Hyperglycemic Stress Induces Expression, Degradation, and Nuclear Association of Rho GDP Dissociation Inhibitor 2 (RhoGDIß) in Pancreatic ß-Cells.

Small G proteins (e.g., Rac1) play critical regulatory roles in islet ß-cell function in health (physiological insulin secretion) and in metabolic stress (cell dysfunction and demise). Multiple regulatory factors for these G proteins, such as GDP dissociation inhibitors (GDIs), have been implicated in the functional regulation of these G proteins. The current set of investigations is aimed at understanding impact of chronic hyperglycemic stress on the expression and subcellular distribution of three known isoforms of RhoGDIs (RhoGDIα, RhoGDIß, and RhoGDIγ) in insulin-secreting ß-cells. The data accrued in these studies revealed that the expression of RhoGDIß, but not RhoGDIα or RhoGDIγ, is increased in INS-1 832/13 cells, rat islets, and human islets. Hyperglycemic stress also promoted the cleavage of RhoGDIß, leading to its translocation to the nuclear compartment. We also report that RhoGDIα, but not RhoGDIγ, is associated with the nuclear compartment. However, unlike RhoGDIß, hyperglycemic conditions exerted no effects on RhoGDIα's association with nuclear fraction. Based on these observations, and our earlier findings of the translocation of Rac1 to the nuclear compartment under the duress of metabolic stress, we conclude that the RhoGDIß-Rac1 signaling module promotes signals from the cytosolic to the nucleus, culminating in accelerated ß-cell dysfunction under metabolic stress.

Molecular mechanism of regulation of RhoA GTPase by phosphorylation of RhoGDI.

Rho-specific guanine nucleotide dissociation inhibitors (RhoGDIs) play a crucial role in the regulation of Rho family GTPases. They act as negative regulators that prevent the activation of Rho GTPases by forming complexes with the inactive GDP-bound state of GTPase. Release of Rho GTPase from the RhoGDI-bound complex is necessary for Rho GTPase activation. Biochemical studies provide evidence of a "phosphorylation code," where phosphorylation of some specific residues of RhoGDI selectively releases its GTPase partner (RhoA, Rac1, Cdc42, etc.). This work attempts to understand the molecular mechanism behind this specific phosphorylation-induced reduction in binding affinity. Using several microseconds long atomistic molecular dynamics simulations of the wild-type and phosphorylated states of the RhoA-RhoGDI complex, we propose a molecular-interaction-based mechanistic model for the dissociation of the complex. Phosphorylation induces major structural changes, particularly in the positively charged polybasic region (PBR) of RhoA and the negatively charged N-terminal region of RhoGDI that contribute most to the binding affinity. Molecular mechanics Poisson-Boltzmann surface area binding energy calculations show a significant weakening of interaction on phosphorylation at the RhoA-specific site of RhoGDI. In contrast, phosphorylation at a Rac1-specific site does not affect the overall binding affinity significantly, which confirms the presence of a phosphorylation code. RhoA-specific phosphorylation leads to a reduction in the number of contacts between the PBR of RhoA and the N-terminal region of RhoGDI, which manifests a reduction of the binding affinity. Using hydrogen bond occupancy analysis and energetic perturbation network, we propose a mechanistic model for the allosteric response, i.e., long-range signal propagation from the site of phosphorylation to the PBR and buried geranylgeranyl group in the form of rearrangement and rewiring of hydrogen bonds and salt bridges. Our results highlight the crucial role of specific electrostatic interactions in manifestation of the phosphorylation code.

[The mechanism of OC-STAMP overexpression induced actin cytoskeleton remodeling in promoting epithelial-mesenchymal transition in the alveolar type â ¡ epithelial cell].

