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Links between Borg proteins and cytoskeletal regulation and cell shape. Cartoon showing the cellular localisations of Borg proteins. See text for details.
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Despite being discovered more than 15 years ago, the Borg (binder of Rho GTPases) family of Cdc42 effector proteins (Cdc42EP1–5) remains largely uncharacterised and relatively little is known about their structure, regulation and role in development and disease. Recent studies are starting to unravel some of the key functional and mechanistic aspec...
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Context 1
... septin-binding property has since been extended to the rest of the members of the family [11,[21][22][23]. Not surprisingly, most Borg proteins have been shown to form filamentous structures that can co-localise with actin fibres and septin filaments (Figure 2). In normal mammary cells, Cdc42EP4 can co-localise with actin stress fibres and induce filopodia and stress fibre formation [24]. ...
Citations
... Our previous studies suggest that the Rho GTPase Cdc42 and its effector proteins Borg regulate septin assembly during the formation of toxin-induced protrusions (12) and during the formation of septin barriers against P. aeruginosa cellular uptake (13). Studies found that expression of constitutively active or -inactive Cdc42 as well as Cdc42-binding deficient Borg2 induces disassembly of filamentous septin structures that coalign with actin fibers in melanoma cells and cancer-associated fibroblasts (11,14,15). Thus, there is a connection between Cdc42, downstream Borg proteins, septins and changes in actin organization. ...
... However, Borg3 seems to be no classical Cdc42 effector, as it might not exert an enzymatic function upon activation. Instead, active Cdc42 might be solely necessary to localize Borg3 (11,14,25). ...
Septin GTPases polymerize into higher-order structures as a part of the cytoskeleton and are involved in vital cellular functions. Among these, they play a role in the formation of primary cilia. However, the regulation of septin assembly in the confined ciliary compartment has not been studied in detail. Here we show, that Borg3 also known as Cdc42EP5, a regulator of septin assembly is involved in the formation of cilia. Borg3 accumulation in cilia is facilitated by switching the Rho-GTPase Cdc42 between an inactive and active state at the base of the primary cilium. Knock out of Borg3 as well as dysregulation of Cdc42 results in reduced septin localization to the primary cilium and reduced formation of cilia.
... Although the function of CDC42EP1 and its interplay with CDC42 in enteric pathogen invasion are unknown, CDC42EP1 has been reported to bind SEPTIN family members, GTP-binding cytoskeletal components that form hetero-oligomeric complexes and act as intracellular scaffolds or barriers reported to aid or inhibit bacterial infections (Boddy et al., 2018;Farrugia & Calvo, 2016;Mostowy et al., 2010;Torraca & Mostowy, 2016). In NIH 3T3 ...
... A major goal of this study was to clarify the function of CDC42EP1, particularly how it functions in concert with CDC42 during pathogen infections in IECs. Previously, the function of CDC42EP1 has been poorly described, and it is most well-known as a binding partner of Rho GTPase CDC42 and septins (Farrugia & Calvo, 2016;Hirsch et al., 2001;Joberty et al., 2001). Our present study confirmed the interaction of CDC42EP1 with CDC42, but our results go beyond previous reports to show that this interaction is enhanced when CDC42 is in its active conformation and possesses its prenylation modification. ...
... It is currently unknown how CDC42EP1 regulates later pathogenic events. With no known enzymatic domains, and domains at which it binds to CDC42 and certain septins(Farrugia & Calvo, 2016;Hirsch et al., 2001;Joberty et al., 1999), CDC42EP1 may potentially function as a scaffolding protein. We speculate that CDC42EP1 may be recruited to the bacteria to form a scaffold for the acquisition of early endosomal or lysosomal markers that assist in forming SCVs.Further research may reveal CDC42EP1 as a regulator of Culture of human cell lines HeLa (ATCC: CCL2) cells and HEK293T (ATCC: CRL-3216) were grown and cultured in Dulbecco's Modified Eagle Medium (DMEM), supplemented with 10% fetal bovine serum (FBS) (Sigma) and 1% penicillin-streptomycin. Caco-2 (ATCC: HTB-37) and C2Bbe1 (ATCC: CRL-2102) cells were grown and cultured in DMEM, supplemented with 20% FBS and 1% penicillin-streptomycin. ...
