GTPase Signaling Research Articles

Genomic Analysis of Abnormal DNAM Methylation in Parathyroid Tumors.

Background Parathyroid tumors are common endocrine neoplasias associated with primary hyperparathyroidism. Although numerous studies have studied the subject, the predictive value of gene biomarkers nevertheless remains low. Methods In this study, we performed genomic analysis of abnormal DNA methylation in parathyroid tumors. After data preprocessing, differentially methylated genes were extracted from patients with parathyroid tumors by using t-tests. Results After refinement of the basic differential methylation, 28241 unique CpGs (634 genes) were identified to be methylated. The methylated genes were primarily involved in 7 GO terms, and the top 3 terms were associated with cyst morphogenesis, ion transport, and GTPase signal. Following pathway enrichment analyses, a total of 10 significant pathways were enriched; notably, the top 3 pathways were cholinergic synapses, glutamatergic synapses, and oxytocin signaling pathways. Based on PPIN and ego-net analysis, 67 ego genes were found which could completely separate the diseased group from the normal group. The 10 most prominent genes included POLA1, FAM155 B, AMMECR1, THOC2, CCND1, CLDN11, IDS, TST, RBPJ, and GNA11. SVM analysis confirmed that this grouping approach was precise. Conclusions This research provides useful data to further explore novel genes and pathways as therapeutic targets for parathyroid tumors.

真核细胞运动接触抑制行为的力学-化学耦合模拟

Contact inhibition of locomotion (CIL) drives various biological phenomena, including cell dispersion, collective cell migration, and cancer invasion. Based on our previously proposed cell migration model with multilayered signaling cascades, a mechanical-chemical coupling model for CIL was developed by further incorporating cell-cell contact-dependent signaling pathways to guide the cytoskeleton remodeling. The cell structure model incorporates a discrete actin filament network within a cycle. The entire mathematical model is composed of nonlinear diffusion-reaction equations coupled with force-balance equations and solved numerically by the in-house built LBP-D1Q3 method. Numerical simulations indicated that the manifestation of distinct CIL behavior depends on the cooperation between FilGAP-FLNa and N-cadherin signaling pathways in terms of Rho GTPase signaling. After achieving a suitable inhibitory effect of FilGAP, the cells retain moderate polarity, which can be reversed in response to N-cadherin signaling, leading to normal CIL behavior. The lack of FilGAP transforms the cells to a fully polarized state, which is difficult to reverse, thus leading to the “bypass” behavior. The lack of N-cadherin signaling prevents cells from repolarizing in response to cell-cell contact, leading to static cell-cell adhesion. These simulations provide a theoretical basis for understanding the effect of contact inhibition signaling on cell migration.

Autism-associated chromatin remodeler CHD8 regulates erythroblast cytokinesis and fine-tunes the balance of Rho GTPase signaling.

SUMMARYCHD8 is an ATP-dependent chromatin-remodeling factor whose monoallelic mutation defines a subtype of autism spectrum disorders (ASDs). Previous work found that CHD8 is required for the maintenance of hematopoiesis by integrating ATM-P53-mediated survival of hematopoietic stem/progenitor cells (HSPCs). Here, by using Chd8F/FMx1-Cre combined with a Trp53F/F mouse model that suppresses apoptosis of Chd8−/− HSPCs, we identify CHD8 as an essential regulator of erythroid differentiation. Chd8−/− P53−/− mice exhibited severe anemia conforming to congenital dyserythropoietic anemia (CDA) phenotypes. Loss of CHD8 leads to drastically decreased numbers of orthochromatic erythroblasts and increased binucleated and multinucleated basophilic erythroblasts with a cytokinesis failure in erythroblasts. CHD8 binds directly to the gene bodies of multiple Rho GTPase signaling genes in erythroblasts, and loss of CHD8 results in their dysregulated expression, leading to decreased RhoA and increased Rac1 and Cdc42 activities. Our study shows that autism-associated CHD8 is essential for erythroblast cytokinesis.

Abstract 2430: The role of cellular iron and the homeostatic iron regulator (HFE) in high-grade brain tumor cell migration