Objective: To explore the mechanism of osteoclast stimulatory transmembrane protein (OC-STAMP) overexpression on epithelial-mesenchymal transition (EMT) . Methods: In April 2021, mice alveolar type Ⅱ epithelial cells MLE-12 were divided into five groups: overexpression control group (NC group), Ocstamp overexpression group (over-Ocstamp group), Fasudil intervention group (over-Ocstamp+Fasudil group), silence control group (si-NC group), Ocstamp silence group (si-Ocstamp group). The protein expressions of OC-STAMP, epithelial marker protein-E-cadherin (E-cad), interstitial marker protein-α-smooth muscle actin (α-SMA), Ras homolog gene family member A (RhoA), Rho GDP dissociation inhibitor α (Rho GDIα), Rho-associated protein kinase (ROCK), phosphate myosin phosphatase (p-MYPT) were examined by Western blotting and Immunocytochemical staining. The filamentous actin (F-actin) was detected by Phalloidin method. t test was used to compare the relative expression of each protein between the two groups. Results: Western blotting and Immunocytochemical staining showed that compared with the NC group, the expression level of E-cad was down-regulated, while the expression levels of α-SMA, Rho GDIα, RhoA, ROCK, p-MYPT were increased, and F-actin expression was enhanced in the over-Ocstamp group. The differences were statistically significant (P<0.05). There were no significant differences in E-cad and α-SMA protein expression in si-Ocstamp group compared with si-NC group (P>0.05). Compared with over-Ocstamp group, the expression level of E-cad protein in over-Ocstamp+Fasudil group was up-regulated, the expression levels of α-SMA, Rho GDIα, RhoA, ROCK and p-MYPT protein were decreased, and F-actin expression was weakened, with statistical significance (P<0.05) . Conclusion: OC-STAMP overexpression in alveolar type Ⅱ epithelial cells may induce actin cytoskeleton remodeling through activation of Rho GDIα/RhoA/ROCK signaling pathway, thus promoting EMT.

Phosphorylation of RhoGDI1, a Rho GDP dissociation inhibitor, regulates root hair development in Arabidopsis under salt stress.

To ensure optimal growth, plants actively regulate their growth and development based on environmental changes. Among these, salt stress significantly influences growth and yield. In this study, we demonstrate that the growth of root hairs of salt-stressed Arabidopsis thaliana seedlings is regulated by the SALT OVERLY SENSITIVE 2 (SOS2)-GUANOSINE NUCLEOTIDE DIPHOSPHATE DISSOCIATION INHIBITOR 1 (RhoGDI1)-Rho GTPASE OF PLANTS 2 (ROP2) module. We show here that the kinase SOS2 is activated by salt stress and subsequently phosphorylates RhoGDI1, a root hair regulator, thereby decreasing its stability. This change in RhoGDI1 abundance resulted in a fine-tuning of polar localization of ROP2 and root hair initiation followed by polar growth, demonstrating how SOS2-regulated root hair development is critical for plant growth under salt stress. Our results reveal how a tissue-specific response to salt stress balances the relationship of salt resistance and basic growth.

The Rho guanine dissociation inhibitor α inhibits skeletal muscle Rac1 activity and insulin action.

The molecular events governing skeletal muscle glucose uptake have pharmacological potential for managing insulin resistance in conditions such as obesity, diabetes, and cancer. With no current pharmacological treatments to target skeletal muscle insulin sensitivity, there is an unmet need to identify the molecular mechanisms that control insulin sensitivity in skeletal muscle. Here, the Rho guanine dissociation inhibitor α (RhoGDIα) is identified as a point of control in the regulation of insulin sensitivity. In skeletal muscle cells, RhoGDIα interacted with, and thereby inhibited, the Rho GTPase Rac1. In response to insulin, RhoGDIα was phosphorylated at S101 and Rac1 dissociated from RhoGDIα to facilitate skeletal muscle GLUT4 translocation. Accordingly, siRNA-mediated RhoGDIα depletion increased Rac1 activity and elevated GLUT4 translocation. Consistent with RhoGDIα's inhibitory effect, rAAV-mediated RhoGDIα overexpression in mouse muscle decreased insulin-stimulated glucose uptake and was detrimental to whole-body glucose tolerance. Aligning with RhoGDIα's negative role in insulin sensitivity, RhoGDIα protein content was elevated in skeletal muscle from insulin-resistant patients with type 2 diabetes. These data identify RhoGDIα as a clinically relevant controller of skeletal muscle insulin sensitivity and whole-body glucose homeostasis, mechanistically by modulating Rac1 activity.

