Regulation Of Signal Transduction Research Articles

Sphingosine-1-phosphate controls endothelial sphingolipid homeostasis via ORMDL.

Disruption of sphingolipid homeostasis and signaling has been implicated in diabetes, cancer, cardiometabolic, and neurodegenerative disorders. Yet, mechanisms governing cellular sensing and regulation of sphingolipid homeostasis remain largely unknown. In yeast, serine palmitoyltransferase, catalyzing the first and rate-limiting step of sphingolipid de novo biosynthesis, is negatively regulated by Orm1 and 2. Lowering sphingolipids triggers Orms phosphorylation, upregulation of serine palmitoyltransferase activity and sphingolipid de novo biosynthesis. However, mammalian orthologs ORMDLs lack the N-terminus hosting the phosphosites. Thus, which sphingolipid(s) are sensed by the cells, and mechanisms of homeostasis remain largely unknown. Here, we identify sphingosine-1-phosphate (S1P) as key sphingolipid sensed by cells via S1PRs to maintain homeostasis. The increase in S1P-S1PR signaling stabilizes ORMDLs, restraining SPT activity. Mechanistically, the hydroxylation of ORMDLs at Pro137 allows a constitutive degradation of ORMDLs via ubiquitin-proteasome pathway, preserving SPT activity. Disrupting S1PR/ORMDL axis results in ceramide accrual, mitochondrial dysfunction, impaired signal transduction, all underlying endothelial dysfunction, early event in the onset of cardio- and cerebrovascular diseases. Our discovery may provide the molecular basis for therapeutic intervention restoring sphingolipid homeostasis.

CD6-mediated inhibition of T cell activation via modulation of Ras

BackgroundCD6 is one of many cell surface receptors known to regulate signal transduction upon T cell activation. However, whether CD6 mediates costimulatory or inhibitory signals is controversial. When T cells engage with antigen presenting cells (APCs), CD6 interacts with its ligand CD166 at the cell–cell interface while the cytosolic tail assembles a complex signalosome composed of adaptors and effector enzymes, that may either trigger activating signaling cascades, or instead modulate the intensity of signaling. Except for a few cytosolic adaptors that connect different components of the CD6 signalosome, very little is known about the mechanistic effects of the cytosolic effectors that bind CD6.MethodsJurkat model T cells were transfected to express wild-type (WT) CD6, or a cytoplasmic truncation, signaling-disabled mutant, CD6Δcyt. The two resulting cell lines were directly activated by superantigen (sAg)-loaded Raji cells, used as APCs, to assess the net signaling function of CD6. The Jurkat cell lines were further adapted to express a FRET-based unimolecular HRas biosensor that reported the activity of this crucial GTPase at the immunological synapse.ResultsWe show that deletion of the cytosolic tail of CD6 enhances T-cell responses, indicating that CD6 restrains T-cell activation. One component of the CD6-associated inhibitory apparatus was found to be the GTPase activating protein of Ras (RasGAP), that we show to associate with CD6 in a phosphorylation-dependent manner. The FRET HRas biosensor that we developed was demonstrated to be functional and reporting the activation of the T cell lines. This allowed to determine that the presence of the cytosolic tail of CD6 results in the down-regulation of HRas activity at the immunological synapse, implicating this fundamental GTPase as one of the targets inhibited by CD6.ConclusionsThis study provides the first description of a mechanistic sequence of events underlying the CD6-mediated inhibition of T-cell activation, involving the modulation of the MAPK pathway at several steps, starting with the coupling of RasGAP to the CD6 signalosome, the repression of the activity of Ras, and culminating in the reduction of ERK1/2 phosphorylation and of the expression of the T-cell activation markers CD69 and IL-2R α chain.9f2SuWv7Lo8KDpPx-hd1D5Video abstract

Network pharmacology and in vivo experiment-based strategy to investigate mechanisms of JingFangFuZiLiZhong formula for ulcerative colitis