Abstract High-grade brain tumors such as grade III astrocytoma and glioblastoma represent among the most difficult to manage malignancies facing oncological practice. Diffuse migration and invasion into adjacent healthy brain tissue yields complete surgical resection of these tumors unfeasible. As a result, despite receiving maximal surgical resection and an aggressive course of chemoradiation, the majority of high-grade brain tumor patients suffer from recurrent disease. Increased expression levels of the homeostatic iron regulator gene (HFE) in high-grade brain tumors have been correlated with poorer outcomes. HFE is known to influence cellular iron metabolism by inhibiting transferrin-mediated iron uptake yet little is known regarding how HFE or iron impact the migratory capabilities of high-grade brain tumor cells. In order to better understand how HFE expression and cellular iron metabolism influence cell migration in high grade brain tumors, we utilized brain tumor cell lines that had been genetically manipulated to express different levels of HFE. We observed that knocking down HFE in KR158 or LN229 glioma cell lines resulted in significantly decreased migratory capacity. Since HFE is known to inhibit transferrin mediated iron uptake, we studied how directly modulating the iron status of glioma cells impacted their ability to migrate. Treatment of a panel of glioma cell lines: LN229, T98G, U87, KR158, with iron in the form of hemin or ferric ammonium citrate resulted in significantly reduced migration. Furthermore, the iron-induced reduction in migration could be rescued by the addition of deferoxamine, an iron chelator. Cell viability in response to the iron treatments was assayed and found to not be significantly altered - suggesting that cellular iron status was influencing migratory capacity independent of cell viability. To gain mechanistic insights into HFE and iron-induced effects on cell migration, we analyzed the Chinese Glioma Genome Atlas (CGGA) for correlations between HFE and the Rho GTPases RHOA, RAC1, and CDC42 – genes which are known to play a crucial role in determining the migratory capacity of cancer cells. Interestingly, we found statistically significant correlations between the Rho GTPases RHOA, RAC1, CDC42 and HFE in both grade III astrocytoma and glioblastoma patient cohorts. Immunoblotting of iron treated glioma cell lines demonstrated that expression of RhoA and Cdc42 was reduced suggesting that alterations in Rho GTPase expression and signaling may play a role in iron-induced effects on cell migration. Our results demonstrate that targeting cancer cell iron metabolism as an addition to existing treatment regimens may be a promising avenue for further investigation. Citation Format: Ganesh Shenoy, Katie Troike, Kondaiah Palsa, Madison Kuhn, Quinn Wade, Becky Slagle-Webb, Amanda Snyder, Chachrit Khunsriraksakul, Justin D. Lathia, Dhimant Desai, Hong-Gang Wang, Elizabeth Proctor, James R. Connor. The role of cellular iron and the homeostatic iron regulator (HFE) in high-grade brain tumor cell migration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2430.

Abstract 5971: DEPDC1B promotes melanoma angiogenesis and metastasis through sequestration of ubiquitin ligase CDC16 to stabilize secreted SCUBE3

Abstract The ability of melanoma to acquire metastasis through the induction of angiogenesis is one of the major causes of skin cancer death. Here, we find that high transcript levels of DEPDC1B in cutaneous melanomas are significantly associated with a poor prognosis. Tissue microarray analysis indicates DEPDC1B expression is positively correlated with SOX10 in the different stages of melanoma. Consistently, DEPDC1B is both required and sufficient for melanoma growth, metastasis, angiogenesis, and functions as a direct downstream target of SOX10 to partly mediate its oncogenic activity. In contrast to other tumor types, the DEPDC1B-mediated enhancement of melanoma metastatic potential is not dependent on the activities of RHO GTPase signaling and canonical Wnt signaling, but is acquired through secretion of SCUBE3, which is crucial for promoting angiogenesis in vitro and in vivo. Mechanistically, DEPDC1B regulates SCUBE3 protein stability through the competitive association with ubiquitin ligase CDC16 to prevent SCUBE3 from undergoing degradation via the ubiquitin-proteasome pathway. Importantly, expression of SOX10, DEPDC1B, and SCUBE3 are positively correlated with microvessel density in the advanced stage of melanomas. In conclusion, we reveal a SOX10-DEPDC1B-SCUBE3 regulatory axis promotes melanoma angiogenesis and metastasis, which suggests targeting secreted SCUBE3 can be a therapeutic strategy against metastatic melanoma. Citation Format: Feng Hu, Ki On Fong, May P.L Cheung, Jessica A.J Liu, Rui Liang, Tsz Wai Li, Rakesh Sharma, Philip P.C Ip, Xintao Yang, Martin Cheung. DEPDC1B promotes melanoma angiogenesis and metastasis through sequestration of ubiquitin ligase CDC16 to stabilize secreted SCUBE3 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5971.

Abstract 2013: Integrated proteomic analysis identifies four distinct subtypes of high-risk neuroblastoma