Molecular dynamics simulations reveal the inhibition mechanism of Cdc42 by RhoGDI1.

Cell division control protein 42 homolog (Cdc42), which controls a variety of cellular functions including rearrangements of the cell cytoskeleton, cell differentiation and proliferation, is a potential cancer therapeutic target. As an endogenous negative regulator of Cdc42, the Rho GDP dissociation inhibitor 1 (RhoGDI1) can prevent the GDP/GTP exchange of Cdc42 to maintain Cdc42 into an inactive state. To investigate the inhibition mechanism of Cdc42 through RhoGDI1 at the atomic level, we performed molecular dynamics (MD) simulations. Without RhoGDI1, Cdc42 has more flexible conformations, especially in switch regions which are vital for binding GDP/GTP and regulators. In the presence of RhoGDI1, it not only can change the intramolecular interactions of Cdc42 but also can maintain the switch regions into a closed conformation through extensive interactions with Cdc42. These results which are consistent with findings of biochemical and mutational studies provide deep structural insights into the inhibition mechanisms of Cdc42 by RhoGDI1. These findings are beneficial for the development of novel therapies targeting Cdc42-related cancers.

RhoGDIα regulates spermatogenesis through Rac1/cofilin/F-actin signaling.

Spermatogenesis is an extremely complex process, and any obstruction can cause male infertility. RhoGDIα has been identified as a risk of male sterility. In this study, we generate RhoGDIα knockout mice, and find that the males have severely low fertility. The testes from RhoGDIα-/- mice are smaller than that in WT mice. The numbers of spermatogonia and spermatocytes are decreased in RhoGDIα-/- testis. Spermatogenesis is compromised, and spermatocyte meiosis is arrested at zygotene stage in RhoGDIα-/- mice. Acrosome dysplasia is also observed in sperms of the mutant mice. At the molecular level, RhoGDIα deficiency activate the LIMK/cofilin signaling pathway, inhibiting F-actin depolymerization, impairing testis and inducing low fertility in mouse. In addition, the treatment of RhoGDIα-/- mice with Rac1 inhibitor NSC23766 alleviate testis injury and improve sperm quality by inhibiting the LIMK/cofilin/F-actin pathway during spermatogenesis. Together, these findings reveal a previously unrecognized RhoGDIα/Rac1/F-actin-dependent mechanism involved in spermatogenesis and male fertility.

A direct interaction between RhoGDIα/Tau alleviates hyperphosphorylation of Tau in Alzheimer's disease and vascular dementia.

RhoGDIα is an inhibitor of RhoGDP dissociation that involves in Aß metabolism and NFTs production in Alzheimer's disease (AD) by regulating of RhoGTP enzyme activity. Our previous research revealed that RhoGDIα, as the target of Polygala saponin (Sen), might alleviate apoptosis of the nerve cells caused by hypoxia/reoxygenation (H/R). To further clarify the role of RhoGDIα in the generation of NFTs, we explored the relationship between RhoGDIα and Tau. We found out that RhoGDIα and Tau can bind with each other and interact by using coimmunoprecipitation (Co-IP) and GST pulldown methods in vitro. This RhoGDIα-Tau partnership was further verified by using immunofluorescence colocalization and fluorescence resonance energy transfer (FRET) approaches in PC12 cells. Using the RNA interference (RNAi) technique, we found that the RhoGDIα may be involved in an upstream signaling pathway for Tau. Subsequently, in Aß25-35- and H/R-induced PC12 cells, forced expression of RhoGDIα via cDNA plasmid transfection was found to reduce the hyperphosphorylation of Tau, augment the expression of bcl-2 protein, and inhibit the expression of Bax protein (reducing the Bax/bcl-2 ratio) and the activity of caspase-3. In mouse AD and VaD models, forced expression of RhoGDIα via injection of a viral vector (pAAV-EGFP-RhoGDIα) into the lateral ventricle of the brain alleviated the pathological symptoms of AD and VaD. Finally, GST pulldown confirmed that the binding sites on RhoGDIα for Tau were located in the range of the ΔC33 fragment (aa 1-33). These results indicate that RhoGDIα is involved in the phosphorylation of Tau and apoptosis in AD and VaD. Overexpression of RhoGDIα can inhibit the generation of NFTs and delay the progress of these two types of dementia.