Human enterocytes are primary targets of infection by invasive bacterium Salmonella Typhimurium, and studies using nonintestinal epithelial cells established that S. Typhimurium activates Rho family GTPases, primarily CDC42, to modulate the actin cytoskeletal network for invasion. The host intracellular protein network that engages CDC42 and influences the pathogen's invasive capacity are relatively unclear. Here, proteomic analyses of canonical and variant CDC42 interactomes identified a poorly characterized CDC42 interacting protein, CDC42EP1, whose intracellular localization is rapidly redistributed and aggregated around the invading bacteria. CDC42EP1 associates with SEPTIN-7 and Villin, and its relocalization and bacterial engagement depend on host CDC42 and S. Typhimurium's capability of activating CDC42. Unlike CDC42, CDC42EP1 is not required for S. Typhimurium's initial cellular entry but is found to associate with Salmonella-containing vacuoles after long-term infections, indicating a contribution to the pathogen's intracellular growth and replication. These results uncover a new host regulator of enteric Salmonella infections, which may be targeted to restrict bacterial load at the primary site of infection to prevent systemic spread.
... Despite these limitations, we note that Axl2 shares similar binding partners with Gic1, a yeast analog of the mammalian Borg proteins involved in the septin organization (Farrugia and Calvo, 2016;Iwase et al., 2006;Sadian et al., 2013). Axl2 can interact with Cdc42 in bud3 cells (this study), Axl2 may also function in septin recruitment independently of the axial landmark, as previously suggested . ...
Cell polarization generally occurs along a single axis that is directed by a spatial cue. Cells of the budding yeast Saccharomyces cerevisiae undergo polarized growth and oriented cell division in a spatial pattern by selecting a specific bud site. Haploid a or α cells bud in the axial pattern in response to a transient landmark that includes Bud3, Bud4, Axl1, and Axl2. Septins, a family of filament-forming GTP-binding proteins, are also involved in axial budding and recruited to an incipient bud site, but the mechanism of recruitment remains unclear. Here, we show that Axl2 interacts with Bud3 and the Cdc42 GTPase in its GTP-bound state. Axl2 also interacts with Cdc10, a septin subunit, promoting efficient recruitment of septins near the cell division site. Furthermore, a cdc42 mutant defective in the axial budding pattern at a semi-permissive temperature had a reduced interaction with Axl2 and compromised septin recruitment in the G1 phase. We thus propose that active Cdc42 brings Axl2 to the Bud3-Bud4 complex and that Axl2 then interacts with Cdc10, linking septin recruitment to the axial landmark.
... Although the formation of filaments is essential for the functions performed by septins, the mechanisms behind their formation, as well as the existence and role of possible regulatory proteins, are far from being well understood. A family of proteins called Borg's (binders of Rho GTPases) has been identified as essential for the regulation of the septin cytoskeleton and its architecture (43)(44)(45)(46)(58)(59)(60)(61)(62). In humans, there are five members of the Borg family (Borg1-Borg5), whose sequences present the following characteristic regions: a polybasic region; a CRIB domain (Cdc42/Rac interactive binding motif); homology domain 1 (BD1); homology domain 2 (BD2, not present in Borg3); homology domain 3 (BD3); a region predicted to interact with actin (only present in Borg3) and a proline-rich region (only present in Borg5) (43). ...
... A family of proteins called Borg's (binders of Rho GTPases) has been identified as essential for the regulation of the septin cytoskeleton and its architecture (43)(44)(45)(46)(58)(59)(60)(61)(62). In humans, there are five members of the Borg family (Borg1-Borg5), whose sequences present the following characteristic regions: a polybasic region; a CRIB domain (Cdc42/Rac interactive binding motif); homology domain 1 (BD1); homology domain 2 (BD2, not present in Borg3); homology domain 3 (BD3); a region predicted to interact with actin (only present in Borg3) and a proline-rich region (only present in Borg5) (43). The BD3 region is reported to be able to interact with septins, while BD1 and BD2 are of unknown function (43). ...