Abstract Neuroblastoma is the most common solid extracranial tumor in children. Despite intensive multi-modal therapy, 5-year survival rates for patients with high-risk disease remain at approximately 50%. Neuroblastomas typically have low mutational burden and harbor few recurrent mutations making the identification of new therapeutic targets challenging. To characterize the proteomic landscape of high-risk neuroblastoma, we performed liquid chromatography-mass spectrometry-based deep expression proteomics and phosphoproteomics on 30 neuroblastoma tumors from 26 patients with high-risk disease. Our integrated analysis identified four distinct proteomic subgroups of high-risk neuroblastoma that were not otherwise apparent based on clinical or genomic features. The four subgroups were named based on their defining proteomic signature: C1-Mixed, C2-Neuronal, C3-Functional MYCN and C4-Stromal. Only one third of C3-Functional MYCN tumors had amplification of MYCN. Segmental chromosomal losses and gains were enriched in, but not exclusive to, C1-Mixed and C3-Functional MYCN tumors. The activities of multiple kinases including CDK2, CDK7 and MEK2 differed significantly between subgroups. C3-Functional MYCN and C4-Stromal tumors were enriched for immune and stromal cells, respectively. Focal adhesion signaling was specifically upregulated in C4-Stromal tumors suggesting increased extracellular matrix interactions in this tumor subgroup. C2-Neuronal tumors were enriched for axon guidance and neurotrophin signaling pathways. Rho family GTPase signaling was also evident in multiple tumor subgroups. C1-Mixed and C3-Functional MYCN tumors had elevated expression of RNA processing proteins which was associated with increased alternative splicing. Splicing analysis also identified multiple novel protein coding splice events that were shared amongst multiple neuroblastoma tumors and outliers compared to both the Genotype-Tissue Expression (GTEx) dataset and a panel of commercial reference tissues. Protein domain analysis of these novel splice variants suggested that these novel protein isoforms may have aberrant functions that contribute to tumorigenesis. In conclusion, phosphoproteomic analysis can identify candidate pathways for the development of new therapies for patients with high-risk neuroblastoma. Citation Format: Kristin L. Leskoske, Sara A. Byron, Seema Plaisier, Apurva M. Hegde, Krystine Garcia-Mansfield, Ritin Sharma, Genevieve Bergendahl, Abhinav Nagulapally, William Ferguson, Jaqueline Kraveka, Javier Oesterheld, William P. Hendricks, Giselle L. Saulnier Sholler, Jeffrey M. Trent, Patrick Pirrotte. Integrated proteomic analysis identifies four distinct subtypes of high-risk neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2013.

P-188 Transgelin/PARP1 regulates colorectal cancer metastasis through Rho GTPase pathway

Our previous studies showed that transgelin may promoted the migration of HT29 and HCT116 colon cancer cells through nuclear translocation. Proteomics analysis demonstrated that transgelin interacted with 297 proteins, while the gene expression profile showed that TAGLN affected the expression of 256 downstream genes. Bioinformatics analysis identified 7 key genes, the Rho GTPase signaling pathway as the key pathway, and suggested that PARP1 may participate in the regulation of the key genes with transgelin. This study aims to verify the interaction between PARP1 and transgelin, and explore the mechanism of transgelin/PARP1 in regulating the metastasis of colorectal cancer. Co-immunoprecipitation and immunoblotting were used to verify the interaction between PARP1 and transgelin. The expression of PARP1 in colorectal cancer and non-cancerous tissues were analyzed with the mRNA data from GEO microarrays and TCGA database, and were also examined with a tissue microarray by immunohistochemistry. Kaplan-Meier Analysis was performed to analyze the influence of PARP1 expression on overall survival. We then explored the nuclear localization of transgelin in siRNA-mediated knockdown of PARP1 in colon cancer cells by immunoblotting and immunofluorescence. The proliferation, migration and invasion of PARP1-knockdown SW480 and RKO cells were detected by CCK8, wound healing and transwell invasion assays. An expression plasmid containing RAC1 which was one of the key genes identified from the TAGLN-overexpression cDNA microarray analysis, and also a key member of the Rho GTPase family was constructed. This plasmid was co-transfected with PARP1-siRNA into SW480 and RKO cells to replenish the expression of RAC1. The proliferation, migration and invasion assays of the co-transfected colon cancer cells were also performed. Immunoprecipitation and immunoblotting showed that PARP1 interacted with transgelin in HCT116 cells. The expression of PARP1 in colon cancer was increased as compared to the non-tumor samples p < 0.05). The expression of PARP1 was correlated with disease-free survival (p < 0.05) and overall survival p < 0.05. Knockdown of PARP1 reduced the nuclear transgelin in RKO and SW480 cells, and simultaneously decreased the proliferation migration and invasion of colon cancer cells, respectively. Overexpression of TAGLN led to increased expression of RAC1, while knockdown of PARP1 resulted in reduced expression of RAC1 in colon cancer cells. While the PARP1-knockdown cells were re-introduced with RAC1 expression, their proliferation migration and invasion ability were re-gained as compared to the control groups. Transgelin interacted with PARP1 in HCT116 cells. PARP1 was an adverse prognostic marker in colorectal cancer and participated in the nuclear translocation of transgelin. Transgelin / PARP1 may regulate the metastasis of colorectal cancer through RAC1 of the Rho GTPase signaling pathway.