TGFß1 induces myofibroblast transdifferentiation via increasing Smad-mediated RhoGDI-RhoGTPase signaling.

This study serves to investigate the effects of the Smad pathway on TGFß1-mediated RhoGDI expression and its binding to RhoGTPases in myofibroblast transdifferentiation. Myofibroblast transdifferentiation was induced by TGFß1 in vitro. Cells were pretreated with different siRNAs or inhibitors. Myofibroblast transdifferentiation was detected by immunohistochemistry. Immunofluorescence was used to observe the nuclear translocation of Smad4, and PSR (Picrositius Red) staining was used to measure collagen concentration. TGFß1 induced the phosphorylation of Smad2/3 and the nuclear translocation of Smad4 in human aortic adventitial fibroblasts (HAAFs). Furthermore, TGFß1 increased the expression of RhoGDI and its binding to RhoGTPases. Nevertheless, inhibition of Smad2/3 phosphorylation decreased TGFß1-induced RhoGDI1/2 expressions and RhoGDI2-RhoGTPases interactions. These data suggested that the inhibition of Smad phosphorylation attenuates myofibroblast transdifferentiation by inhibiting TGFß1-induced RhoGDI1/2 expressions and RhoGDI-RhoGTPases signaling.

MLL regulates the actin cytoskeleton and cell migration by stabilising Rho GTPases via the expression of RhoGDI1.

Attainment of proper cell shape and the regulation of cell migration are essential processes in the development of an organism. The mixed lineage leukemia (MLL or KMT2A) protein, a histone 3 lysine 4 (H3K4) methyltransferase, plays a critical role in cell-fate decisions during skeletal development and haematopoiesis in higher vertebrates. Rho GTPases - RhoA, Rac1 and CDC42 - are small G proteins that regulate various key cellular processes, such as actin cytoskeleton formation, the maintenance of cell shape and cell migration. Here, we report that MLL regulates the homeostasis of these small Rho GTPases. Loss of MLL resulted in an abnormal cell shape and a disrupted actin cytoskeleton, which lead to diminished cell spreading and migration. MLL depletion affected the stability and activity of Rho GTPases in a SET domain-dependent manner, but these Rho GTPases were not direct transcriptional targets of MLL. Instead, MLL regulated the transcript levels of their chaperone protein RhoGDI1 (also known as ARHGDIA). Using MDA-MB-231, a triple-negative breast cancer cell line with high RhoGDI1 expression, we show that MLL depletion or inhibition by small molecules reduces tumour progression in nude mice. Our studies highlight the central regulatory role of MLL in Rho/Rac/CDC42 signalling pathways. This article has an associated First Person interview with the first author of the paper.

RhoGDI1 interacts with PHLDA2, suppresses the proliferation, migration, and invasion of trophoblast cells, and participates in the pathogenesis of preeclampsia.

Preeclampsia (PE) is a pregnancy-associated disease, which is the major cause of mortality on maternity and perinatal infants. It is hypothesized that PE is a consequence of the dysfunction of the trophoblast cells. Pleckstrin homology-like domain, family A, member 2 (PHLDA2) was shown to inhibit the proliferation, migration, and invasion of trophoblast cells in our previous studies. However, the mechanism by which PHLDA2 affects trophoblast cell function has not been clarified. In the current study, co-immunoprecipitation (Co-IP) with mass spectroscopy analysis was used to explore the proteins that interacted with PHLDA2. A total of 291 candidate proteins were found to be associated with PHLDA2. The interaction between PHLDA2 and Rho guanine nucleotide dissociation inhibitor (RhoGDI) 1 was identified by Co-IP and immunofluorescence staining. Western blot analysis indicated that overexpression of PHLDA2 resulted in upregulation of the RhoGDI1 protein levels, which were stabilized in the presence of cycloheximide. Similarly, overexpression of RhoGDI1 promoted PHLDA2 expression and its stability. Furthermore, pull-down and Co-IP results indicated that PHLDA2 repressed the activity of Rho guanosine triphosphate hydrolase family proteins by regulating RhoGDI1 expression. In addition, RhoGDI1 expression was upregulated in the placental tissues of patients with PE. The effects of the suppression of PHLDA2 expression on proliferation, migration, and invasion of trophoblast cells were partly abrogated following knockdown of RhoGDI1. Taken together, the data indicated that RhoGDI1 mediated regulation of PHLDA2 on the biological behavior of trophoblast cells and may participate in the pathophysiology of PE.