Background Ulcerative colitis (UC), a chronic inflammatory disease, often cause carcinogenesis, disability, and intestinal perforation. The JingFangFuZiLiZhong formula (JFFZLZ) shows a good effect against UC in the clinic. Hence, we aim to investigate the mechanisms between JFFZLZ and UC via network pharmacology data mining and in vivo experiments. Methods We obtained active constituents and related targets from public databases. The overlapped genes between JFFZLZ and UC targets were further analysed by enrichment analysis. The active constituents and hub targets were used to construct molecule docking analysis. We finally screened out nine hub targets and their expressions were verified in the Gene Expression Omnibus database and UC rats’ colon tissues after JFFZLZ treatment. Results The results implied that JFFZLZ mainly regulated signal transduction, metabolites production, and inflammation pathways. The expression of STAT3, CXCL8, IL6, CXCL12, TNF, TP53, and PTPN11 were both upregulated in colon tissues of UC patients and UC rats. While RELA, EGFR, and TP53 were downregulated in UC patients, but upregulated in UC rats. Furthermore, JFFZLZ could repair UC rats’ colon mucosal damage and promote the healing of ulcers via regulating the hub targets. Conclusion These results elucidated that the anti-UC effect of JFFZLZ was closely related to the inhibition of inflammatory response, inhibition of oxidative stress, and repairing colon mucosal damage through different signal pathways. The findings could contribute to a better understanding of the regulation mechanisms in JFFZLZ against UC. Key messages JFFZLZ could reduce the inflammatory infiltration and repair UC rats’ colon mucosal damage. Through the network pharmacology-based strategy and public database mining, we obtained the hub targets and key pathways between JFFZLF and UC. The mechanism of JFFZLZ against UC was inhibition of inflammatory response and oxidative stress by regulating the expression of the hub targets.

Aging-Related Gene Expression Signatures Define Likelihood of Disease Progression in Patients with Lower-Risk Myelodysplastic Syndromes

Myelodysplastic syndromes (MDS) are a heterogeneous clonal bone marrow disorders occurring mostly in the elderly. The Revised International Prognostic System (IPSS-R) groups MDS patients according to the risk of transformation into acute myeloid leukemia. For treatment decision-making, patients are usually divided into two groups: lower- and higher-risk with the cutpoint of 3.5 according to the IPSS-R. However, part of lower-risk MDS patients (LR-MDS) progresses rapidly. Recently, we demonstrated that DNA damage response (DDR) activation and the ensuing senescence form an anti-tumor barrier in CD34+ cells of LR-MDS patients, and that this barrier can be breached by RUNX1 mutations (Kaisrlikova M, et al., Leukemia. 2022; 36:1898). As an extension, we analyzed transcriptomes of LR-MDS patients regardless of their driver mutation, with and without rapid progression (within 2 years of diagnosis), to determine molecular mechanisms underlying the progression. Because MDS is associated with aging, and many anti-tumor barrier mechanisms (DDR, checkpoint activation, senescence) lead to a pro-aging phenotype, we questioned whether aging-related gene expression signatures define the likelihood of disease progression in patients with LR-MDS. We performed RNA-seq on CD34+ cells of 61 LR-MDS diagnostic samples (53 patients without rapid progression (NRP) and 8 patients with rapid progression (RP)) with a median age of 65 years. The differential expression analysis showed 1,701 up- and 1,569 downregulated genes in NRP (FDR<0.01). According to the GO Biological Processes (GO BP) and REACTOME databases, the most significantly upregulated pathways were related to macromolecular metabolic processes such as DNA repair, replication, protein metabolism, cell cycle, chromosome organization, including regulation of gene expression, and telomere organization (Fig. 1). The pathways of rRNA expression regulation, splicing, and RNA metabolism were deregulated as well as the pathways related to apoptosis, cellular senescence and cellular response to stress, immune response, and mitochondrial metabolism. Furthermore, we found deregulation of crucial signaling pathways mediated by NOTCH, RHO GTPases, NF-kB, MAPK, b-catenin, and WNT. Although 1,569 genes were upregulated in RP, only seventeen GO BP terms consisted of regulation of metabolic processes, cytoskeleton organization, phosphorylation, and signal transduction were upregulated. The NRP results are consistent with the hallmarks of aging (López-Otín C, et al., Cell. 2013; 153:1194). In GSEA, 36/37 and 120/143 gene sets were upregulated in NRP for the datasets consisting of gene sets chosen according to the keyword 'aging' and 'stress', respectively (Fig. 2). Among them, gene sets associated with replication stress, DNA damage, oxidative stress and unfolded protein response were significant (FDR<0.1, ІNESІ>1.7). Abnormal splicing is associated with cellular aging. Therefore, we conducted a differential alternative splicing analysis using rMATS. We found significant differences in 1,356 alternative splicing events (FDR<0.05, absolute inclusion level difference>10%) between NRP and RP. Finally, we performed analysis of healthy controls (n=7) and age-matched NRP (NRP-age) (n=13) samples. We observed 70 up- and 209 downregulated genes in NRP-age (FDR<0.05). Only the splicing pathways were upregulated and the immune pathways downregulated. In GSEA, no gene sets were significant (FDR<0.1) for aging and stress datasets. To conclude, these results show increased signatures of aging in CD34+ cells from NRP, which correspond to an increased level of the intrinsic ability of CD34+ cells to suppress malignant progression through anti-tumor barrier activation. This is consistent with the current understanding that most aging phenotypes are known to be the products of DDR and tumor suppression. Based on our data, we hypothesize that the progression of LR-MDS patients represents a cell-autonomously and perhaps also non-cell-autonomously-mediated failure of appropriate cellular stress response mechanisms. Our data also show similarity at the transcriptome level between age-matched NRP and healthy controls. Supported by AZV (NV18-03-00227) and (NU21-03-00565), GA CR (N20-19162S), and MH CZ-DRO (UHKT, 00023736). Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Inherited Germline DNA Repair Gene Defects Are Prevalent Among Thai Patients with Myeloid Neoplasms

