The Homeostasis of p62/SQSTM1 Short Isoform Regulates Selective Autophagy

Autophagy, a highly conserved lysosomal degradation pathway, has been shown to play a pivotal role in many physiological and pathological processes. Sequestosome 1 (SQSTM1/p62) which serves as autophagy receptor is a multifunctional protein involved in signal transduction, protein degradation and cell transformation. Human SQSTM1 has two isoforms, p62L and p62S, which are derived from alternative splicing at the 5’donor sites. However, few studies focus on p62S, and its function needs to be further explored. Here we found that p62S, but not p62L is mainly degrades by ubiquitin-proteasome signaling pathway. E3 ligase TRIM72 was identified as an interacting partner for p62S, and promotes the ubiquitination and degradation of p62S. Furthermore, we demonstrate that p62S competes with the autophagy receptor p62L which binds to ubiquitinated autophagy cargoes, thus playing a dominant negative role in autophagy regulation, while this inhibitory effect could be attenuated by TRIM72-dependent ubiquitination of p62S. Delineation of the mechanism and regulatory roles of p62S sheds a new light on the proposed pathological implications of p62 in cell physiology. TRIM72 and p62S are promising therapeutic targets for autophagy-related diseases.

Correlation Between SMADs and Colorectal Cancer Expression, Prognosis, and Immune Infiltrates

Background: In current years, the incidence and mortality of colorectal cancer (CRC) are increasing, and the 5-year survival rate of advanced metastatic CRC is poor. The Small mothers against decapentaplegic (SMAD) superfamily is an intracellular signal transduction protein associated with the development and prognosis of a variety of tumors. At present, no study has systematically analyzed the relationship between SMADs and CRC.Methods: Here, R3.6.3 was used to analyze the expression of SMADs in pan-cancer and CRC. Protein expression of SMADs were analyzed by HPA. GEPIA was used to evaluate the correlation between SMADs and tumor stage in CRC. The effect of R language and GEPIA on prognosis was analyzed. Mutation rates of SMADs in CRC were determined by c-BioPortal and potentially related genes were predicted using GeneMANIA. R analysis was used for correlation with immune cell infiltration in CRC.Results: Both SMAD1 and SMAD2 were found to be weak expression in CRC and correlated with immune invasion level. SMAD1 was correlated with patient prognosis, and SMAD2 was correlated with tumor stage. SMAD3, SMAD4 and SMAD7 were all low expressed in CRC and associated with a variety of immune cells. SMAD3 and SMAD4 proteins were also low expressed, and SMAD4 had the highest mutation rate. SMAD5 and SMAD6 were overexpressed in CRC, and SMAD6 was also associated with patient OS and CD8+ T cells, macrophages and neutrophils. Conclusions: Our results reveal innovative and strong evidence that SMADs can be used as biomarkers for the treatment and prognosis of CRC.

Increasing the Ascomycin Yield by Relieving the Inhibition of Acetyl/Propionyl-CoA Carboxylase by the Signal Transduction Protein GlnB

Ascomycin (FK520) is a multifunctional antibiotic produced by Streptomyces hygroscopicus var. ascomyceticus. In this study, we demonstrated that the inactivation of GlnB, a signal transduction protein belonging to the PII family, can increase the production of ascomycin by strengthening the supply of the precursors malonyl-CoA and methylmalonyl-CoA, which are produced by acetyl-CoA carboxylase and propionyl-CoA carboxylase, respectively. Bioinformatics analysis showed that Streptomyces hygroscopicus var. ascomyceticus contains two PII family signal transduction proteins, GlnB and GlnK. Protein co-precipitation experiments demonstrated that GlnB protein could bind to the α subunit of acetyl-CoA carboxylase, and this binding could be disassociated by a sufficient concentration of 2-oxoglutarate. Coupled enzyme activity assays further revealed that the interaction between GlnB protein and the α subunit inhibited both the activity of acetyl-CoA carboxylase and propionyl-CoA carboxylase, and this inhibition could be relieved by 2-oxoglutarate in a concentration-dependent manner. Because GlnK protein can act redundantly to maintain metabolic homeostasis under the control of the global nitrogen regulator GlnR, the deletion of GlnB protein enhanced the supply of malonyl-CoA and methylmalonyl-CoA by restoring the activity of acetyl-CoA carboxylase and propionyl-CoA carboxylase, thereby improving the production of ascomycin to 390 ± 10 mg/L. On this basis, the co-overexpression of the β and ε subunits of propionyl-CoA carboxylase further increased the ascomycin yield to 550 ± 20 mg/L, which was 1.9-fold higher than that of the parent strain FS35 (287 ± 9 mg/L). Taken together, this study provides a novel strategy to increase the production of ascomycin, providing a reference for improving the yield of other antibiotics.

