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Myo9a is an actin-based molecular motor with a RhoGAP domain in its C-terminal tail. It plays a role in a variety of biological processes, such as in regulating the immune response, neuron development, and cancer progression, and its deregulation can lead to the development of disease conditions. Myo9a acts mainly via its RhoGAP domain. In the current study, we used a pET32a vector with an N-terminal Trx-His6 tag to express Myo9a-RhoGAP in a soluble form. High-purity Myo9a-RhoGAP protein was obtained after two rounds of Ni2+ affinity and size-exclusion chromatography. We mixed Myo9a-RhoGAP and RhoA in equimolar ratios in the presence of 5 mM MgCl2 and 20 mM NaF to achieve a stable RhoA GTP hydrolysis transition state complex. Analytical gel filtration and SDS-PAGE were used to verify complex formation. ITC and GAP assays suggested that Myo9a-RhoGAP could bind to RhoA and accelerate RhoA GTP hydrolysis in vitro. We purified the soluble Myo9a-RhoGAP protein with GAP activity and achieved the Myo9a-RhoGAP/RhoA·GDP/MgF3− complex assembly in vitro for the first time. The data may provide novel insights into Myo9a structure and function.

Background Our study aims to explore the relationship, shared gene signature, and the underlying mechanisms that connect rheumatoid arthritis (RA) to colorectal cancer (CRC). Methods Mendelian randomization (MR) analysis was conducted to assess the causality between RA and CRC. Summary statistic data-based Mendelian randomization (SMR) leveraging eQTL data was employed to identify the CRC-related causal genes. Integrated analyses of single-cell RNA sequencing and bulk RNA sequencing were employed to comprehensively investigate the shared gene signature and potential mechanisms underlying the pathogenesis of both RA and CRC. Predictive analysis of the shared hub gene in CRC immunotherapy response was performed. Pan-cancer analyses were conducted to explore the potential role of MYO9A in 33 types of human tumors. Results MR analysis suggested that RA might be associated with a slight increased risk of CRC (Odds Ratio = 1.04, 95% Confidence Interval = 1.01–1.07, P  = 0.005). SMR analysis combining transcriptome analyses identified MYO9A as a causal gene in CRC and a shared gene signature in both RA and CRC. MYO9A may contribute to tumor suppression, while downregulation of MYO9A may impact CRC tumorigenesis by disrupting epithelial polarity and architecture, resulting in a worse prognosis in CRC. Additionally, MYO9A shows promise as a powerful predictive biomarker for cancer prognosis and immunotherapy response in CRC. Pan-cancer analyses demonstrated MYO9A may have a protective role in the occurrence and progression of various human cancers. Conclusion RA might be associated with a slight increased risk of CRC. MYO9A is a shared gene signature and a potential immune-related therapeutic target for both CRC and RA. Targeting the MYO9A-mediated loss of polarity and epithelial architecture could be a novel therapeutic approach for CRC.

The molecular pathogenesis of diabetic kidney disease progression is complex and remains unresolved. Rho-GAP MYO9A was recently identified as a novel podocyte protein and a candidate gene for monogenic FSGS. Myo9A involvement in diabetic kidney disease has been suggested. Here, we examined the effect of diabetic milieu on Myo9A expression in vivo and in vitro . We determined that Myo9A undergoes S-nitrosylation, a post-translational modification dependent on nitric oxide (NO) availability. Diabetic mice with nodular glomerulosclerosis and severe proteinuria associated with doxycycline-induced, podocyte-specific VEGF 164 gain-of-function showed markedly decreased glomerular Myo9A expression and S-nitrosylation, as compared to uninduced diabetic mice. Immortalized mouse podocytes exposed to high glucose revealed decreased Myo9A expression, assessed by qPCR, immunoblot and immunocytochemistry, and reduced Myo9A S-nitrosylation (SNO-Myo9A), assessed by proximity link assay and biotin switch test, functionally resulting in abnormal podocyte migration. These defects were abrogated by exposure to a NO donor and were not due to hyperosmolarity. Our data demonstrate that high-glucose induced decrease of both Myo9A expression and SNO-Myo9A is regulated by NO availability. We detected S-nitrosylation of Myo9A interacting proteins RhoA and actin, which was also altered by high glucose and NO dependent. RhoA activity inversely related to SNO-RhoA. Collectively, data suggest that dysregulation of SNO-Myo9A, SNO-RhoA and SNO-actin may contribute to the pathogenesis of advanced diabetic kidney disease and may be amenable to therapeutic targeting.