The Pseudo-Natural Product Rhonin Targets RHOGDI.

For the discovery of novel chemical matter generally endowed with bioactivity, strategies may be particularly efficient that combine previous insight about biological relevance, e.g., natural product (NP) structure, with methods that enable efficient coverage of chemical space, such as fragment-based design. We describe the de novo combination of different 5-membered NP-derived N-heteroatom fragments to structurally unprecedented "pseudo-natural products" in an efficient complexity-generating and enantioselective one-pot synthesis sequence. The pseudo-NPs inherit characteristic elements of NP structure but occupy areas of chemical space not covered by NP-derived chemotypes, and may have novel biological targets. Investigation of the pseudo-NPs in unbiased phenotypic assays and target identification led to the discovery of the first small-molecule ligand of the RHO GDP-dissociation inhibitor 1 (RHOGDI1), termed Rhonin. Rhonin inhibits the binding of the RHOGDI1 chaperone to GDP-bound RHO GTPases and alters the subcellular localization of RHO GTPases.

miR-497 promotes the migration and invasion of human medulloblast Daoy cell line through targeted regulation of ARHGDIA in vitro.

OBJECTIVES: To further investigate the effects of miR-497 on the biological behavior of human medulloblastoma cell line in vitro. METHODS: Human medulloblastoma cell lines, Daoy and D341, were used in this study, and the miR-497 expression in the cells was measured by Quantitative PCR with fluorescence. The Daoy cells were divided into the mimics group (Daoy cells treated with mimics), inhibitor group (Daoy cells treated with inhibitor), normal Daoy cells, ARHGDIA siRNA group (Daoy cells transfected with ARHGDIA siRNA), ARHGDIA control group (Daoy cells did not receive any treatment), and negative control group (normal cells transfected with ARHGDIA siRNA). The expression of miR-497 and ARHGDIA mRNA was measured by Quantitative PCR with fluorescence, while the level of ARHGDIA protein was measured by Western blot. The binding capability of ARHGDIA and miR-497 was assessed by luciferase assay, the migration of cells was assessed by wound healing assay, and the invasion of cells was assessed by Transwell assay. RESULTS: Compared to D341 cells, the miR-497 level was significantly higher in the Daoy cells (P < 0.01). The dual-luciferase reporter assay showed that miR-497 targets ARHGDIA. Transfecting the normal Daoy cells with miR-497 mimics significantly reduced the expression of ARHGDIA protein (P < 0.05), while transfecting normal Daoy cells with miR-497 inhibitor significantly increased the expression of ARHGDIA protein (P < 0.05). Consequently, the migration and invasion capability of cells increased significantly after transfection with miR-497 mimic (P < 0.05), and decreased significantly after transfection with miR-497 inhibitor (P < 0.05). In addition, the migration and invasion capabilities of the cells also increased significantly after transfection with ARHGDIA siRNA (P < 0.05). CONCLUSIONS: miR-497/ARHGDIA axis participates in the in vitro migration and invasion of human medulloblastoma cell lines.

Macrothrombocytopenia of Takenouchi-Kosaki syndrome is ameliorated by CDC42 specific- and lipidation inhibitors in MEG-01 cells.