Certain germline mutations increase the risk of developing myeloid neoplasms (MNs) accompanied by second hit somatic events, such as therapy-related or oxidation-induced DNA damages. Aberrancy in DNA damage response (DDR) pathways leads to genomic instability giving rise to cancer. Previous data showed that the prevalence of pathogenic germline mutations in young adult Caucasian MN patients was 13-19%.1, 2 The most commonly affected pathways were involved in telomere biology and transcription factors, while the defects in DDRs were found in 15-25%. However, data in Asians are limited. In this study, we investigated the prevalence and landscape of germline variants in cancer predisposition genes in a cohort of Thai patients diagnosed with MNs. Targeted next generation sequencing of 23 germline predisposition genes was performed using the SureSelect custom kit and the Illumina HiSeq 2500 system. The total of 127 unselected adult (> 15 years old) patients from 3 medical centers comprising MDS (66%), MDS/MPN (14%) secondary AML (sAML, 20%) were enrolled. The median age was 66 years old (range 16-93) and 50% were male. Variants reported in COSMIC, the frequency of higher than 0.01% in the Genome Aggregation Database (gnomAD) or Thai exome database (consisted of 1084 Thai exome controls) and variant allelic frequency of less than 30% were excluded. New or exceeding rare nonsense variants or indels, as well as missense variants predicted to be deleterious by at least 4/6 in silico software, were included. The variants were classified according to American College of Medical Genetics (ACMG) classification. 14pathogenic and likely pathogenic germline variants were identified in 15 patients (15/127; 12%). One patient had 2 variants including heterozygous CEBPAP75fs and MSH2A604G. Of the 16 variants, there were 8 (50%) missense, 7 (44%) frameshift, and 1 (6%) nonsense, with a median VAF of 51%, ranging 36-83%. The variants affected the DDR pathway (8/16; 50%), transcriptional regulation pathway (4/16; 25%) and signal transduction pathway (2/16; 13%). Two variants were recurrent i.e.,FANCAAla1219GlyfsTer59 and BRCA2Glu626delinsAspPro and the others were identified in BRCA1/2, MSH2, MSH6, GATA2, CEBPA, DDX41, CBL and NF1. The median age of patients harboring germline variants was younger (58 vs. 66 years old) than those who did not. Half of the cases (6/12) were characterized by normal cytogenetics without enrichment of -7 or complex karyotype, comparable to patients with no germline variants. Of note, 28% (4/14) patients had previous history of gastrointestinal stromal tumor, Graves’ disease, Immune thrombocytopenia, and aplastic anemia (FANCA mutation) for years before MNs were diagnosed. None of the patients reported family history of hematological malignancies. In conclusion, the prevalence of germline predisposition to MNs in Southeast Asian populations is as high as in Caucasians and the identified variants are more commonly involved in the DDR pathway. The defects in the DDR pathway suggested an important role of genomic instability in leukemogenesis. This subset of patients should preferably receive non-cytotoxic treatments to avoid oxidative damages that possibly lead to further clonal evolution. ReferencesKeel SB, Scott A, Sanchez-Bonilla M, Ho PA, Gulsuner S, Pritchard CC, et al. Genetic features of myelodysplastic syndrome and aplastic anemia in pediatric and young adult patients. Haematologica 2016 Nov; 101(11): 1343-1350. Feurstein S, Churpek JE, Walsh T, Keel S, Hakkarainen M, Schroeder T, et al. Germline variants drive myelodysplastic syndrome in young adults. Leukemia 2021 2021/08/01; 35(8): 2439-2444.

The Role of Copper Homeostasis in Brain Disease.