... In humans, there are five members of the Borg family (Borg1-Borg5), whose sequences present the following characteristic regions: a polybasic region; a CRIB domain (Cdc42/Rac interactive binding motif); homology domain 1 (BD1); homology domain 2 (BD2, not present in Borg3); homology domain 3 (BD3); a region predicted to interact with actin (only present in Borg3) and a proline-rich region (only present in Borg5) (43). The BD3 region is reported to be able to interact with septins, while BD1 and BD2 are of unknown function (43). The CRIB domain derives its name from its predicted interaction with Cdc42/Rac proteins, which are small GTPases (43). ...
The molecular basis for septin filament assembly has begun to emerge over recent years. These filaments are essential for many septin functions which depend on their association with biological membranes or components of the cytoskeleton. Much less is known about how septins specifically interact with their binding partners. Here we describe the essential role played by the C-terminal domains in both septin polymerization and their association with the BD3 motif of the Borg family of Cdc42 effector proteins. We provide a detailed description, at the molecular level, of a previously reported interaction between BD3 and the NC-interface between SEPT6 and SEPT7. Upon ternary complex formation, the heterodimeric coiled coil formed by the C-terminal domains of the septins becomes stabilized and filament formation is promoted under conditions of ionic strength/protein concentration which are not normally permissible, likely by favouring hexamers over smaller oligomeric states. This demonstrates that binding partners, such as Borg's, have the potential to control filament assembly/disassembly in vivo in a way which can be emulated in vitro by altering the ionic strength. Experimentally validated models indicate that the BD3 peptide lies antiparallel to the coiled coil and is stabilized by a mixture of polar and apolar contacts. At its center, an LGPS motif, common to all human Borg sequences, interacts with charged residues from both helices of the coiled coil (K368 from SEPT7 and the conserved E354 from SEPT6) suggesting a universal mechanism which governs Borg-septin interactions.
... ARP2/3 can also be activated via the WASP complex, which in turn is regulated by CDC42, another member of the RHO GTPase family. Our data indicate that the CDC42 effector protein CDC42EP1 is an LPA-upregulated phosphoprotein with functions in cell shape regulation [95], potentially providing a further link between LPA-signaling and F-actin organization. F-actin crosslinking is further promoted by MARCKS [96], which is also among the proteins with upregulated phosphosites in our screen (Tables 2 and S8). ...
Lysophosphatidic acid (LPA) species accumulate in the ascites of ovarian high-grade serous cancer (HGSC) and are associated with short relapse-free survival. LPA is known to support metastatic spread of cancer cells by activating a multitude of signaling pathways via G-protein-coupled receptors of the LPAR family. Systematic unbiased analyses of the LPA-regulated signal transduction network in ovarian cancer cells have, however, not been reported to date.
Methods: LPA-induced signaling pathways were identified by phosphoproteomics of both patient-derived and OVCAR8 cells, RNA sequencing, measurements of intracellular Ca²⁺ and cAMP as well as cell imaging. The function of LPARs and downstream signaling components in migration and entosis were analyzed by selective pharmacological inhibitors and RNA interference.
Results: Phosphoproteomic analyses identified > 1100 LPA-regulated sites in > 800 proteins and revealed interconnected LPAR1, ROCK/RAC, PKC/D and ERK pathways to play a prominent role within a comprehensive signaling network. These pathways regulate essential processes, including transcriptional responses, actomyosin dynamics, cell migration and entosis. A critical component of this signaling network is MYPT1, a stimulatory subunit of protein phosphatase 1 (PP1), which in turn is a negative regulator of myosin light chain 2 (MLC2). LPA induces phosphorylation of MYPT1 through ROCK (T853) and PKC/ERK (S507), which is majorly driven by LPAR1. Inhibition of MYPT1, PKC or ERK impedes both LPA-induced cell migration and entosis, while interference with ROCK activity and MLC2 phosphorylation selectively blocks entosis, suggesting that MYPT1 figures in both ROCK/MLC2-dependent and -independent pathways. We finally show a novel pathway governed by LPAR2 and the RAC-GEF DOCK7 to be indispensable for the induction of entosis.