Macrothrombocytopenia is a common pathology of missense mutations in genes regulating actin dynamics. Takenouchi-Kosaki syndrome (TKS) harboring the c.191A > G, Tyr64Cys (Y64C) variant in Cdc42 exhibits a variety of clinical manifestations, including immunological and hematological anomalies. In the present study, we investigated the functional abnormalities of the Y64C mutant in HEK293 cells and elucidated the mechanism of macrothrombocytopenia, one of the symptoms of TKS patients, by monitoring the production of platelet-like particles (PLP) using MEG-01 cells. We found that the Y64C mutant was concentrated at the membrane compartment due to impaired binding to Rho-GDI and more active than the wild-type. The Y64C mutant also had lower association with its effectors Pak1/2 and N-WASP. Y64C mutant-expressing MEG-01 cells demonstrated short cytoplasmic protrusions with aberrant F-actin and microtubules, and reduced PLP production. This suggested that the Y64C mutant facilitates its activity and membrane localization, resulting in impaired F-actin dynamics for proplatelet extension, which is necessary for platelet production. Furthermore, such dysfunction was ameliorated by either suppression of Cdc42 activity or prenylation using chemical inhibitors. Our study may lead to pharmacological treatments for TKS patients.

The RHO Family GTPases: Mechanisms of Regulation and Signaling.

Much progress has been made toward deciphering RHO GTPase functions, and many studies have convincingly demonstrated that altered signal transduction through RHO GTPases is a recurring theme in the progression of human malignancies. It seems that 20 canonical RHO GTPases are likely regulated by three GDIs, 85 GEFs, and 66 GAPs, and eventually interact with >70 downstream effectors. A recurring theme is the challenge in understanding the molecular determinants of the specificity of these four classes of interacting proteins that, irrespective of their functions, bind to common sites on the surface of RHO GTPases. Identified and structurally verified hotspots as functional determinants specific to RHO GTPase regulation by GDIs, GEFs, and GAPs as well as signaling through effectors are presented, and challenges and future perspectives are discussed.

Downregulation of miR­483­5p inhibits TGF­ß1­induced EMT by targeting RhoGDI1 in pulmonary fibrosis.

Transforming growth factor­ß1 (TGF­ß1)­induced epithelial­mesenchymal transition (EMT) serves a significant role in pulmonary fibrosis (PF). Increasing evidence indicates that microRNAs (miRNAs or miRs) contribute to PF pathogenesis via EMT regulation. However, the role of miR­483­5p in PF remains unclear. Therefore, the present study investigated the potential effect of miR­483­5p on TGF­ß1­induced EMT in PF. It was found that the expression of miR­483­5p was upregulated in both PF tissue and A549 cells treated with TGF­ß1, whereas expression of Rho GDP dissociation inhibitor 1 (RhoGDI1) was downregulated. miR­483­5p mimic transfection promoted TGF­ß1­induced EMT; by contrast, miR­483­5p inhibitor inhibited TGF­ß1­induced EMT. Also, miR­483­5p mimic decreased RhoGDI1 expression, whereas miR­483­5p inhibitor increased RhoGDI1 expression. Furthermore, dual­luciferase reporter gene assay indicated that miR­483­5p directly regulated RhoGDI1. Moreover, RhoGDI1 knockdown eliminated the inhibitory effect of the miR­483­5p inhibitor on TGF­ß1­induced EMT via the Rac family small GTPase (Rac)1/PI3K/AKT pathway. In conclusion, these data indicated that miR­483­5p inhibition ameliorated TGF­ß1­induced EMT by targeting RhoGDI1 via the Rac1/PI3K/Akt signaling pathway in PF, suggesting a potential role of miR­483­5p in the prevention and treatment of PF.

A Complete Survey of RhoGDI Targets Reveals Novel Interactions with Atypical Small GTPases.

