SH2 Domain-containing Protein Research Articles

A Novel Missense Variant in SORBS2 Is Causative With Familial Alzheimer's Disease.

Alzheimer's disease (AD) is a common neurodegenerative disorder with a substantial genetic component. Despite advances in elucidating the genetic underpinnings of AD, much of its heritability remains unexplained. Discovering novel genetic variants and understanding their pathogenic roles are crucial challenges in AD research. This study aimed to identify pathogenic genes and elucidate their role in familial early-onset AD (EOAD). Blood samples from an EOAD pedigree and Sorbin and SH3 Domain-Containing Protein 2 (SORBS2) T189M transgenic mice were analyzed. Cognitive function was assessed via the Morris water maze (MWM). Protein expression was evaluated by western blotting, while amyloid-β (Aβ) levels were quantified via immunohistochemistry and enzyme-linked immunosorbent assay. Inflammatory markers were measured using immunofluorescence and quantitative reverse transcription polymerase chain reaction (PCR). Neuronal morphology, including dendritic and spine alterations, was examined using Golgi staining. We identified a novel SORBS2 variant (c. 566C>T, p. T189M) in a Han Chinese family, segregating with AD in a Mendelian fashion. SORBS2 T189M transgenic mice exhibited cognitive deficits, cortical Aβ accumulation, and an increased Aβ42/Aβ40 ratio. Additionally, elevated levels of interleukin (IL)-1β, IL-6, tumor necrosis factor α (TNF-α), and ionized calcium-binding adaptor molecule 1 (Iba1)-positive microglia, along with neuronal loss, were observed in the brains of T189M mice. Our study suggest that the SORBS2 T189M variant is a novel candidate causal mutation associated with familial AD in a Chinese pedigree, contributing to AD pathogenesis by promoting neuroinflammation and neuronal injury. Notably, this study is the first to establish a link between SORBS2 mutations and AD.

SH2B1 promotes apoptosis in diabetic cataract via p38 MAPK pathway.

Patients with diabetes face an increased risk of developing cataracts, with unclear mechanisms. Our study illuminates these mechanisms by identifying differentially expressed proteins in the lens anterior capsule of patients with diabetic cataract (DC) and age-related cataract using quantitative proteomics. We found SH2 domain-containing adapter protein B1 (SH2B1) to be crucial in DC progression. Reduced SH2B1 expression was confirmed through PCR and western blotting in patient samples, diet-induced obese mice, and high-glucose (HG)-cultured human lens epithelial cells. Under HG conditions, cell proliferation decreased, while migration and apoptosis, alongside changes in Bcl2 and caspase-3 expression, increased. Overexpressing SH2B1 alleviated these changes and influenced the p38 mitogen-activated protein kinase (MAPK) signaling pathway. This suggests SH2B1 and the p38 MAPK pathway as significant in DC pathogenesis and potential therapeutic targets. Clinically, this could lead to therapies aimed at halting or slowing DC progression.

SHEP1 alleviates cardiac ischemia reperfusion injury via targeting G3BP1 to regulate macrophage infiltration and inflammation

The macrophage-associated inflammation response plays an important role in myocardial ischemia-reperfusion injury (MIRI). SHEP1(SH2 domain-containing Eph receptor-binding protein 1) has been implicated in adhesion and migration of inflammatory cells. However, the role and molecular mechanism of SHEP1 regulating macrophage remains unclear during MIRI. Here, the expression of SHEP1 was increased in macrophages co-cultured with hypoxia-reoxygenated cardiomyocytes and within ischemia-reperfusion injured myocardium at the early stage of injury. Cell migration and inflammation were also enhanced in SHEP1 knock-out macrophages and macrophage-specific deficiency of SHEP1 mice under MIRI, which further led to deteriorated cardiac injury and cardiac function in vivo. Mechanistically, macrophage-derived SHEP1 competitively bound to G3BP1 to suppress inflammation via the MAPK pathway. In addition, administrating inhibitor of G3BP1 could improve cardiac function in macrophage-specific deficiency of SHEP1 mice under MIRI. Our results demonstrate that SHEP1 deficiency in macrophages exacerbates MIRI through G3BP1-dependent signaling pathway. SHEP1-G3BP1 interaction are therefore indispensable for SHEP1 regulated- infiltration and proinflammatory responses of macrophages, which provided a potential and clinically significant therapeutic target for MIRI.