Conclusion: We have identified a comprehensive LPA-induced signal transduction network controlling LPA-triggered cytoskeletal changes, cell migration and entosis in HGSC cells. Due to its pivotal role in this network, MYPT1 may represent a promising target for interfering with specific functions of PP1 essential for HGSC progression.
... GO analysis revealed that a number of genes potentially affected by differential methylation upon exposure of FBs to the preconditioned media from SMCs (treated with the preconditioned media from +/− oscillatory shear stress-exposed ECs) played roles in such processes as negative regulation of GTPase activity (hit names: GPS1, IQGAP2, PTPRN2, RCC2), protein glycosylation (B3GALT6, DOLK, GALNT1, GALNT17, MAN2A2, RFNG, TET2, TMTC2, For instance, it was shown that activation of CDC42 is involved in the hypoxia-induced production of angiogenesis-promoting factors such as vascular endothelial growth factor (VEGF) [18], as well as in actin filament polymerization [19]. Proteins of this family also play roles in cytoskeletal remodeling and signaling, cell shape, directed migration and differentiation, and pathological fibroblast activation [20]. The aforementioned HIF1AN, participating in the oxidation-reduction process, is also present in FBs. ...
Epigenomic changes in the venous cells exerted by oscillatory shear stress towards the endothelium may result in consolidation of gene expression alterations upon vein wall remodeling during varicose transformation. We aimed to reveal such epigenome-wide methylation changes. Primary culture cells were obtained from non-varicose vein segments left after surgery of 3 patients by growing the cells in selective media after magnetic immunosorting. Endothelial cells were either exposed to oscillatory shear stress or left at the static condition. Then, other cell types were treated with preconditioned media from the adjacent layer’s cells. DNA isolated from the harvested cells was subjected to epigenome-wide study using Illumina microarrays followed by data analysis with GenomeStudio (Illumina), Excel (Microsoft), and Genome Enhancer (geneXplain) software packages. Differential (hypo-/hyper-) methylation was revealed for each cell layer’s DNA. The most targetable master regulators controlling the activity of certain transcription factors regulating the genes near the differentially methylated sites appeared to be the following: (1) HGS, PDGFB, and AR for endothelial cells; (2) HGS, CDH2, SPRY2, SMAD2, ZFYVE9, and P2RY1 for smooth muscle cells; and (3) WWOX, F8, IGF2R, NFKB1, RELA, SOCS1, and FXN for fibroblasts. Some of the identified master regulators may serve as promising druggable targets for treating varicose veins in the future.
... CDC42EP3, one of five CDC42 effector proteins, acts as a key regulator of the activities of CDC42 (Farrugia and Calvo, 2017). Previous studies have indicated that the bio functional roles of CDC42EP3 in regulating cell shape change, actomyosin contractility and pathological fibroblast activation (Farrugia and Calvo, 2016). Additionally, CDC42EP3 is also associated with the occurrence and progression of human cancers, such as colorectal cancer (Feng et al., 2021), ovarian cancer (Yan et al., 2021), and glioma (Yang et al., 2022). ...
Background
Parkinson’s disease (PD) is the second most common progressive neurodegenerative disorder and the leading cause of disability in the daily activities. In the management of PD, accurate and specific biomarkers in blood for the early diagnosis of PD are urgently needed. DNA methylation is one of the main epigenetic mechanisms and associated with the gene expression and disease initiation of PD. We aimed to construct a methylation signature for the diagnosis of PD patients, and explore the potential value of DNA methylation in therapeutic options.