Nuclear Vav3 is required for polycomb repression complex-1 activity in B-cell lymphoblastic leukemogenesis

Acute B-cell lymphoblastic leukemia (B-ALL) results from oligo-clonal evolution of B-cell progenitors endowed with initiating and propagating leukemia properties. The activation of both the Rac guanine nucleotide exchange factor (Rac GEF) Vav3 and Rac GTPases is required for leukemogenesis mediated by the oncogenic fusion protein BCR-ABL. Vav3 expression becomes predominantly nuclear upon expression of BCR-ABL signature. In the nucleus, Vav3 interacts with BCR-ABL, Rac, and the polycomb repression complex (PRC) proteins Bmi1, Ring1b and Ezh2. The GEF activity of Vav3 is required for the proliferation, Bmi1-dependent B-cell progenitor self-renewal, nuclear Rac activation, protein interaction with Bmi1, mono-ubiquitination of H2A(K119) (H2AK119Ub) and repression of PRC-1 (PRC1) downstream target loci, of leukemic B-cell progenitors. Vav3 deficiency results in de-repression of negative regulators of cell proliferation and repression of oncogenic transcriptional factors. Mechanistically, we show that Vav3 prevents the Phlpp2-sensitive and Akt (S473)-dependent phosphorylation of Bmi1 on the regulatory residue S314 that, in turn, promotes the transcriptional factor reprogramming of leukemic B-cell progenitors. These results highlight the importance of non-canonical nuclear Rho GTPase signaling in leukemogenesis.

Strategies for Targeting KRAS: A Challenging Drug Target.

In the developed world, cancer is the most common cause of death. Among the 36 human genes of the RAS family, KRAS, NRAS, and HRAS play a prominent role in human cancer. KRAS belongs to the Ras superfamily of proteins and is a small GTPase signal transduction protein. Among the RAS isoform, KRAS is the dominant mutant that induces approximately 86% of the RAS mutations. The most frequently mutated KRAS isoform is KRAS4B. About 90% of pancreatic cancer, 30-40% of colon cancer, and 15 to 20% of lung cancers are caused by mutations KRAS4B isoform. Liver cancer, bladder cancer, breast cancer, and myeloid leukaemia are also caused by mutations in KRAS but are rare. The FDA has recently approved sotorasib for the treatement of KRASG12C-mutated advanced non-small cell lung cancer (NSCLC) patients. However, no FDAapproved drugs are available for other KRAS-driven cancer. As the KRAS proteins lack a druggable pocket accessible to the chemical inhibitors, the cancer-causing mutant proteins are almost identical to their essential wild-type counterparts. Therefore, they are considered undruggable. The new insights into the structure and function of RAS have changed this understanding and encouraged the development of many drug candidates. This review provides information about the different strategies for targeting KRAS, a challenging drug target that might be valuable for the scientific community.

Quantitative phosphoproteomics reveals diverse stimuli activate distinct signaling pathways during neutrophil activation

Neutrophils display functional heterogeneity upon responding diversely to physiological and pathological stimulations. During type 2 diabetes (T2D), hyperglycemia constitutively activates neutrophils, leading to reduced response to infections and on the other hand, elevated metabolic intermediates such as homocysteine induce bidirectional activation of platelets and neutrophils leading to thrombosis. Hence, in the context of T2D-associated complications, we examined the influence of high glucose, homocysteine, and LPS representing effector molecules of hyperglycemia, thrombosis, and infection, respectively, on human neutrophil activation to identify distinct signaling pathways by quantitative phosphoproteomics approach. High glucose activated C-Jun-N-Terminal Kinase, NTRK1, SYK, and PRKACA kinases associated with Rho GTPase signaling and phagocytosis, whereas LPS induced AKT1, SRPK2, CSNK2A1, and TTN kinases involved in cytokine signaling and inflammatory response. Homocysteine treatment led to activatation of LRRK2, FGR, MAPK3, and PRKCD kinases which are associated with neutrophil degranulation and cytoskeletal remodeling. Diverse inducers differentially modulated phosphorylation of proteins associated with neutrophil functions such as oxidative burst, degranulation, extracellular traps, and phagocytosis. Further validation of phosphoproteomics data on selected kinases revealed neutrophils pre-cultured under high glucose showed impeded response to LPS to phosphorylate p-ERK1/2Thr202/Tyr204, p-AKTSer473, and C-Jun-N-Terminal KinaseSer63 kinases. Our study provides novel phosphoproteome signatures that may be explored to understand neutrophil biology in T2D-associated complications.

Identification of LINC00654-NINL Regulatory Axis in Diffuse Large B-Cell Lymphoma In Silico Analysis.