SASH1 is a novel binding partner to disassemble Caskin1 tandem SAM homopolymer through heterogeneous SAM-SAM interaction.

Calcium/calmodulin-dependent serine protein kinase (CASK) interaction protein 1/2 (Caskin1/2) is essential neuronal synaptic scaffold protein in nervous system development. Knockouts of Caskin1/2 display severe deficits in novelty recognition and spatial memory. The tandem sterile alpha motif (SAM) domains of Caskin1/2, also conserved in their Drosophila homolog Ckn, are known to form homopolymers, yet their dynamic regulation mechanism remains unclear. In this study, SAM and SH3 domain-containing protein 1 (SASH1) was first identified as a novel binding partner of Caskin1/2 through yeast two-hybrid (Y2H) screening. The SAM-SAM interaction between SASH1 and Caskin1 was biochemically characterized by size-exclusion chromatography (SEC), isothermal titration calorimetry (ITC), and glutathione-S-transferase (GST) pull-down and co-immunoprecipitation (co-IP) assays. Structural insights from AlphaFold2-predicted models of the Caskin1-SAMs/SASH1-SAM1 complex, along with mutagenesis validations, revealed key residues at the end-helix (EH)/mid-loop (ML) interface for this interaction. More interestingly, the Caskin1-SAMs homopolymer can be disrupted by the SAM-SAM interaction, which was consistently verified by using sedimentation, transmission electron microscopy (TEM), and immunofluorescence (IF) staining in heterologous cell lines. In summary, our findings provide a solid biochemical basis for the Caskin1/SASH1 interaction and propose a potential mechanism for regulating Caskin1/2 homopolymerization via SAM-SAM interactions. More importantly, the principle governing SAM homopolymer depolymerization is generalized via suggesting two distinct types of heterogeneous SAM-SAM interactions, offering fresh insights into SAM domain-mediated homopolymerization and depolymerization.

Study on the Role of microRNA-138-5p Through Sorbin and SH3 Domain-Containing Protein 2 in Breast Cancer.

Breast cancer (BRCA) is one of the major threats to women's health worldwide. This study focuses on the roles of sorbin and SH3 domain-containing protein 2 (SORBS2) protein and microRNA-138-5p in the progression of BRCA, analyzing their regulatory effects on cancer cell proliferation, cell cycle, and migration ability. Using bioinformatics tools and experimental methods, the study found that SORBS2 is commonly underexpressed in BRCA. Additionally, it demonstrated that microRNA-138-5p can significantly inhibit tumor growth by suppressing SORBS2 expression, providing new molecular targets for BRCA treatment.

Aloe Emodin Alleviates Radiation-Induced Heart Disease via Blocking P4HB Lactylation and Mitigating Kynurenine Metabolic Disruption.

Aloe emodin is an anthraquinone of traditional Chinese medicine monomer, which plays a protective action in cardiovascular diseases. However, the regulatory mechanisms of aloe emodin in the protection of radiation-induced heart damage (RIHD) are unclear. As a novel post-translational modification, lactylation is considered as a critical mediator in inflammatory cascade and cardiac injury. Here, using a cross of differential omics and 4D label-free lactylation omics, protein disulfide-isomerase (P4HB) is identified as a novel target for lactylation, and aloe emodin inhibits the binding of lactate to the K311 site of P4HB. Aloe emodin stabilizes kynurenine metabolism through inhibition of aspartate aminotransferase (GOT2) accumulation on damaged mitochondria. Mechanistically, aloe emodin inhibits phosphorylated glycogen synthase kinase 3B (p-GSK3B) transcription in the nucleus to repress the interaction of prostaglandin G/H synthase 2 (PTGS2) with SH3 domain of SH3 domain-containing GRB2-like protein B1 (SH3GLB1), thereby disrupting the functions of mitochondrial complexes and reducing SH3GLB1-mediated mitoROS accumulation, eventually suppressing calcium-binding and coiled-coil domain-containing protein 2 (NDP52)-induced mitophagy. This study unveils the regulatory role of aloe emodin in RIHD alleviation through PTGS2/SH3GLB1/NDP52 axis, indicates aloe emodin stabilizes GOT2-mediated kynurenine metabolism through P4HB lactylation. Collectively, this study provides novel insights into the regulatory mechanisms underlying the protective role of aloe emodin in cardiac injury, and opens new avenues for therapeutic strategies of aloe emodin in preventing RIHD by regulating lactylation.

