmacotherapy in modern gastroenterology

Introduction. Modern gastroenterology is characterized by the combined (comorbid) nature of the diseases. In treatment, this promotes polypharmacy and increases complications (drug lesions, allergic reactions, exacerbation of diseases of other organs and systems), and, importantly, increases the cost of pharmacotherapy.Aim. To compare two pharmacotherapy options for patients with gallstone disease at the stage of biliary sludge and patients with biliary sludge combined with irritable bowel syndrome.Materials and methods. In the work, based on the experience of treating 170 patients, two options for pharmacotherapy are considered, which may well turn out to be rational in all respects. Option 1 - monotherapy aimed at one of the components that form a complex pathogenetic symptom complex. The basis for offering this treatment option is the biological concept of the “regulatory cascade”. Option 2 – “stepwise” (stepwise) therapy with the choice of the “base” drug for the first step. Evaluation of the effectiveness and rational correction for the second step of treatment and subsequent ones – if necessary. Results. The biliary sludge was eliminated or reduced in patients who received the UDCA monotherapy against the background of recovery of gastrointestinal motility. The overall treatment effect (for each nosology) in patients with biliary sludge and irritable bowel syndrome using the complex therapy (UDCA and mebeverin) was 84 and 87.8% respectively.Conclusions. Both options are rational today: 1st requires further study; 2nd – active use. Both options exclude polypharmacy and other adverse effects.

A histidine kinase and a response regulator provide phage resistance to Marinomonas mediterranea via CRISPR-Cas regulation

CRISPR-Cas systems are used by many prokaryotes to defend against invading genetic elements. In many cases, more than one CRISPR-Cas system co-exist in the same cell. Marinomonas mediterranea MMB-1 possesses two CRISPR-Cas systems, of type I–F and III-B respectively, which collaborate in phage resistance raising questions on how their expression is regulated. This study shows that the expression of both systems is controlled by the histidine kinase PpoS and a response regulator, PpoR, identified and cloned in this study. These proteins show similarity to the global regulators BarA/UvrY. In addition, homologues to the sRNAs CsrB and CsrC and the gene coding for the post-transcriptional repressor CsrA have been also identified indicating the conservation of the elements of the BarA/UvrY regulatory cascade in M. mediterranea. RNA-Seq analyses have revealed that all these genetics elements are regulated by PpoS/R supporting their participation in the regulatory cascade. The regulation by PpoS and PpoR of the CRISPR-Cas systems plays a role in phage defense since mutants in these proteins show an increase in phage sensitivity.

Circ_0045714/miR-331-3p interaction affects IL-1β-evoked human articular chondrocyte injury through regulating PIK3R3 in a ceRNA regulatory cascade

Background Osteoarthritis (OA) is characterized by joint pain and joint function limitation. Hsa_circ_0045714 (circ_0045714) is a novel OA-related circular RNA. However, its repertoire remains to be further clarified in joint chondrocytes. Methods RNA and protein expression levels and inflammatory factor levels were detected by real-time quantitative polymerase chain reaction, western blotting and enzyme-linked immunosorbent assay. Cell proliferation and apoptosis were determined by colony formation assay, cell counting kit-8 assay and apoptosis assay. Direct interaction was predicted by bioinformatics method and confirmed by dual-luciferase reporter assay. Results Expression of circ_0045714 and phosphoinositide-3-kinase (PI3K) regulatory subunit 3 (PIK3R3) was declined, and microRNA (miR)-331-3p was promoted in knee articular cartilages and cells from OA patients, as well as interleukin (IL)-1β-challenged human articular chondrocytes (HAC) cell line. In stimulation of IL-1β, HAC cells showed a loss of colony formation ability, cell viability and expression of Bcl-2 and Collagen II, allied with an increase in apoptosis rate and levels of IL-6, IL-8 and tumor necrosis factor-α, Bcl-2-associated X protein, cleaved caspase-3, and ADAM with thrombospondin motif-5. Noticeably, overexpressing circ_0045714 and inhibiting miR-331-3p could suppress IL-1β-evoked these effects, and both were through up-regulating PIK3R3, a key gene in PI3K/AKT signaling pathway. Mechanically, circ_0045714 functioned as competing endogenous RNA (ceRNA) for miR-331-3p and further regulated expression of the downstream target gene PIK3R3. Conclusion There was a novel circ_0045714/miR-331-3p/PIK3R3 ceRNA axis in HAC, and its inhibition might be one mechanism of HAC injury in OA.