BackgroundThe long non-coding RNA (lncRNA)-mRNA regulation network plays an important role in the development of diffuse large B-cell lymphoma (DLBCL). This study uses bioinformatics to find an innovative regulation axis in DLBCL that will provide a positive reference for defining the mechanism of disease progression.MethodsBatch Cox regression was used to screen prognosis-related lncRNAs, and a random forest model was used to identify hub lncRNA. The clinical value of the lncRNA was evaluated and Spearman correlation analysis was used to predict the candidate target genes. Gene Oncology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment were used to define the biological function of the lncRNA. A batch Cox regression model, expression validation, and Spearman correlation analysis were used to select the best downstream target genes. The expression and prognostic value validation of this gene was conducted using public data. Gene Set Enrichment Analysis (GSEA) was performed to explore potential mechanisms for this gene in DLBCL.ResultsLINC00654 was identified as the hub lncRNA and 1443 mRNAs were selected as downstream target genes of the lncRNA. The target genes were enriched in the regulation of GTPase and Notch signaling pathways. After validation, the ninein-like (NINL) gene was selected as the potential target of LINC00654 and the LINC00654-NINL axis was constructed. Patients with better responses to therapy were shown to have high NINL gene expression (p-value = 0.036). NINL also had high expression in the DB cell line and low expression in the OCILY3 cell line. Survival analysis showed that high NINL expression was a risk factor for overall survival (OS) and disease-specific survival (DSS) within older patients and those with advanced-stage cancer. GSEA results showed that NINL may be involved in neutrophil-mediated immunity and NF-κB signaling.ConclusionThis study identified a novel LncRNA00654-NINL regulatory axis in DLBCL, which could provide a favorable reference for exploring the possible mechanisms of disease progression.

Structural and biophysical properties of farnesylated KRas interacting with the chaperone SmgGDS-558

KRas is a small GTPase and membrane-bound signaling protein. Newly synthesized KRas is post-translationally modified with a membrane-anchoring prenyl group. KRas chaperones are therapeutic targets in cancer due to their participation in trafficking oncogenic KRas to membranes. SmgGDS splice variants are chaperones for small GTPases with basic residues in their hypervariable domain (HVR), including KRas. SmgGDS-607 escorts pre-prenylated small GTPases, while SmgGDS-558 escorts prenylated small GTPases. We provide a structural description of farnesylated and fully processed KRas (KRas-FMe) in complex with SmgGDS-558 and define biophysical properties of this interaction. Surface plasmon resonance measurements on biomimetic model membranes quantified the thermodynamics of the interaction of SmgGDS with KRas, and small-angle x-ray scattering was used to characterize complexes of SmgGDS-558 and KRas-FMe structurally. Structural models were refined using Monte Carlo and molecular dynamics simulations. Our results indicate that SmgGDS-558 interacts with the HVR and the farnesylated C-terminus of KRas-FMe, but not its G-domain. Therefore, SmgGDS-558 interacts differently with prenylated KRas than prenylated RhoA, whose G-domain was found in close contact with SmgGDS-558 in a recent crystal structure. Using immunoprecipitation assays, we show that SmgGDS-558 binds the GTP-bound, GDP-bound, and nucleotide-free forms of farnesylated and fully processed KRas in cells, consistent with SmgGDS-558 not engaging the G-domain of KRas. We found that the dissociation constant, Kd, for KRas-FMe binding to SmgGDS-558 is comparable with that for the KRas complex with PDEδ, a well-characterized KRas chaperone that also does not interact with the KRas G-domain. These results suggest that KRas interacts in similar ways with the two chaperones SmgGDS-558 and PDEδ. Therapeutic targeting of the SmgGDS-558/KRas complex might prove as useful as targeting the PDEδ/KRas complex in KRas-driven cancers.

Control of Fibrotic Gene Expression in Cardiac Myocytes and Fibroblasts via the Rap1 GTPase

Fibrosis describes the excessive deposition of extracellular matrix components by cardiac myofibroblasts (CFs), a process which frequently promotes the adverse effects of pathological conditions damaging cardiac muscle, such as myocardial infarction and arrhythmogenic cardiomyopathy. Roughly 1 million Americans suffer a myocardial infarction each year, after which necrotic cardiac myocytes (CMs) are replaced by CF‐derived extracellular matrix (ECM), in response to (a) biochemical signaling cues from necrotic CMs and (b) mechanical cues from the contracting myocardium and ventricle cavity pressure. Accumulation of ECM proteins (such as collagen) within the infarct scar is initially critical to prevent further tissue damage, but chronic and excessive collagen deposition is detrimental to regeneration of CMs and healing of the myocardium. While the physiological outcomes of cardiac fibrosis are well studied, the biochemical and mechano‐responsive signaling mechanisms which regulate fibrotic gene expression remain poorly investigated. The RhoA GTPase signaling pathway has been shown to play a central role in regulating fibrotic gene expression and is activated via many different pro‐fibrotic signaling factors and biomechanical stimuli (i.e., changes in ECM composition, organization and rigidity). The GTPase Rap1 is also very well‐known to mediate both inside‐out and outside‐in signaling via attachment of integrins to ECM proteins. In certain contexts, Rap1 has also been shown to antagonize RhoA signaling, but very little is known about its role in fibrotic gene expression. We therefore hypothesized that expression of Rap1 would inhibit fibrotic gene expression in cardiac myocytes and fibroblasts. Expression of either wildtype (WT) or constitutively active (63E) GFP‐tagged Rap1A constructs in cardiac fibroblasts resulted in a dramatic decrease in mRNA levels of ACTA2 (smooth muscle alpha‐actin), a commonly used marker of myofibroblast activation. Further, expression of WT or 63E Rap1A in either cardiac myocytes or fibroblasts resulted in a significant decrease in mRNA levels of ECM proteins (such as COL1A1, COL3A1, COL4A1 and FN1), as well as other common markers of fibrotic gene expression (CTGF and EGR1). Taken together, these data indicate that Rap1 signaling inhibits fibrotic gene expression and myofibroblast activation. Interestingly, expression of WT or 63E Rap1A in cardiac fibroblasts caused a dramatic increase in inflammatory markers (such as TNF, IL6, NFKB1A) and matrix remodeling enzymes (MMP3 and MMP9), genes which have been shown to play a role in increasing collagen turnover, reducing ECM rigidity/stiffness and repair of damaged myocardial tissue. Altogether, these data point to a significant role for Rap1 GTPase in reducing fibrotic gene expression and myofibroblast activation and suggest possible new therapeutic strategies to combat the damaging effects of cardiac fibrosis.