Materials and methods
Whole blood DNA methylation and gene expression data of PD patients as well as healthy controls were extracted from Gene Expression Omnibus database. Next, differentially expressed genes (DEGs) and differentially methylated genes (DMGs) between PD patients and healthy controls were identified. Least absolute shrinkage and selection operator cox regression analysis was carried out to construct a diagnostic signature based on the overlapped genes. And, the receiver operating characteristic (ROC) curves were drawn and the area under the curve (AUC) was used to assess the diagnostic performance of the signature in both the training and testing datasets. Finally, gene ontology and gene set enrichment analysis were subsequently carried out to explore the underlying mechanisms.
Results
We obtained a total of 9,596 DMGs, 1,058 DEGs, and 237 overlapped genes in the whole blood between PD patients and healthy controls. Eight methylation-driven genes (HIST1H4L, CDC42EP3, KIT, GNLY, SLC22A1, GCM1, INO80B, and ARHGAP26) were identified to construct the gene expression signature. The AUCs in predicting PD patients were 0.84 and 0.76 in training dataset and testing dataset, respectively. Additionally, eight methylation-altered CpGs were also identified to construct the CpGs signature which showed a similarly robust diagnostic capability, with AUCs of 0.8 and 0.73 in training dataset and testing dataset, respectively.
Conclusion
We conducted an integrated analysis of the gene expression and DNA methylation data, and constructed a methylation-driven genes signature and a methylation-altered CpGs signature to distinguish the patients with PD from healthy controls. Both of them had a robust prediction power and provide a new insight into personalized diagnostic and therapeutic strategies for PD.
... MXD4 is a MYC antagonist known to increase the fraction of cells in the G0/G1 phase in hematopoietic differentiation 68 , and could be a master regulator of entry into the quiescence-like state. CDC42EP5 is a small Rho-GTPase belonging to the Borg family and is involved in cell shape regulation and lamellipodia formation 69 . Similarly, CLIP3 (or CLIPR-59) is a CAP-Gly domain-containing linker protein with a poorly-specified function, perhaps modulating the compartmentalization of the AKT kinase family 70 . ...
Despite the fact that the cell cycle is a fundamental process of life, a detailed quantitative understanding of gene regulation dynamics throughout the cell cycle is far from complete. Single-cell RNA-sequencing (scRNA-seq) technology gives access to these dynamics without externally perturbing the cell. Here, by generating scRNA-seq libraries in different cell systems, we observe cycling patterns in the unspliced-spliced RNA space of cell cycle-related genes. Since existing methods to analyze scRNA-seq are not efficient to measure cycling gene dynamics, we propose a deep learning approach (DeepCycle) to fit these patterns and build a high-resolution map of the entire cell cycle transcriptome. Characterizing the cell cycle in embryonic and somatic cells, we identify major waves of transcription during the G1 phase and systematically study the stages of the cell cycle. Our work will facilitate the study of the cell cycle in multiple cellular models and different biological contexts. Single-cell RNA-sequencing technology gives access to cell cycle dynamics without externally perturbing the cell. Here the authors present DeepCycle,a robust deep learning method to infer the cell cycle state in single cells from scRNA-seq data.
... The affected Cdc42 downstream effectors included Pak kinases (Pak1, Pak2, and Pak6), Map3k7, Cdc42-binding protein kinases (Cdc42bpa, Cdc42bpb), and Cdc42 effector protein (Cdc42ep4) ( Figure 5, Tables S1-S5). were arranged according to [94,101,107,111,114,117,121,124,128,129,133,137,140,144,145,[150][151][152][153][154][155][156][157]. Abbreviations: Cblb: E3 ubiquitin-protein ligase CBL-B; Cdc42bpa: Cdc42-binding protein kinase α; Cdc42bpb: Cdc42-binding protein kinase β; Cdc42ep4: Cdc42 effector protein 4; Git1: ARF GTPase-activating protein GIT1; Plekhg3: pleckstrin homology domain-containing family G member 3; Scrib: protein scribble homolog. ...