bFGF Activates Integrin β1 and Subsequently Upregulates Collagens via the p-MEK1/2/p-ERK1/2 Signaling Pathway in L929 Cells

The substantial loss of collagen in the supporting tissues of the pelvic floor is characteristic of pelvic organ prolapse (POP). A bFGF-induced collagen increase has been widely recognized by scholars, but the role of bFGF in pelvic floor dysfunction and the mechanism by which bFGF promotes collagen have not been reported. Here, we elucidated this mechanism. After bFGF stimulation, L929 cells showed significantly increased expression of collagen, integrin β1, and MEK1/2 signaling proteins. Our previous studies showed that integrin β1 plays an important role in electric stimulation-induced collagen expression, which suggests an underlying mechanism. By overexpressing and silencing integrin β1, we proved that integrin β1 is also an important signal transduction protein of bFGF that promotes collagen through the MEK1/2 pathway, which is a classic collagen-promoting pathway. In summary, these findings suggest that bFGF can stimulate the expression of collagen through the integrin β1/p-MEK1/2/p-ERK/1/2 signaling pathway in L929 cells. Keyword Pelvic organ prolapse; bFGF; integrinβ1; ERK1/2; Collagen Special project for Chinese women's pelvic floor dysfunction prevention and treatment (no. 201817092) and the Natural Science Foundation of Hubei Province（no.2019CFB149）.

RGS4 negatively modulates Nociceptin/Orphanin FQ opioid receptor signaling: implication for L-Dopa induced dyskinesia

Background and purpose: Regulator of G-protein signal 4 (RGS4) is a signal transduction protein that accelerates intrinsic GTPase activity of Gαi/o and Gαq subunits, suppressing GPCR signaling. Here we investigate whether RGS4 modulates nociceptin/orphanin FQ opioid (NOP) receptor signaling and whether this modulation has relevance for L-Dopa induced dyskinesia. Experimental approach: HEK293T cells transfected with NOP, NOP/RGS4 or NOP/RGS19 were challenged with N/OFQ and the small molecule NOP agonist AT-403, using D1-stimulated cAMP levels as a readout. Primary rat striatal neurons and adult mouse striatal slices were challenged with N/OFQ or AT-403 in the presence of the RGS4 inhibitor, CCG-203920, and D1-stimulated cAMP or pERK responses were monitored. In vivo, CCG-203920 was co-administered with AT-403 and levodopa to 6-hydroxydopamine hemilesioned rats, and dyskinetic movements, striatal biochemical correlates of dyskinesia (pERK and pGluR1 levels) and striatal RGS4 levels were measured. Key results: RGS4 expression reduced NOFQ and AT-403 potency and efficacy in HEK293T cells. CCG-203920 increased N/OFQ potency in primary rat striatal neurons, and potentiated AT-403 response in mouse striatal slices. CCG-203920 enhanced AT-403 mediated inhibition of dyskinesia and its biochemical correlates, without compromising its motor-improving effects. Unilateral dopamine depletion caused bilateral reduction of RGS4 levels which was reversed by levodopa. Levodopa acutely upregulated RGS4 in the lesioned striatum. Conclusions and Implications: RGS4 physiologically inhibits NOP receptor signaling and an RGS4 inhibitor enhances NOP responses. Furthermore, an RGS4 inhibitor improved the antidyskinetic potential of NOP receptor agonists, mitigating the effects of upregulation of striatal RGS4 levels occurring during dyskinesia expression.

Comparative transcriptome analysis of leaves during early stages of chilling stress in two different chilling-tolerant brown-fiber cotton cultivars

Chilling stress generates significant inhibition of normal growth and development of cotton plants and lead to severe reduction of fiber quality and yield. Currently, little is known for the molecular mechanism of brown-fiber cotton (BFC) to respond to chilling stress. Herein, RNA-sequencing (RNA-seq)-based comparative analysis of leaves under 4°C treatment in two different-tolerant BFC cultivars, chilling-sensitive (CS) XC20 and chilling-tolerant (CT) Z1612, was performed to investigate the response mechanism. A total of 72650 unigenes were identified with eight commonly used databases. Venn diagram analysis identified 1194 differentially expressed genes (DEGs) with significant up-regulation in all comparison groups. Furthermore, enrichment analyses of COG and KEGG, as well as qRT-PCR validation, indicated that 279 genes were discovered as up-regulated DEGs (UDEGs) with constant significant increased expression in CT cultivar Z1612 groups at the dimensions of both each comparison group and treatment time, locating in the enriched pathways of signal transduction, protein and carbohydrate metabolism, and cell component. Moreover, the comprehensive analyses of gene expression, physiological index and intracellular metabolite detections, and ascorbate antioxidative metabolism measurement validated the functional contributions of these identified candidate genes and pathways to chilling stress. Together, this study for the first time report the candidate key genes and metabolic pathways responding to chilling stress in BFC, and provide the effective reference for understanding the regulatory mechanism of low temperature adaptation in cotton.