Transcriptional regulation of Rab21 GTPase in leukemia

Rab GTPase family proteins have critical roles in membrane trafficking. Rab21 is a member of the Ras oncogene family, whose dysregulation has been associated with drug resistance in cancer. Regulation of Rab21 expression is still unknown. Here we present data that RAB21 expression is regulated by Casein Kinase II (CK2) and by the Ikaros protein in B‐cell acute lymphoblastic leukemia (B‐ALL). Ikaros is a DNA‐binding protein, encoded by the Ikzf1 gene, which acts as a tumor suppressor in leukemia. Analysis of global DNA binding showed increased occupancy of Ikaros at promoter of the Rab21 gene in several B‐ALL cell lines and primary B‐ALL cells. The role of Ikaros in transcriptional regulation of RAB21 expression in B‐ALL was determined by gain‐of‐function and loss‐of‐function experiments. Overexpression of Ikaros in B‐ALL via retroviral transduction resulted in increased Ikaros binding to the RAB21 promoter and decreased RAB21 transcription. In contrast, shRNA‐mediated knockdown of Ikaros inhibited Ikaros binding to the RAB21 promoter and resulted in an increased RAB21 expression. These data suggest that Ikaros directly repress RAB21 transcription. Our previous studies showed that CK2 directly phosphorylates Ikaros and reduces its DNA‐binding affinity. We tested whether CK2 affects Ikaros’ ability to regulate RAB21 expression in B‐ALL. Treatment of B‐ALL cells with a specific CK2 inhibitors, CX‐4945, resulted in increased Ikaros binding at the RAB21 promoter and reduced RAB21 expression. The use of luciferase reporter assay showed that both wildtype Ikaros and Ikaros phosphoresistant mutants strongly repress Rab21 transcription, while phopshomimetic Ikaros mutants at CK2 phosphorylation sites lost the ability to repress RAB21 transcription. Together, these data demonstrate that Ikaros and CK2 regulate transcription and overall expression of RAB21 in B‐cell ALL. Results uncovered an interplay between CK2 and Rab GTPase signaling pathways and suggest a mechanism by which CK2 regulates Rab21 activity in leukemia.

The role of cytoskeleton regulators in embryonic facial morphogenesis

Small GTPases are downstream of the non‐canonical WNT JNK‐PCP pathway. The most well studied members of this group are RhoA, Rac1 and CDC42. These small GTPases regulate cell shape, orientation, and migration through regulating the actomyosin cytoskeleton. In addition, components of this pathway have been linked to non‐syndromic cleft lip with or without cleft palate. The objective of this study is to test whether small GTPase signaling is required and sufficient for facial morphogenesis at stages where lip fusion is taking place.MethodsThe faces of E4.5 chicken embryos were dissected to include the two facial processes involved in lip fusion, the frontonasal and maxillary processes (Fig. 1). After a dose‐response experiment to find the optimal concentrations, the organ cultures were treated with either an antagonist of RhoA signaling (ROCKi 20µM) or an antagonist of Rac1 (Rac1i 100µM) or an agonist (RhoA/ Rac1/CDC42 activator 0.5µg/ml). Organ cultures were photographed and measured at 0 and 24hrs. Percentage growth differences were analyzed using Prism Graphpad software (v. 9.2).ResultsIn the control cultures, frontonasal mass width decreased by 23% and increased in cranio‐caudal height by 17% over 24‐hour period. The nasal slits did not change. The maxillary prominences increased in height and width by 12% and 37% respectively over 24‐hour period similar to growth in vivo. The ROCKi treated cultures prevented the narrowing of the frontonasal mass and increased the length of the nasal slits. ROCKi treatment had no effect on the height of the frontonasal mass nor maxillary prominences. In contrast, the RAC1i had no effect on facial growth. The agonist significantly reduced frontonasal mass width compared to ROCKi. In addition, the agonist significantly decreased growth of the maxillary prominences.ConclusionsThe changes in facial morphogenesis may be explained by active cytoskeletal remodeling, particularly in the RhoA pathway. We excluded a requirement for RAC1 in facial morphogenesis in our system. Our data also suggest that different regions of the face have different requirements for specific small GTPase signaling. The frontonasal mass is dependent on RhoA signaling but not the maxillary prominences. Over activation of all GTPases shows that correct levels are necessary for normal morphogenesis. Overall, we have revealed a complex interplay between small GTPAses in midfacial morphogenesis.