In the human body, copper is an important trace element and is a cofactor for several important enzymes involved in energy production, iron metabolism, neuropeptide activation, connective tissue synthesis, and neurotransmitter synthesis. Copper is also necessary for cellular processes, such as the regulation of intracellular signal transduction, catecholamine balance, myelination of neurons, and efficient synaptic transmission in the central nervous system. Copper is naturally present in some foods and is available as a dietary supplement. Only small amounts of copper are typically stored in the body and a large amount of copper is excreted through bile and urine. Given the critical role of copper in a breadth of cellular processes, local concentrations of copper and the cellular distribution of copper transporter proteins in the brain are important to maintain the steady state of the internal environment. The dysfunction of copper metabolism or regulatory pathways results in an imbalance in copper homeostasis in the brain, which can lead to a myriad of acute and chronic pathological effects on neurological function. It suggests a unique mechanism linking copper homeostasis and neuronal activation within the central nervous system. This article explores the relationship between impaired copper homeostasis and neuropathophysiological progress in brain diseases.

Covalent Warheads Targeting Cysteine Residue: The Promising Approach in Drug Development.

Cysteine is one of the least abundant amino acids in proteins of many organisms, which plays a crucial role in catalysis, signal transduction, and redox regulation of gene expression. The thiol group of cysteine possesses the ability to perform nucleophilic and redox-active functions that are not feasible for other natural amino acids. Cysteine is the most common covalent amino acid residue and has been shown to react with a variety of warheads, especially Michael receptors. These unique properties have led to widespread interest in this nucleophile, leading to the development of a variety of cysteine-targeting warheads with different chemical compositions. Herein, we summarized the various covalent warheads targeting cysteine residue and their application in drug development.

ZC3H11A loss of function enhances NF-κB signaling through defective IκBα protein expression.

ZC3H11A is a cellular protein associated with the transcription export (TREX) complex that is induced during heat-shock. Several nuclear-replicating viruses exploit the mRNA export mechanism of ZC3H11A protein for their efficient replication. Here we show that ZC3H11A protein plays a role in regulation of NF-κB signal transduction. Depletion of ZC3H11A resulted in enhanced NF-κB mediated signaling, with upregulation of numerous innate immune related mRNAs, including IL-6 and a large group of interferon-stimulated genes. IL-6 upregulation in the absence of the ZC3H11A protein correlated with an increased NF-κB transcription factor binding to the IL-6 promoter and decreased IL-6 mRNA decay. The enhanced NF-κB signaling pathway in ZC3H11A deficient cells correlated with a defect in IκBα inhibitory mRNA and protein accumulation. Upon ZC3H11A depletion The IκBα mRNA was retained in the cell nucleus resulting in failure to maintain normal levels of the cytoplasmic IκBα mRNA and protein that is essential for its inhibitory feedback loop on NF-κB activity. These findings indicate towards a previously unknown mechanism of ZC3H11A in regulating the NF-κB pathway at the level of IkBα mRNA export.

Abstract 12552: Causal Effect of Exposure to Elevated Lipoprotein(a) Levels on the Blood Transcriptome : A Mendelian Randomization Study

INTRODUCTION: Lipoprotein(a) [Lp(a)] is one of the strongest genetic risk factors for atherosclerotic cardiovascular diseases (ASCVD). Several ex vivo studies have revealed a pro-inflammatory and pro-atherogenic role of Lp(a) in the etiology of ASCVD, mainly through induction of inflammation, metabolic processes, and apoptosis. However, whether Lp(a) causally influences the human transcriptome is unknown. Here, we aimed at identifying whether genetically-predicted Lp(a) levels could also affect the whole blood expression of genes involved in metabolic- and inflammatory-related pathways. Methods: We performed a series of inverse-variance weighted mendelian randomization analyses using single-nucleotide polymorphisms located in the LPA locus (±500kb; p&lt;5x10 -8 ) from the UK Biobank GWAS on Lipoprotein(a) quantiles as exposure (n=377,590 and whole blood expression levels of 14,288 coding gene from eQTLGen as outcomes (n=31,684 from 37 studies). We then used the Functional Mapping and Annotation of Genome-Wide Association Studies tool (FUMA) to identify gene sets for cardiometabolic- and inflammatory-related pathways from gene ontology biological processes. Results: The MR analyses identified 1363 genes that may be causally influenced by exposure to high Lp(a) levels (with p&lt;0.05). The 10 most strongly associated genes with genetically-predicted Lp(a) levels are FSTL3 , PRKC1 , PDE6C , PLXNB3 , SESN1 , SASH1 , CD1D , CCDC102A , SS18L1 and TCF12 . These genes are also differentially expressed in the heart. We identified 16 cardiometabolic-related pathways, mostly related to immune response, signaling, apoptosis, cardiovascular and vasculature development, cell proliferation, metabolic process, and catalytic activity. The 5 most significant pathways are regulation of cell population proliferation, regulation of intracellular signal transduction, positive regulation of cell population proliferation, cardiovascular system development and TOR signaling. Conclusions: Results of this study highlight a potentially causal effect of exposure to elevated Lp(a) levels on several key genes involved in metabolic processes and inflammation and provide mechanistic insight on the pathobiological role of Lp(a) in the etiology of ASCVD.