Regulation of RHOV signaling by interaction with SH3 domain-containing adaptor proteins and phosphorylation by PKA

RHOV and RHOU are considered atypical Rho-family small GTPases because of the existence of N- and C-terminal extension regions, abnormal GDP/GTP cycling, and post-translational modification. Particularly, RHOV and RHOU both have a proline-rich (PR) motif in the N-terminal region. It has been reported that the PR motif of RHOU interacts with GRB2, a SH3 domain-containing adaptor protein, and regulates its activity through EGF receptor signaling. However, it is unknown whether RHOV, like RHOU, interacts with SH3 domain-containing adaptor proteins. In this study, we investigated the interactions between RHOV and SH3 domain-containing adaptor proteins, including GRB2 and NCK2. The RHOV-induced serum response factor (SRF)-dependent gene transcriptional activity was attenuated in cells co-expressing either GRB2 or NCK2 compared to cells expressing RHOV alone. From the results of experiments using various gene mutants of RHOV and GRB2, it appears that the PR motif of the N-terminal region of RHOV is the crucial binding site for the SH3 domain-containing proteins. Furthermore, we found that Ser25 in the N-terminal region of RHOV is phosphorylated by PKA and that its phosphorylation is suppressed by interaction with NCK2 but not GRB2. We have found a novel regulatory mechanism for the phosphorylation of RHOV and its interaction with SH3 domain-containing adaptor proteins.

Supplemental Psyllium Fiber Increases Antimicrobial Proteins via the Tuft Cell-ILC2 Circuit and Type II Immune Response in the Mouse Small Intestine

Dietary fibers regulate intestinal barrier function; however, the precise mechanisms remain unclear. This study investigated the effects of psyllium fibers on antimicrobial protein expression, focusing on the type II immunity and tuft cell-group 2 innate lymphoid cell (ILC2) circuit in the small intestine of the mouse. Supplemental psyllium fiber upregulated antimicrobial proteins, such as small proline-rich protein 2A (SPRR2A) and resistin-like beta (RELMβ), in mouse small intestine, evidently affecting cecal microbiota composition. The psyllium fibers also increased the RNA and protein expression of molecules related to ILC2 and tuft cells, such as IL-13, IL-25, DCLK1, Gfi-1b, SH2 domain-containing protein 3C, and Spi-B. In addition, ILC2 inhibitor (disulfiram) and bitter taste receptor blocker administration reduced psyllium-induced SPRR2A and RELMβ expression. Collectively, psyllium supplementation upregulates antimicrobial proteins such as SPRR2A and RELMß via the type II immune response and tuft cell-ILC2 circuit in the mouse small intestine.