Biochemical Mechanisms that Control the Effects of RhoA Small GTPase Signaling on Synaptic Stability and Cognition

Dendritic spines are the site of most excitatory connections in the brain. Small GTPases act as binary switches that link cell surface receptors to persistent modifications in the morphogenesis of dendritic spines. Small GTPases are active when bound to GTP and inactive when GDP bound. The direct activation of small GTPases is accomplished by guanine nucleotide exchange factors (GEFs). The small GTPase RhoA, is enriched in dendritic spines and its activation can destabilize the actin cytoskeleton within spines reducing dendritic spine stability. Rodent studies suggest that excessive RhoA signaling causes cognitive impairment in part via its ability to reduce dendritic spine density in forebrain neurons. Despite the broad therapeutic potential of targeting the RhoA pathway as a means of enhancing cognition, the specific biochemical mechanisms that control its activity in neurons remain largely unknown. PDZ‐RhoGEF is a RhoA‐specific guanine nucleotide exchange factor that shows an unusually potent ability to activate RhoA. We show that PDZ‐RhoGEF destabilizes dendritic spines in cortical neurons and causes cortical‐dependent cognitive impairment. Ongoing studies are extending this analysis to determine if PDZ‐RhoGEF is similarly important for activity‐mediated remodeling of dendritic spines after NMDA receptor activation. Despite the importance of PDZ‐RhoGEF to synaptic and cognitive phenotypes, owing to a lack of studies, minimal is known about the biochemical mechanisms that regulate PDZ‐RhoGEF. Our recent findings indicate that PDZ‐RhoGEF activity and cellular localization is controlled by its interaction with the DISC1 scaffolding‐like protein, such that DISC1 helps localize PDZ‐RhoGEF to cellular membranes yet inhibits PDZ‐RhoGEF’s ability to activate RhoA. Experiments utilizing structured illumination microscopy are being conducted to further assess the functional consequences of the interaction between PDZ‐RhoGEF and DISC1 in neurons, and in particular, within dendritic spines. We theorize that the knockdown of DISC1 in neurons will result in the improper synaptic localization of PDZ‐RhoGEF with a concomitant increase in PDZ‐RhoGEF‐mediated RhoA activity. Collectively, these findings identify PDZ‐RhoGEF as a potent regulator of synaptic stability and cognition and highlight a previously unrecognized involvement for DISC1 in controlling RhoA activity via PDZ‐RhoGEF.

The Giardia ventrolateral flange is a lamellar membrane protrusion that supports attachment.

Attachment to the intestinal epithelium is critical to the lifestyle of the ubiquitous parasite Giardia lamblia. The ventrolateral flange is a sheet-like membrane protrusion at the interface between parasites and attached surfaces. This structure has been implicated in attachment, but its role has been poorly defined. Here, we identified a novel actin associated protein with putative WH2-like actin binding domains we named Flangin. Flangin complexes with Giardia actin (GlActin) and is enriched in the ventrolateral flange making it a valuable marker for studying the flanges’ role in Giardia biology. Live imaging revealed that the flange grows to around 1 μm in width after cytokinesis, then remains uniform in size during interphase, grows in mitosis, and is resorbed during cytokinesis. A flangin truncation mutant stabilizes the flange and blocks cytokinesis, indicating that flange disassembly is necessary for rapid myosin-independent cytokinesis in Giardia. Rho family GTPases are important regulators of membrane protrusions and GlRac, the sole Rho family GTPase in Giardia, was localized to the flange. Knockdown of Flangin, GlActin, and GlRac result in flange formation defects. This indicates a conserved role for GlRac and GlActin in forming membrane protrusions, despite the absence of canonical actin binding proteins that link Rho GTPase signaling to lamellipodia formation. Flangin-depleted parasites had reduced surface contact and when challenged with fluid shear force in flow chambers they had a reduced ability to remain attached, confirming a role for the flange in attachment. This secondary attachment mechanism complements the microtubule based adhesive ventral disc, a feature that may be particularly important during mitosis when the parental ventral disc disassembles in preparation for cytokinesis. This work supports the emerging view that Giardia’s unconventional actin cytoskeleton has an important role in supporting parasite attachment.