Physiological and transcriptomic analysis reveal the regulatory mechanism underlying grain quality improvement induced by rice ratooning.

Ratoon rice cropping has been introduced for increased rice production in southern China and, as a result, has been becoming increasingly popular. However, only a few studies have addressed the regulatory mechanism underlying grain quality improvement induced by rice ratooning. In this study, parameters of rice quality, including head rice yield, chalky grain percentage, grain chalkiness degree, hardness and taste value, were shown to be much improved in the ratooning season rice as compared to its counterparts main and late cropping season rice, indicating that such an improvement was irrespective of seasonal effects. In addition, the nutritional components of grains varied greatly between main-cropping season rice, ratooning season rice and late-cropping season rice and displayed a significant correlation with rice quality. Finally, the regulatory mechanism underlying rice quality improvement revealed that gibberellin-dominated regulation and plant hormone signal transduction jointly contributed to a decrease in formation of chalky grains. This work improves our knowledge on rice quality improvement under rice ratooning, particularly on the regulatory mechanism of plant hormones. © 2022 Society of Chemical Industry.

Identification of MicroRNAs Related to Phytohormone Signal Transduction and Self-incompatibility of Rabbiteye Blueberry Pollen

MicroRNAs (miRNAs) related to phytohormone signal transduction and self-incompatibility may play an important role in the xenia effect. However, associated research in this area is still lacking in rabbiteye blueberry (Vaccinium ashei). In this study, we identified miRNAs, predicted their target genes, performed functional enrichment analysis of the target genes, and screened for miRNAs related to phytohormone signaling and self-incompatibility. A total of 491 miRNAs were identified, of which 27 and 67 known miRNAs as well as 274 and 416 new miRNAs were found in the rabbiteye blueberry cultivars Brightwell and Premier, respectively. Compared with ‘Premier’, 31 miRNAs were upregulated and 62 miRNAs were downregulated in ‘Brightwell’. Kyoto Encyclopedia of Genes and Genomes (KEGG) functional enrichment analysis indicated that the 4985 target genes predicted were involved in biosynthesis of amino acids, plant–pathogen interaction, and spliceosome pathways. A total of 10, one, one, five, two, five, and two candidate miRNAs related to auxin, cytokinin, gibberellin, abscisic acid, ethylene, brassinosteroid, and salicylic acid signaling, respectively, in rabbiteye blueberry pollen were identified. Further analysis indicated that novel_miR_49 was a candidate miRNA related to self-incompatibility, and their target gene was maker-VaccDscaff21-snap-gene-21.37. In addition, the KEGG enrichment analysis of the target genes of novel_miR_49 showed that they were involved in the ribosome, aminoacyl-tRNA biosynthesis, and glycosylphosphatidylinositol-anchor biosynthesis pathways. The results revealed that the microRNAs of rabbiteye blueberry pollen regulated to phytohormone signal transduction and self-incompatibility signal transduction based on related to auxin, cytokinin, gibberellin, abscisic acid, ethylene, brassinosteroid, and salicylic acid signaling. Results suggest that more research of the effects of miRNAs on regulation of hormone signal transduction and self-incompatibility is necessary for elucidating the molecular mechanism of the xenia effect.

OR04-6 Comprehensive Profiling of the MELK Inhibitor OTSSP167's Action and Its Effect on Sensitization to Wee1 Inhibitor AZD1775 in a Spectrum of ACC Models