SORBS1 expression as a biomarker for obesity and breast cancer prognosis: Insights from genomic analysis and survival outcomes.

e13050 Background: Obesity is a known risk factor for breast cancer development and poorer survival. Correlation between the expression of the adaptor protein SORBS1 (Sorbin and SH3 domain-containing protein 1) and body mass index (BMI). Methods: Bioinformatics analysis. Results: We analyzed published datasets and plotted SORBS1 expression against patient BMI. Our findings show a negative correlation between SORBS1 expression and BMI in both datasets (R = -0.42, p = 0.00087, and R = -0.45 p = 0.00083, respectively). We also observed a negative correlation between SORBS1 expression and Homeostatic Model Assessment of Insulin Resistance (HOMA-IR) (R = -0.43, p = 0.00016). Additionally, we examined BMI-associated variants of the SORBS1 gene in the Qatar Biobank cohort of 15k individuals. We identified a total of 26 variants that were associated with an elevated risk for BMI in this population at a nominal significance. The top risk variant associated with the BMI is a missense variant (rs35480352; beta=0.058, p-value = 3.3e-03) with a frequency of 9.1% for the risk allele in our population. These results suggest that SORBS1 expression decreases with obesity and diabetes. This is significant because obesity is a known risk factor for breast cancer development and poorer survival. We also examined the SORBS1 gene expression in breast adenocarcinoma using UCSC TOIL RNA-seq recompute and TCGA-BRCA genomic and methylation data. Out of the 963 patients analyzed, only 12 (1.2%) showed any SORBS1 gene alteration, such as mutation (0.7%), amplification (0.2%), or deep deletion (0.3%), indicating a low occurrence of genomic alterations. Furthermore, our findings revealed no significant difference in SORBS1 methylation levels between the tumor and matched normal samples, indicating that the SORBS1 gene is not hypermethylated in breast carcinoma. Interestingly, our comparison of SORBS1 expression values between tumor and normal samples indicated significant under-expression in BRCA (log2FC = -2.7, p = 1.07E-127). SORBS1 expression was found to vary across various clinical stages (p = 0.0082, Kruskal-Wallis rank test), exhibiting a decline in advanced cancer stages. Moreover, we detected a significant difference in SORBS1 expression among different molecular subtypes (p < 2E-16, Kruskal-Wallis rank test), with a lower expression level in more aggressive subtypes like HER2. Patients were classified into two groups according to their SORBS1 expression levels, using the median expression as a threshold: high and low. Conclusions: Our findings showed that the SORBS1 low group had substantially lower overall survival (log-rank p = 0.0024) and progression-free survival rates (log-rank p = 0.028). Silencing of SORBS1 in MCF-7 and MDA-MB-231 cells increased cell proliferation, migration, and invasion, underscoring its importance in breast cancer prognosis and as a target for intervention.

Nucleotide-binding leucine-rich repeat receptor homologs Pib and PibH8 interact and contribute to immunity in rice.

Plant nucleotide-binding leucine-rich repeat receptors (NLRs) sense pathogen effectors and activate effector-triggered immunity (ETI). Many plant NLRs form pairs with other NLRs to recognize effectors and initiate ETI. PIRICULARIA ORYZAE RESISTANCE IN BL1 (Pib), an NLR protein in rice (Oryza sativa), activates resistance by recognizing the rice blast effector AvrPib. The activation of Pib is suppressed by SH3 DOMAIN-CONTAINING PROTEIN 2 (OsSH3P2) in the absence of AvrPib. However, how Pib triggers defense responses and whether Pib pairs with another NLR are not clear. In this study, we identified Pib by map-based cloning and showed that a homolog of Pib, PIB HOMOLOGUE 8 (PibH8), interacts with Pib. Pib and PibH8 mediate resistance to the Magnaporthe oryzae isolate Guy11, a rice blast strain carrying AvrPib. Interestingly, the pib/pibh8 double mutant exhibited enhanced susceptibility to Guy11 compared to the single mutant. Furthermore, PibH8 can oligomerize through its coiled-coil (CC) domain, which also contributes to the Pib-PibH8 interaction, suggesting that Pib and PibH8 may form a complex to recognize AvrPib. OsSH3P2 inhibited the interaction of Pib and PibH8 through association with the CC domain of PibH8. Taken together, these results indicate that both Pib and PibH8 are required for rice blast resistance to M. oryzae carrying AvrPib, which is negatively regulated by OsSH3P2. This study not only identifies an NLR that functions in rice blast resistance but also reveals a possible complex immune strategy in which homologous NLR proteins may regulate the complete activation of plant immunity.