Arabidopsis SYP121 acts as an ROP2 effector in the regulation of root hair tip growth

Tip growth is an extreme form of polarized cell expansion that occurs in all eukaryotic kingdoms to generate highly elongated tubular cells with specialized functions, including fungal hyphae, animal neurons, plant pollen tubes, and root hairs (RHs). RHs are tubular structures that protrude from the root epidermis to facilitate water and nutrient uptake, microbial interactions, and plant anchorage. RH tip growth requires polarized vesicle targeting and active exocytosis at apical growth sites. However, how apical exocytosis is spatially and temporally controlled during tip growth remains elusive. Here, we report that the Qa-Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) SYP121 acts as an effector of Rho of Plants 2 (ROP2), mediating the regulation of RH tip growth. We show that active ROP2 promotes SYP121 targeting to the apical plasma membrane. Moreover, ROP2 directly interacts with SYP121 and promotes the interaction between SYP121 and the R-SNARE VAMP722 to form a SNARE complex, probably by facilitating the release of the Sec1/Munc18 protein SEC11, which suppresses the function of SYP121. Thus, the ROP2-SYP121 pathway facilitates exocytic trafficking during RH tip growth. Our study uncovers a direct link between an ROP GTPase and vesicular trafficking and a new mechanism for the control of apical exocytosis, whereby ROP GTPase signaling spatially regulates SNARE complex assembly and the polar distribution of a Q-SNARE.

Hippocampal proteins discovery of diabetes-induced central neuropathy based on proteomics.

Growing evidence suggests that diabetes can cause multifactorial damage to the central nervous system (CNS) and may lead to dementia. However, the underlying mechanism of diabetes-induced central neuropathy remains sparse. In recent years, proteomics has provided better methods and means in analyzing the molecular mechanisms of disease. We applied proteomics to investigate the changes of hippocampal proteins in diabetic rats, with a view to discover the biomarkers of diabetes-induced central neuropathy and elucidated the potential biological relationships. Male Wistar rats were randomly divided into the control group and model group. The model group rats were injected intraperitoneally with streptozotocin. Morris water maze test was performed to evaluate the learning and memory of rats, and the hippocampus was taken out. Proteomics were adopted to investigate the changes of differentially expressed proteins. Compared with the control group, the escape latency of the diabetic rats was significantly increased (P < 0.01, P < 0.05). It was presented that four differentially expressed proteins might be the potential biomarkers of diabetes-induced central neuropathy: septin 5, GRB2 related binding protein 2 (GAB2), casein kinase 1ε (CK1ε), aquaporin 4 (AQP4). These differentially expressed proteins were mainly involved in the following signaling pathways: apoptosis, glycine/serine/threonine metabolic and GTPase signaling pathway. These findings provided reference insights into the underlying molecular pathogenesis of diabetes-induced CNS neuropathy.

AC-265347 Inhibits Neuroblastoma Tumor Growth by Induction of Differentiation without Causing Hypocalcemia.

Neuroblastoma is the most common extracranial solid tumor of childhood, with heterogeneous clinical manifestations ranging from spontaneous regression to aggressive metastatic disease. The calcium-sensing receptor (CaSR) is a G protein-coupled receptor (GPCR) that senses plasmatic fluctuation in the extracellular concentration of calcium and plays a key role in maintaining calcium homeostasis. We have previously reported that this receptor exhibits tumor suppressor properties in neuroblastoma. The activation of CaSR with cinacalcet, a positive allosteric modulator of CaSR, reduces neuroblastoma tumor growth by promoting differentiation, endoplasmic reticulum (ER) stress and apoptosis. However, cinacalcet treatment results in unmanageable hypocalcemia in patients. Based on the bias signaling shown by calcimimetics, we aimed to identify a new drug that might exert tumor-growth inhibition similar to cinacalcet, without affecting plasma calcium levels. We identified a structurally different calcimimetic, AC-265347, as a promising therapeutic agent for neuroblastoma, since it reduced tumor growth by induction of differentiation, without affecting plasma calcium levels. Microarray analysis suggested biased allosteric modulation of the CaSR signaling by AC-265347 and cinacalcet towards distinct intracellular pathways. No upregulation of genes involved in calcium signaling and ER stress were observed in patient-derived xenografts (PDX) models exposed to AC-265347. Moreover, the most significant upregulated biological pathways promoted by AC-265347 were linked to RHO GTPases signaling. AC-265347 upregulated cancer testis antigens (CTAs), providing new opportunities for CTA-based immunotherapies. Taken together, this study highlights the importance of the biased allosteric modulation when targeting GPCRs in cancer. More importantly, the capacity of AC-265347 to promote differentiation of malignant neuroblastoma cells provides new opportunities, alone or in combination with other drugs, to treat high-risk neuroblastoma patients.