Abstract Adrenocortical carcinoma (ACC) is an aggressive cancer with a significant risk of recurrence and high mortality rates. ACC has limited response to currently used treatments and there is an unmet clinical need to identify new treatment options. Disease rarity coupled with absence of preclinical models of disease heterogeneity contributed to limited therapeutic progress in ACC. We previously established new in vitro and in vivo ACC models (CUACC1, CUACC2, CUACC9) with variable genomic alterations commonly seen in ACC tumors. Prior work demonstrated that maternal leucine embryonic kinase (MELK) is an oncogenic kinase in ACC, and that it can be targeted with the MELK inhibitor OTSSP167. To further evaluate the anti-tumor efficacy and mode of action of OTSSP167, this study used genetically diverse ACC cell lines (CUACC1, CUACC2, H295R), patient derived xenografts (PDX) (CUACC1 and CUACC9) and a cell line xenograft model (H295R) of ACC. ACC PDXs treated with 10 mg/kg OTSSP167 had significantly reduced MELK expression, tumor growth inhibition of more than 80%, reduced mitosis, and increased tumor tissue necrosis. Transcriptomic and phospho-proteomic analysis at multiple treatment timepoints in in vitro models revealed that OTSSP167 induced wide transcriptomic and proteomic changes involving effectors of signal transduction, cell cycle, protein translation, and epigenetic regulation to exert its anti-tumorigenic effects. Evaluation of OTSSP167's role in protein synthesis via a puromycin based SUnSET (surface sensing of translation) and kinase assay established that OTSSP167 inhibits de-novo protein synthesis, and targets RSK1 and its substrate rpS6 (S235/S236). Varying the degree of MELK silencing in H295R cell lines using a doxycycline inducible shRNA construct, inhibited RSK1 phosphorylation at T573 and S380 indicating a MELK-RSK1 axis in ACC. Additionally, OTSSP167's effect on the DNA damage pathway was validated, which activated the G2/M checkpoint protein Wee1, increased inhibitory phosphorylation of CDK1, causing an accumulation of cyclin B1 and G2/M arrest independent of TP53 status. Combination treatment with the Wee1 inhibitor AZD1775 and OTSSP167 enhanced OTSSP167 mediated cytotoxic effects in in-vitro models with an increase in mitotic entry and caspase mediated apoptosis. In summary, OTSSP167 exerted its anti-tumorigenic effects via inhibition of the protein translation machinery, DNA damage and cell cycle dysregulation causing drug sensitization. Results from these studies are a first demonstration of the anti-cancer effects of OTSSP167 in sensitization to Wee1 inhibition in ACC. These data suggest that OTSSP167 is a potential therapeutic option for patients with ACC and could be combined with Wee1 inhibitors in a clinical trial using a synthetic lethal approach. Presentation: Saturday, June 11, 2022 12:45 p.m. - 1:00 p.m.

Hybrid in vitro/in silico analysis of low-affinity protein-protein interactions that regulate signal transduction by Sema6D.

Semaphorins constitute a large family of secreted and membrane-bound proteins that signal through cell-surface receptors, plexins. Semaphorins generally use low-affinity protein-protein interactions to bind with their specific plexin(s) and regulate distinct cellular processes such as neurogenesis, immune response, and organogenesis. Sema6D is a membrane-bound semaphorin that interacts with class A plexins. Sema6D exhibited differential binding affinities to class A plexins in prior cell-based assays, but the molecular mechanism underlying this selectivity is not well understood. Therefore, we performed hybrid in vitro/in silico analysis to examine the binding mode of Sema6D to class A plexins and to identify residues that give rise to the differential affinities and thus contribute to the selectivity within the same class of semaphorins. Our biophysical binding analysis indeed confirmed that Sema6D has a higher affinity for Plexin-A1 than for other class A plexins, consistent with the binding selectivity observed in the previous cell-based assays. Unexpectedly, our present crystallographic analysis of the Sema6D-Plexin-A1 complex showed that the pattern of polar interactions is not interaction-specific because it matches the pattern in the prior structure of the Sema6A-Plexin-A2 complex. Thus, we performed in silico alanine scanning analysis and discovered hotspot residues that selectively stabilized the Sema6D-Plexin-A1 pair via Van der Waals interactions. We then validated the contribution of these hotspot residues to the variation in binding affinity with biophysical binding analysis and molecular dynamics simulations on the mutants. Ultimately, our present results suggest that shape complementarity in the binding interfaces is a determinant for binding selectivity.

A Whole New Comprehension about ncRNA-Encoded Peptides/Proteins in Cancers.

It is generally considered that non-coding RNAs do not encode proteins; however, more recently, studies have shown that lncRNAs and circRNAs have ORFs which are regions that code for peptides/protein. On account of the lack of 5'cap structure, translation of circRNAs is driven by IRESs, m6A modification or through rolling amplification. An increasing body of evidence have revealed different functions and mechanisms of ncRNA-encoded peptides/proteins in cancers, including regulation of signal transduction (Wnt/β-catenin signaling, AKT-related signaling, MAPK signaling and other signaling), cellular metabolism (Glucose metabolism and Lipid metabolism), protein stability, transcriptional regulation, posttranscriptional regulation (regulation of RNA stability, mRNA splicing and translation initiation). In addition, we conclude the existing detection technologies and the potential of clinical applications in cancer therapy.

Brain O-GlcNAcylation: From Molecular Mechanisms to Clinical Phenotype.