Expression of several deubiquitinase and tyrosine phosphatase genes involved in inflammatory response in lymphoma

Lymphoma represents a heterogeneous group of cancers affecting the lymphatic system, encompassing non-Hodgkin lymphoma (NHL) and Hodgkin lymphoma (HL). The majority of NHL cases originate from B cells. HL is characterized by the presence of rare Hodgkin and Reed-Sternberg cells. Within the context of the inflammatory response, two SH2 domain-containing protein tyrosine phosphatases (SHPs), namely SHP-1 and SHP-2, serve as negative regulators. Deubiquitinating enzymes (DUBs) play a crucial role in inhibiting NF-κB activation in response to various stimuli. This study focuses on the DUBs OTUB1, OTUB2, and Cezanne. Blood samples were collected from 50 NHL patients, 26 HL patients, and 50 healthy individuals. Quantitative RT-PCR was employed to assess the mRNA expression of OTUB1, OTUB2, Cezanne, SHP-1,and SHP-2, while ELISA was used to determine IL-6 and CA125 concentrations. The results revealed that the mRNA level of OTUB1 was significantly downregulated in NHL patients but not in HL patients. Notably, Cezanne expression was downregulated in lymphoma patients, with significantly lower levels observed in HL compared to NHL patients. Furthermore, SHP-1 mRNA expression was significantly lower in the NHL group compared to the HL group or healthy individuals. Conversely, SHP-2 gene expression was upregulated in NHL patients but remained unchanged in HL patients. In conclusion, these findings highlight significant differences in the expressions of DUB and SHP genes and the inflammatory response in lymphoma patients. This study provides a foundation for further investigation into the roles of DUBs and tyrosine phosphatases in regulating the functional activation of lymphoma cells.

Repolarization of Immunosuppressive Macrophages by Targeting SLAMF7-Regulated CCL2 Signaling Sensitizes Hepatocellular Carcinoma to Immunotherapy.

CCL2 upregulation caused by SLAMF7 deficiency in hepatocellular carcinoma cells induces immunosuppressive macrophage polarization and confers resistance to immune checkpoint blockade, providing potential biomarkers and targets to improve immunotherapy response in patients.

Hepatitis B virus evades the immune system by suppressing the NF-κB signaling pathway with DENND2A.

Overcoming hepatitis B virus (HBV) is a challenging problem because HBV deceives the host immune system. We have found that DENN domain-containing 2A (DENND2A) was essential for HBV maintenance, although its role remains unclear. In this study, we elucidate its function by screening a novel DENND2A-binding peptide, DENP4-3S. DENP4-3S exhibits homology to SAM and SH3 domain-containing protein 1 (SASH1), a scaffold protein involved in Toll-like receptor signaling that promotes proinflammatory cytokine production. We confirmed that DENND2A interacts with SASH1 specifically. Overexpression and knockdown experiments showed that overexpression of DENND2A suppressed the transcriptional activity of NF-κB, and the knockdown of DENND2A promoted it and the production of cytokines and interferons. Here, we constructed a fusion protein (10M-DEN3SN) consisting of an anti-asialoglycoprotein receptor antibody and DENP4-3S to deliver the peptide to hepatocytes specifically. 10M-DEN3SN inhibited the interaction between DENND2A and SASH1, and rescued SASH1 trapped by DENND2A, leading to the upregulation of NF-κB and its downstream signaling. In addition, 10M-DEN3SN suppressed HBV proliferation in PXB chimeric mice. These results with the DENND2A-binding peptide delivered into hepatocytes suggested the involvement of DENND2A, SASH, and NF-κB signaling pathway in the HBV infection and onset of hepatitis. In conclusion, this study indicates that HBV utilizes DENND2A and SASH1 to evade the immune system.IMPORTANCEHepatitis B virus (HBV) is a serious liver infection with no established cure, causing an abnormal host immune response. Here, we identified a novel peptide that interacts with DENN domain-containing 2A (DENND2A), a host factor essential for HBV maintenance. The resulting peptide showed sequence homology, revealing an interaction between DENND2A and the immune system regulator SASH1. This study suggests that DENND2A contributes to HBV infection by suppressing the cellular immune system by inhibiting SASH1. The DENND2A-binding peptide, incorporated into our hepatocyte-specific peptide delivery system, inhibited the DENND2A-SASH1 interaction and promoted the production of cytokines and interferons in cultured hepatocytes. As a consequence, the peptide suppressed HBV proliferation in humanized mice. We report new insights into the role of DENND2A and SASH1 in HBV maintenance and highlight the importance of the immune system.