... While Ser454 and Ser460 were hyperphosphorylated in wild-type cells after ligand treatment, knockdown of β-arrestin2 abolished these increased phosphorylations and caused hyperphosphorylation at Thr457 only after treatment with TAL ( Figure 6, Tables S2, S3 and S5). Figure 1. Associations and interactions between proteins were arranged according to [94,101,107,111,114,117,121,124,128,129,133,137,140,144,145,[150][151][152][153][154][155][156][157]. Abbreviations: Cblb: E3 ubiquitin-protein ligase CBL-B; Cdc42bpa: Cdc42-binding protein kinase α; Cdc42bpb: Cdc42-binding protein kinase β; Cdc42ep4: Cdc42 effector protein 4; Git1: ARF GTPase-activating protein GIT1; Plekhg3: pleckstrin homology domaincontaining family G member 3; Scrib: protein scribble homolog. ...
In recent years, thyrotropin-releasing hormone (TRH) and its analogs, including taltirelin (TAL), have demonstrated a range of effects on the central nervous system that represent potential therapeutic agents for the treatment of various neurological disorders, including neurodegenerative diseases. However, the molecular mechanisms of their actions remain poorly understood. In this study, we investigated phosphosignaling dynamics in pituitary GH1 cells affected by TRH and TAL and the putative role of β-arrestin2 in mediating these effects. Our results revealed widespread alterations in many phosphosignaling pathways involving signal transduction via small GTPases, MAP kinases, Ser/Thr- and Tyr-protein kinases, Wnt/β-catenin, and members of the Hippo pathway. The differential TRH- or TAL-induced phosphorylation of numerous proteins suggests that these ligands exhibit some degree of biased agonism at the TRH receptor. The different phosphorylation patterns induced by TRH or TAL in β-arrestin2-deficient cells suggest that the β-arrestin2 scaffold is a key factor determining phosphorylation events after TRH receptor activation. Our results suggest that compounds that modulate kinase and phosphatase activity can be considered as additional adjuvants to enhance the potential therapeutic value of TRH or TAL.
... Interestingly, unlike other Cdc42 effectors, these genes are only present in vertebrates. However, recently researchers have investigated their role in tumor progression, regulation and function (Farrugia and Calvo, 2016;. ...
Small GTPases are the key to actin cytoskeleton signaling, which opens the lock of effector proteins to forward the signal downstream in several cellular pathways. Actin cytoskeleton assembly is associated with cell polarity, adhesion, movement and other functions in eukaryotic cells. Rho proteins, specifically Cdc42 and Rac, are the primary regulators of actin cytoskeleton dynamics in higher and lower eukaryotes. Effector proteins, present in an inactive state gets activated after binding to the GTP bound Cdc42/Rac to relay a signal downstream. Cdc42/Rac interactive binding (CRIB) motif is an essential conserved sequence found in effector proteins to interact with Cdc42 or Rac. A diverse range of Cdc42/Rac and their effector proteins have evolved from lower to higher eukaryotes. The present study has identified and further classified CRIB containing effector proteins in lower eukaryotes, focusing on parasitic protozoans causing neglected tropical diseases and taking human proteins as a reference point to the highest evolved organism in the evolutionary trait. Lower eukaryotes’ CRIB containing proteins fall into conventional effector molecules, PAKs (p21 activated kinase), Wiskoit-Aldrich Syndrome proteins family, and some have unique domain combinations unlike any known proteins. We also highlight the correlation between the effector protein isoforms and their selective specificity for Cdc42 or Rac proteins during evolution. Here, we report CRIB containing effector proteins; ten in Dictyostelium and Entamoeba, fourteen in Acanthamoeba, one in Trypanosoma and Giardia. CRIB containing effector proteins that have been studied so far in humans are potential candidates for drug targets in cancer, neurological disorders, and others. Conventional CRIB containing proteins from protozoan parasites remain largely elusive and our data provides their identification and classification for further in-depth functional validations. The tropical diseases caused by protozoan parasites lack combinatorial drug targets as effective paradigms. Targeting signaling mechanisms operative in these pathogens can provide greater molecules in combatting their infections.