O-GlcNAc is the attachment of β-N-acetylglucosamine to the hydroxyl group of serine and threonine in nuclear and cytoplasmic proteins. It is generally not further elongated but exists as a monosaccharide that can be rapidly added or removed. Thousands of proteins involved in gene transcription, protein translation and degradation as well as the regulation of signal transduction contain O-GlcNAc. Brain is one of the tissues where O-GlcNAc is the most highly expressed and deletion of neuronal O-GlcNAc leads to death early in development. O-GlcNAc is also important for normal adult brain function, where dynamic processes like learning and memory at least in part depend on the modification of specific proteins by O-GlcNAc. Conversely, too much or too little O-GlcNAc in the brain contributes to several disorders including obesity, intellectual disability and Alzheimer's disease. In this chapter, we describe the expression and regulation of O-GlcNAc in the nervous system.

Roles of non-coding RNAs in the hormonal and nutritional regulation in nodulation and nitrogen fixation.

Symbiotic nitrogen fixation is an important component in the nitrogen cycle and is a potential solution for sustainable agriculture. It is the result of the interactions between the plant host, mostly restricted to legume species, and the rhizobial symbiont. From the first encounter between the host and the symbiont to eventual successful nitrogen fixation, there are delicate processes involved, such as nodule organogenesis, rhizobial infection thread progression, differentiation of the bacteroid, deregulation of the host defense systems, and reallocation of resources. All these processes are tightly regulated at different levels. Recent evidence revealed that non-coding RNAs (ncRNAs), including microRNAs (miRNAs), long non-coding RNAs (lncRNAs), and circular RNAs (circRNAs), participate in these processes by controlling the transcription and translation of effector genes. In general, ncRNAs are functional transcripts without translation potential and are important gene regulators. MiRNAs, negative gene regulators, bind to the target mRNAs and repress protein production by causing the cleavage of mRNA and translational silencing. LncRNAs affect the formation of chromosomal loops, DNA methylation, histone modification, and alternative splicing to modulate gene expression. Both lncRNAs and circRNAs could serve as target mimics of miRNA to inhibit miRNA functions. In this review, we summarized and discussed the current understanding of the roles of ncRNAs in legume nodulation and nitrogen fixation in the root nodule, mainly focusing on their regulation of hormone signal transduction, the autoregulation of nodulation (AON) pathway and nutrient homeostasis in nodules. Unraveling the mediation of legume nodulation by ncRNAs will give us new insights into designing higher-performance leguminous crops for sustainable agriculture.

Engineering the Signal Transduction between CdTe and CdSe Quantum Dots for in Situ Ratiometric Photoelectrochemical Immunoassay of Cry1Ab Protein.

Controllable modulation of a response mode is extremely attracting to fabricate biosensor with programmable analytical performances. Here, we reported a proof-of-concept ratiometric photoelectrochemical (PEC) immunoassay of Cry1Ab protein based on the signal transduction regulation at the sensing interface. A sandwich-type PEC structure was designed so that gold nanorods sensitized quantum dots to fix primary antibody (Au NRs/QDs-Ab1) and methylene blue sensitized QDs to combine a second antibody (MB/QDs-Ab2), which served as photoelectric substrate and signal amplifier, respectively. Unlike common recognition element, such a sandwich-type PEC structure allowed for the in situ generation of two specific response signals. For analysis, Cry1Ab captured by Au NRs/QDs-Ab1 led to a decreased photocurrent (ICry1Ab), while the subsequently anchored MB/QDs-Ab2 produced another photocurrent (IMB). Noteworthy, by taking advantage of the different energy band gaps of QDs, varying locations of CdTe and CdSe QDs could realize different signal transduction strategies (i.e., Mode 1 and Mode 2). Investigations on data analysis of ICry1Ab and IMB via different routes demonstrated the superior analytical performances of ratiometry (Mode 1). Consequently, the ratiometric PEC immunosensor offered a linear range of 0.01-100 ng mL-1 with a detection limit of 1.4 pg mL-1. This work provides an efficient strategy for in situ collection of multiple photocurrents to design ratiometric PEC sensors.

Whole-exome sequencing analysis identifies distinct mutational profile and novel prognostic biomarkers in primary gastrointestinal diffuse large B-cell lymphoma