Stereotactic body radiation therapy suppresses myeloid-derived suppressor cells by regulating miR-21/Sorbin and SH3 Domain-containing Protein 1 axis.

Stereotactic body radiation therapy (SBRT) is a targeted form of radiotherapy used to treat early-stage cancers. Despite its effectiveness, the impact of SBRT on myeloid-derived suppressor cells (MDSCs) is not well understood. In this study, we examined how SBRT affects the differentiation and survival of MDSCs, as well as delved into the molecular mechanisms involved. SBRT was utilized on bone marrow (BM)-derived MDSCs to investigate its impact on the differentiation and survival of MDSCs using flow cytometry. An animal model of lung cancer was created to assess the anti-cancer properties of SBRT and the role of miR-21 expression in MDSCs. The interplay of miR-21 and Sorbin and SH3 domain-containing protein 1 (SORBS1) in MDSC differentiation was explored through dual luciferase activity assay, RT-qPCR, and Western blot analysis. The findings suggest that SBRT led to an increase in miR-21 levels, inhibited MDSC differentiation, and triggered cell apoptosis in BM cells. Inhibition of miR-21 reversed the effects of SBRT on MDSC differentiation and apoptosis. Additionally, it was revealed that SORBS1 was a downstream target of miR-21 in BM cells, and the miR-21/SORBS1 axis played a role in regulating MDSC differentiation and apoptosis induced by SBRT. Modulating miR-21 levels in vivo impinged on the response to SBRT treatment and the quantity of MDSCs in a mouse model of lung cancer. Our data indicate that the upregulation of miR-21 induced by SBRT may contribute to the inhibition of MDSC expansion in a lung cancer model.

Effect of adenovirus-mediated shRNA down-regulates SHP2 expression on the apoptosis of human hepatic stellate cells LX-2

Objective: To investigate the effect of adenovirus-mediated short hairpin RNA (shRNA) downregulating SH2 domain-containing protein tyrosine phosphatase 2 (SHP2) on the apoptosis of human hepatic stellate cells LX-2 cultured in vitro. Methods: The recombinant adenovirus Ad-shRNA/SHP2 carrying shRNA targeted SHP2 and expressing green fluorescent protein (GFP), and the empty control virus Ad-GFP expressing GFP were transfected into LX-2 cells cultured in vitro. Real-time fluorescence quantitative PCR was used to detect SHP2 mRNA expression in LX-2 cells. Western blot was used to detect the protein expressions of SHP2, Bax, and Bcl-2 in LX-2 cells. TUNEL and annexin-V/propidium iodide dual-labeled flow cytometry were used to detect apoptosis in LX-2 cells. Experimental group: (1) Control group: LX-2 cells were transfected with DMEM instead of adenovirus; (2) Ad-GFP group: transfected with empty virus Ad-GFP; (3) Ad-shRNA/SHP2 group: transfected with recombinant adenovirus Ad-shRNA/SHP2. The means between multiple groups were compared using a one-way ANOVA and the LSD test was used for inter group comparisons. Results: shRNA-targeted SHP2 significantly down-regulated the expression of SHP2 protein and mRNA in LX-2 cells (P < 0.05). The TUNEL and annexin-V/propidium iodide dual-labeled flow cytometry results showed that the apoptosis rate of LX-2 cells in the Ad-shRNA/SHP2 group (12.755%±1.606%, 19.340%±2.505%) (P < 0.05) was significantly higher compared to the control group (3.077%±0.731%, 9.438%±0.804%) and the Ad-GFP group (3.250%±0.851%, 8.893%±1.982%), with no statistically significant difference between the control group and the Ad-GFP group (P > 0.05). Western blot analysis of Bax and Bcl-2 protein expression in LX-2 cells of each group revealed that the Bax protein expression was significantly higher in the Ad shRNA/SHP2 group (2.493 ± 0.203) (P < 0.05) compared to the control group and Ad-GFP group (1.989 ± 0.147, 1.999 ± 0.162), with no statistically significant difference between the control group and the Ad-GFP group (P > 0.05), while the Bcl-2 protein was significantly decreased in the Ad-shRNA/SHP2 group (1.042±0.148) compared with the control group and the Ad-GFP group (1.707±0.146, 1.521±0.142), with no statistically significant difference between the control group and the Ad-GFP group (P > 0.05). Conclusions: SHP2 expression down-regulation induces apoptosis of human hepatic stellate cells LX-2 in vitro by reducing Bcl-2/Bax.