There are three RhoGDIs in mammalian cells, which were initially defined as negative regulators of Rho family small GTPases. However, it is now accepted that RhoGDIs not only maintain small GTPases in their inactive GDP-bound form but also act as chaperones for small GTPases, targeting them to specific intracellular membranes and protecting them from degradation. Studies to date with RhoGDIs have usually focused on the interactions between the "typical" or "classical" small GTPases, such as the Rho, Rac, and Cdc42 subfamily members, and either the widely expressed RhoGDI-1 or the hematopoietic-specific RhoGDI-2. Less is known about the third member of the family, RhoGDI-3 and its interacting partners. RhoGDI-3 has a unique N-terminal extension and is found to localize in both the cytoplasm and the Golgi. RhoGDI-3 has been shown to target RhoB and RhoG to endomembranes. In order to facilitate a more thorough understanding of RhoGDI function, we undertook a systematic study to determine all possible Rho family small GTPases that interact with the RhoGDIs. RhoGDI-1 and RhoGDI-2 were found to have relatively restricted activity, mainly binding members of the Rho and Rac subfamilies. RhoGDI-3 displayed wider specificity, interacting with the members of Rho, Rac, and Cdc42 subfamilies but also forming complexes with "atypical" small Rho GTPases such as Wrch2/RhoV, Rnd2, Miro2, and RhoH. Levels of RhoA, RhoB, RhoC, Rac1, RhoH, and Wrch2/RhoV bound to GTP were found to decrease following coexpression with RhoGDI-3, confirming its role as a negative regulator of these small Rho GTPases.

Ang II Promotes SUMO2/3 Modification of RhoGDI1 Through Aos1 and Uba2 Subunits, and then Regulates RhoGDI1 Stability and Cell Proliferation.

PURPOSE: Ang II regulates RhoGDI1 stability and cell proliferation via SUMOylation. However, how Ang II regulates RhoGDI1 SUMOylation remains unknown. In this study, we focused on revealing the effects of E1 subunits (Aos1 and Uba2) on RhoGDI1 SUMOylation in HA-VSMC proliferation. METHODS: The expressions of Aos1, Uba2, and SUMO1 were suppressed by siRNA transfection. HA-VSMCs were treated with Ang II (100 nM) for 24 h. RhoGDI1 SUMOylation and ubiquitination were checked by co-immunoprecipitation. Cell proliferation was detected by EdU assay. RESULTS: Uba2 or Aos1 suppression significantly inhibited Ang II-induced SUMO2/3 modification of RhoGDI1 and cell proliferation, while not affecting SUMO1 modification of RhoGDI1. In addition, Uba2 or Aos1 suppression promoted RhoGDI1 ubiquitination and degradation. These indicate that both Uba2 and Aos1 are necessary for SUMO2/3 modification of RhoGDI1 that participates in cell proliferation by regulating RhoGDI1 ubiquitination and stability. Moreover, SUMO1 suppression did not affect RhoGDI1 ubiquitination and degradation and cell proliferation in Ang II-induced VSMCs, suggesting that SUMO1 modification does not participate in RhoGDI1 stability and cell proliferation. CONCLUSION: This study reveals the differences between SUMO2/3 and SUMO1 modification in regulating RhoGDI1 stability and Ang II-mediated cell proliferation. Schematic summary of roles of SUMO1 and SUMO2/3 modification of RhoGDI1 in regulating RhoGDI1 stability and cell proliferation in Ang II-treated HA-VSMCs.

Comprehensive characterization of protein-protein interactions perturbed by disease mutations.

Technological and computational advances in genomics and interactomics have made it possible to identify how disease mutations perturb protein-protein interaction (PPI) networks within human cells. Here, we show that disease-associated germline variants are significantly enriched in sequences encoding PPI interfaces compared to variants identified in healthy participants from the projects 1000 Genomes and ExAC. Somatic missense mutations are also significantly enriched in PPI interfaces compared to noninterfaces in 10,861 tumor exomes. We computationally identified 470 putative oncoPPIs in a pan-cancer analysis and demonstrate that oncoPPIs are highly correlated with patient survival and drug resistance/sensitivity. We experimentally validate the network effects of 13 oncoPPIs using a systematic binary interaction assay, and also demonstrate the functional consequences of two of these on tumor cell growth. In summary, this human interactome network framework provides a powerful tool for prioritization of alleles with PPI-perturbing mutations to inform pathobiological mechanism- and genotype-based therapeutic discovery.