BackgroundDiffuse large B-cell lymphoma (DLBCL) is the most common aggressive non-Hodgkin lymphoma, and about 10% of DLBCL cases primarily occur in the gastrointestinal tract. Previous reports have revealed that primary gastrointestinal-DLBCL (pGI-DLBCL) harbors different genetic mutations from other nodal or extranodal DLBCL. However, the exonic mutation profile of pGI-DLBCL has not been fully addressed.MethodsWe performed whole-exome sequencing of matched tumor tissues and blood samples from 53 pGI-DLBCL patients. The exonic mutation profiles were screened, and the correlations between genetic mutations and clinicopathological characteristics were analyzed.ResultsA total of 6,588 protein-altering events were found and the five most frequent mutated genes in our pGI-DLBCL cohort were IGLL5 (47%), TP53 (42%), BTG2 (28%), P2RY8 (26%) and PCLO (23%). Compared to the common DLBCL, significantly less or absence of MYD88 (0%), EZH2 (0%), BCL2 (2%) or CD79B (8%) mutations were identified in pGI-DLBCL. The recurrent potential driver genes were mainly enriched in pathways related to signal transduction, infectious disease and immune regulation. In addition, HBV infection had an impact on the mutational signature in pGI-DLBCL, as positive HBsAg was significantly associated with the TP53 and LRP1B mutations, two established tumor suppressor genes in many human cancers. Moreover, IGLL5 and LRP1B mutations were significantly correlated with patient overall survival and could serve as two novel prognostic biomarkers in pGI-DLBCL.ConclusionsOur study provides a comprehensive view of the exonic mutation profile of the largest pGI-DLBCL cohort to date. The results could facilitate the clinical development of novel therapeutic and prognostic biomarkers for pGI-DLBCL.

Destabilization of the SHP2 and SHP1 protein tyrosine phosphatase domains by a non-conserved “backdoor” cysteine

Protein tyrosine phosphatases (PTPs) are critical regulators of cellular signal transduction that catalyze the hydrolytic dephosphorylation of phosphotyrosine in substrate proteins. Among several conserved features in classical PTP domains are an active-site cysteine residue that is necessary for catalysis and a “backdoor” cysteine residue that can serve to protect the active-site cysteine from irreversible oxidation. Curiously, two biologically important phosphatases, Src homology domain-containing PTPs 2 and 1 (SHP2 and SHP1), each contain an additional backdoor cysteine residue at a position of the PTP domain that is occupied by proline in almost all other classical PTPs (position 333 in human SHP2 numbering). Here we show that the presence of cysteine 333 significantly destabilizes the fold of the PTP domains in the SHPs. We find that replacement of cysteine 333 with proline confers increased thermal stability on the SHP2 and SHP1 PTP domains, as measured by temperature-dependent activity assays and differential scanning fluorimetry. Conversely, we show that substantial destabilization of the PTP-domain fold is conferred by introduction of a non-natural cysteine residue in a non-SHP PTP that contains proline at the 333 position. It has previously been suggested that the extra backdoor cysteine of the SHP PTPs may work in tandem with the conserved backdoor cysteine to provide protection from irreversible oxidative enzyme inactivation. If so, our current results suggest that, during the course of mammalian evolution, the SHP proteins have developed extra protection from oxidation at the cost of the thermal instability that is conferred by the presence of their PTP domains’ second backdoor cysteine.

In-Depth Characterization of the Clostridioides difficile Phosphoproteome to Identify Ser/Thr Kinase Substrates

Clostridioides difficile is the leading cause of postantibiotic diarrhea in adults. During infection, the bacterium must rapidly adapt to the host environment by using survival strategies. Protein phosphorylation is a reversible post-translational modification employed ubiquitously for signal transduction and cellular regulation. Hanks-type serine/threonine kinases (STKs) and serine/threonine phosphatases have emerged as important players in bacterial cell signaling and pathogenicity. C. difficile encodes two STKs (PrkC and CD2148) and one phosphatase. We optimized a titanium dioxide phosphopeptide enrichment approach to determine the phosphoproteome of C. difficile. We identified and quantified 2500 proteins representing 63% of the theoretical proteome. To identify STK and serine/threonine phosphatase targets, we then performed comparative large-scale phosphoproteomics of the WT strain and isogenic ΔprkC, CD2148, Δstp, and prkC CD2148 mutants. We detected 635 proteins containing phosphorylated peptides. We showed that PrkC is phosphorylated on multiple sites in vivo and autophosphorylates in vitro. We were unable to detect a phosphorylation for CD2148 in vivo, whereas this kinase was phosphorylated in vitro only in the presence of PrkC. Forty-one phosphoproteins were identified as phosphorylated under the control of CD2148, whereas 114 proteins were phosphorylated under the control of PrkC including 27 phosphoproteins more phosphorylated in the ∆stp mutant. We also observed enrichment for phosphothreonine among the phosphopeptides more phosphorylated in the Δstp mutant. Both kinases targeted pathways required for metabolism, translation, and stress response, whereas cell division and peptidoglycan metabolism were more specifically controlled by PrkC-dependent phosphorylation in agreement with the phenotypes of the ΔprkC mutant. Using a combination of approaches, we confirmed that FtsK was phosphorylated in vivo under the control of PrkC and that Spo0A was a substrate of PrkC in vitro. This study provides a detailed mapping of kinase–substrate relationships in C. difficile, paving the way for the identification of new biomarkers and therapeutic targets.