Convergent gene pair dSH3 and irr regulate Pi and Fe homeostasis in Bradyrhizobium diazoefficiens USDA110 and symbiotic nitrogen fixation efficiency

The nitrogen-fixing bacteroids inhabit inside legume root nodules must manage finely the utilization of P and Fe, the two most critical elements, due to their antagonistic interactions. While the balance mechanism for them remains unclear. A double SH3 domain-containing protein (dSH3) in the Bradyrhizobium diazoefficiens USDA110 was found to inhibit the alkaline phosphatase activity, thereby reducing P supply from organophosphates. The dSH3 gene is adjacent to the irr gene, which encodes the iron response repressor and regulates Fe homeostasis under Fe-limited conditions. Their transcription directions converge to a common intergenic sequence (IGS) region, forming a convergent transcription. Extending the IGS region through Tn5 transposon or pVO155 plasmid insertion significantly down-regulated expression of this gene pair, leading to a remarkable accumulation of P and an inability to grow under Fe-limited conditions. Inoculation of soybean with either of the insertion mutants resulted in N2-fixing failure. However, the IGS-deleted mutant showed no visible changes in N2-fixing efficiency on soybean compared to that inoculated with wild type. These findings reveal a novel regulative strategy in the IGS region and its flanking convergent gene pair for antagonistic utilization of P and Fe in rhizobia and coordination of N2-fixing efficiency.

Snakehead vesiculovirus hijacks SH3RF1 for replication via mediating K63-linked ubiquitination at K264 of the phosphoprotein

Snakehead vesiculovirus (SHVV) is a type of rhabdovirus that causes serious economic losses in snakehead fish culture in China. However, no specific antiviral drugs or vaccines are currently available for SHVV infection. In this study, 4D label-free ubiquitome analysis of SHVV-infected cells revealed dozens of ubiquitinated sites on the five SHVV proteins. We focused on investigating the ubiquitination of phosphoprotein (P), a viral polymerase co-factor involved in viral replication. SHVV-P was proved to be ubiquitinated via K63-linked ubiquitination at lysine 264 (K264). Overexpression of wild-type P, but not its K264R mutant, facilitated SHVV replication, indicating that K264 ubiquitination of the P protein is critical for SHVV replication. RNAi screening of 26 cellular E3 ubiquitin ligases identified five pro-viral factors for SHVV replication, including macrophage erythroblast attacher (MAEA), TNF receptor-associated factor 7 (TRAF7), and SH3 domain-containing ring finger protein 1 (SH3RF1), which interacted with and mediated ubiquitination of SHVV P. TRAF7 and SH3RF1, but not MAEA, mediated K63-linked ubiquitination of SHVV P, while only SH3RF1 mediated K264 ubiquitination of SHVV P. Besides, overexpression of SH3RF1 promoted SHVV replication and maintained the stability of SHVV P. In summary, SH3RF1 mediated K63-linked ubiquitination of SHVV P at K264 to facilitate SHVV replication, providing targets for developing anti-SHVV drugs and live-attenuated SHVV vaccines. Our study provides novel insights into the role of P protein in the replication of single-stranded, negative-sense RNA viruses.