Analysis of Oxoglaucine in the Treatment of Breast Cancer Based on Network Pharmacology and Bioinformatics

To explore the potential molecular mechanism of Oxoglaucine(OG) in the treatment of Breast Cancer(BC) based on network pharmacology and bioinformatics. TCMSP and SwissTargetPrediction databases search for OG Related targets, and GeneCards database finds all BC-related targets. Take the intersection of OG and BC as all potential targets that inhibit BC. All potential targets are topologically analyzed by Cytoscape 3.7.1 software, and finally the core target is obtained. The start analysisi function in the DAVID database performs bioinformatics analysis on all core targets, and further visualizes them with the help of R language tools. As a result, 104 potential targets were obtained, of which SRC, PIK3CA, EGFR, MTOR, ESR1, MAPK1, PTGS2, AR, and NOS3 were the main core targets. OG inhibits the occurrence of BC through Pathways in cancer, PI3K-Akt signaling pathway, Proteoglycans in cancer, ErbB signaling pathway, HIF-1 signaling pathway related pathways, mainly involving signal transduction, protein phosphorylation, negative regulation of apoptotic process, positive regulation of transcription from RNA polymerase II promoter, phosphatidylinositol-mediated signaling biological processes. This study initially reveals the molecular mechanism of OG inhibiting BC, which provides a reference for further research.

Loss of endocytosis-associated RabGEF1 causes aberrant morphogenesis and altered autophagy in photoreceptors leading to retinal degeneration

Rab-GTPases and associated effectors mediate cargo transport through the endomembrane system of eukaryotic cells, regulating key processes such as membrane turnover, signal transduction, protein recycling and degradation. Using developmental transcriptome data, we identified Rabgef1 (encoding the protein RabGEF1 or Rabex-5) as the only gene associated with Rab GTPases that exhibited strong concordance with retinal photoreceptor differentiation. Loss of Rabgef1 in mice (Rabgef1-/-) resulted in defects specifically of photoreceptor morphology and almost complete loss of both rod and cone function as early as eye opening; however, aberrant outer segment formation could only partly account for visual function deficits. RabGEF1 protein in retinal photoreceptors interacts with Rabaptin-5, and RabGEF1 absence leads to reduction of early endosomes consistent with studies in other mammalian cells and tissues. Electron microscopy analyses reveal abnormal accumulation of macromolecular aggregates in autophagosome-like vacuoles and enhanced immunostaining for LC3A/B and p62 in Rabgef1-/- photoreceptors, consistent with compromised autophagy. Transcriptome analysis of the developing Rabgef1-/- retina reveals altered expression of 2469 genes related to multiple pathways including phototransduction, mitochondria, oxidative stress and endocytosis, suggesting an early trajectory of photoreceptor cell death. Our results implicate an essential role of the RabGEF1-modulated endocytic and autophagic pathways in photoreceptor differentiation and homeostasis. We propose that RabGEF1 and associated components are potential candidates for syndromic traits that include a retinopathy phenotype.

Optogenetic control of infection signaling cascade of bacteria by an engineered light-responsive protein

ABSTRACTBacterial pathogens operate by tightly controlling the virulence to facilitate invasion and survival in host. Although pathways regulating virulence have been defined in detail and signals modulating these processes are gradually understood, a lack of controlling infection signaling cascades of pathogens when and whereabouts specificity limits deeper investigating of host-pathogen interactions. Here, we employed optogenetics to reengineer the GacS of Pseudomonas aeruginosa, sensor kinase of GacS/GacA TCS regulates the expression of virulence factors by directly mediating several sRNAs. The resultant protein YGS24 displayed significant light-dependent activity of GacS kinases in Pseudomonas aeruginosa. When introduced in Caenorhabditis elegans host systems, YGS24 stimulated the pathogenicity of PAO1 in BHI and of PA14 in SK medium progressively upon blue-light exposure. This optogenetic system provides an accessible way to spatiotemporally control bacterial pathogenicity in defined host even specific tissues to develop new pathogenesis systems, which may in turn expedite development of innovative therapeutics.IMPORTANCEGacS is a signal transduction protein of the global Gac/Rsm regulatory cascade that is of central importance for the regulation of infection and virulence factors in Pseudomonas aeruginosa. Here, we reprogrammed the input signal specificity of GacS by replacing its input sensor domain with a photosensor domain. The resultant fusion protein YGS24 has the ability of perceiving light signal and, in response, regulates the Gac/Rsm signaling cascade. When tested in host models, this optogenetic system enables the light-dependent pathogenicity switch of bacterial cells and correspondingly tunes the susceptibility of Caenorhabditis elegans to P. aeruginosa-mediated killing. We provide a useful optogenetic tool in the area of pathogenic research that has great demands for precise spatiotemporal control of bacterial pathogenicity.