The cell-end protein Tea4 spatially regulates hyphal branch initiation and appressorium remodeling in the blast fungus Magnaporthe oryzae.

The differentiation of specialized infection cells, called appressoria, from polarized germ tubes of the blast fungus Magnaporthe oryzae, requires remarkable remodeling of cell polarity and architecture, yet our understanding of this process remains incomplete. Here we investigate the behavior and role of cell-end marker proteins in appressorium remodeling and hyphal branch emergence. We show that the SH3 domain-containing protein Tea4 is required for the normal formation of an F-actin ring at Tea1-GFP-labeled polarity nodes, which contributes to the remodeling of septin structures and repolarization of the appressorium. Further, we show that Tea1 localizes to a cortical structure during hyphal septation which, unlike contractile septin rings, persists after septum formation, and, in combination with other polarity determinants, likely spatially regulates branch emergence. Genetic loss of Tea4 leads to mislocalization of Tea1 at the hyphal apex and with it, impaired growth directionality. In contrast, Tea1 is largely depleted from septation events in Δtea4 mutants and branching and septation are significantly reduced. Together, our data provide new insight into polarity remodeling during infection-related and vegetative growth by the blast fungus.

Role of Cytokine-Inducible SH2 Domain-Containing (CISH) Protein in the Regulation of Erythropoiesis.

The cytokine-inducible SH2 domain-containing (CISH) protein was the first member of the suppressor of cytokine signaling (SOCS) family of negative feedback regulators discovered, being identified in vitro as an inducible inhibitor of erythropoietin (EPO) signaling. However, understanding of the physiological role played by CISH in erythropoiesis has remained limited. To directly assess the function of CISH in this context, mice deficient in CISH were characterized with respect to developmental, steady-state, and EPO-induced erythropoiesis. CISH was strongly expressed in the fetal liver, but CISH knockout (KO) mice showed only minor disruption of primitive erythropoiesis. However, adults exhibited mild macrocytic anemia coincident with subtle perturbation particularly of bone marrow erythropoiesis, with EPO-induced erythropoiesis blunted in the bone marrow of KO mice but enhanced in the spleen. Cish was expressed basally in the bone marrow with induction following EPO stimulation in bone marrow and spleen. Overall, this study indicates that CISH participates in the control of both basal and EPO-induced erythropoiesis in vivo.

Identification of SH2 Domain-Containing Protein 3C as a Novel, Putative Interactor of Dipeptidyl Peptidase 3.

Dipeptidyl peptidase 3 (DPP3) is a zinc-dependent exopeptidase with broad specificity for four to eight amino acid residue substrates. It has a role in the regulation of oxidative stress response NRF2-KEAP1 pathway through the interaction with KEAP1. We have conducted stable isotope labeling by amino acids in a cell culture coupled to mass spectrometry (SILAC-MS) interactome analysis of TRex HEK293T cells using DPP3 as bait and identified SH2 Domain-Containing Protein 3C (SH2D3C) as prey. SH2D3C is one of three members of a family of proteins that contain both the SH2 domain and a domain similar to guanine nucleotide exchange factor domains of Ras family GTPases (Ras GEF-like domain), named novel SH2-containing proteins (NSP). NSPs, including SH2D3C (NSP3), are adaptor proteins involved in the regulation of adhesion, migration, tissue organization, and immune response. We have shown that SH2D3C binds to DPP3 through its C-terminal Ras GEF-like domain, detected the colocalization of the proteins in living cells, and confirmed direct interaction in the cytosol and membrane ruffles. Computational analysis also confirmed the binding of the C-terminal domain of SH2D3C to DPP3, but the exact model could not be discerned. This is the first indication that DPP3 and SH2D3C are interacting partners, and further studies to elucidate the physiological significance of this interaction are on the way.