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Models of the regulation of prenylation and trafficking of PBR-containing small GTPases by SmgGDS splice variants. A, SmgGDS-607 may act directly or in cooperation with another protein (X) to stimulate GDP/GTP exchange and promote entry of nonprenylated GTPases into the prenylation pathway . A nonprenylated GTPase may remain bound to SmgGDS-607 until a signal allows it to undergo GDP/GTP exchange and be released to the PTase. Alternatively, a signal may stimulate binding of the nonprenylated GTPase to SmgGDS-607, where it is retained in the nonprenylated form until GDP/GTP exchange occurs. B, inability of a DN GTPase to bind GTP may inhibit its release from SmgGDS-607, thereby diminishing the interaction of the GTPase with the PTase. Overexpression of SmgGDS-607 will generate more complexes of SmgGDS-607 bound to nonprenylated DN GTPases, causing further retention of DN GTPases in the nonprenylated form. C–J, ability of SmgGDS-607 and SmgGDS-558 to selectively bind nonprenylated and prenylated small GTPases, respectively, suggests multiple roles for SmgGDS splice variants in the regulation of small GTPase prenylation and localization. Because prenylation is not reversible , SmgGDS-607 must intercept newly synthesized small GTPases before prenylation occurs. SmgGDS- 607 may then store nonprenylated small GTPases in the cytoplasm as a reserve for rapid prenylation (C) or stimulate nonconventional signaling from cytosolic GTPases (D). SmgGDS-607 may also transfer nonprenylated small GTPases to the PTase (E). SmgGDS-558, which binds only prenylated small GTPases, may facilitate release of newly prenylated GTPases from the PTase (F) or transport GTPases to the ER (G). SmgGDS-558 may also transport fully processed GTPases from the ER to the PM (H) or extract these GTPases from the PM (I), potentially for transport to endomembranes where the small GTPases participate in different signaling pathways (J).
Source publication
Ras and Rho small GTPases possessing a C-terminal polybasic region (PBR) are vital signaling proteins whose misregulation can lead to cancer. Signaling by these proteins depends on their ability to bind guanine nucleotides and their prenylation with a geranylgeranyl or farnesyl isoprenoid moiety and subsequent trafficking to cellular membranes. The...
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The small GTPase Rac1 plays important roles in many processes, including cytoskeletal reorganization, cell migration, cell-cycle progression and gene expression. The initiation of Rac1 signalling requires at least two mechanisms: GTP loading via the guanosine triphosphate (GTP)/guanosine diphosphate (GDP) cycle, and targeting to cholesterol-rich li...
Citations
... 152]SmgGDS, the protein encoded by the oncogene RAP1GDS1, promotes the activity of RhoA and RhoC GTPases; this is associated with poor survival in cancer patients [149]. RAP1GDS1 contains 15 exons and can give rise to two splice variants, namely SmgGDS-558 and SmgGDS-607, which differ in the presence of exon 5, skipped in the former isoform [150,151]. Both variants are oncogenic, but the SmgGDS-607/SmgGDS-558 ratio is higher in breast and lung cancer, so that targeting the switching between isoforms has been proposed as a therapeutic approach in these cancers [152]. ...
Cancer driver genes are either oncogenes or tumour suppressor genes that are classically activated or inactivated, respectively, by driver mutations. Alternative splicing-which produces various mature mRNAs and, eventually, protein variants from a single gene-may also result in driving neoplastic transformation because of the different and often opposed functions of the variants of driver genes. The present review analyses the different alternative splicing events that result in driving neoplastic transformation, with an emphasis on their molecular mechanisms. To do this, we collected a list of 568 gene drivers of cancer and revised the literature to select those involved in the alternative splicing of other genes as well as those in which its pre-mRNA is subject to alternative splicing, with the result, in both cases, of producing an oncogenic isoform. Thirty-one genes fall into the first category, which includes splicing factors and components of the spliceosome and splicing regulators. In the second category, namely that comprising driver genes in which alternative splicing produces the oncogenic isoform, 168 genes were found. Then, we grouped them according to the molecular mechanisms responsible for alternative splicing yielding oncogenic isoforms, namely, mutations in cis splicing-determining elements, other causes involving non-mutated cis elements, changes in splicing factors, and epigenetic and chromatin-related changes. The data given in the present review substantiate the idea that aberrant splicing may regulate the activation of proto-oncogenes or inactivation of tumour suppressor genes and details on the mechanisms involved are given for more than 40 driver genes.
... Perhaps the most recognized example is BCL-X (BCL2L1) 7 , where one isoform is pro-apoptotic (BCL-Xs) while the other is anti-apoptotic (BCL-XL). Additional examples, where different isoforms appear to have more subtle functional differences, include: (1) RAP1GDS1 (also known as SmgGDS) 8 , where its isoforms interact differently with small GTPases 8,9 ; and (2) TRPM3, which encodes cation-selective channels in humans, and can be alternatively spliced into two variants targeting different ions [10][11][12] . RAP1GDS1 and TRPM3 showcase isoforms performing functions that are closely related yet not identical, while BCL-X (BCL2L1) is an excellent example where the isoforms perform entirely opposite functions. ...
Even though alternative RNA splicing was discovered nearly 50 years ago (1977), we still understand very little about most isoforms arising from a single gene, including in which tissues they are expressed and if their functions differ. Human gene annotations suggest remarkable transcriptional complexity, with approximately 252,798 distinct RNA isoform annotations from 62,710 gene bodies (Ensembl v109; 2023), emphasizing the need to understand their biological effects. For example, 256 gene bodies have ≥50 annotated isoforms and 30 have ≥100, where one protein-coding gene ( MAPK10 ) even has 192 distinct RNA isoform annotations. Whether such isoform diversity results from biological redundancy or spurious alternative splicing (i.e., noise), or whether individual isoforms have specialized functions (even if subtle) remains a mystery for most genes. Recent studies by Aguzzoli-Heberle et al., Leung et al., and Glinos et al. demonstrated long-read RNAseq enables improved RNA isoform quantification for essentially any tissue, cell type, or biological condition ( e.g., disease, development, aging, etc.), making it possible to better assess individual isoform expression and function. While each study provided important discoveries related to RNA isoform diversity, deeper exploration is needed. We sought to quantify and characterize real isoform usage across tissues (compared to annotations). We used long-read RNAseq data from 58 GTEx samples across nine tissues (three brain, two heart, muscle, lung, liver, and cultured fibroblasts) generated by Glinos et al. and found considerable isoform diversity within and across tissues. Cerebellar hemisphere was the most transcriptionally complex tissue (22,522 distinct isoforms; 3,726 unique); liver was least diverse (12,435 distinct isoforms; 1,039 unique). We highlight gene clusters exhibiting high tissue-specific isoform diversity per tissue ( e.g., TPM1 expresses 19 in heart’s atrial appendage). We also validated 447 of the 700 new isoforms discovered by Aguzzoli-Heberle et al. and found that 88 were expressed in all nine tissues, while 58 were specific to a single tissue. This study represents a broad survey of the RNA isoform landscape, demonstrating isoform diversity across nine tissues and emphasizes the need to better understand how individual isoforms from a single gene body contribute to human health and disease.
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... Previous work has demonstrated that lipidating proteins can enhance their loading into EVs 8,9,33 . The types and number of lipids added to proteins can drive localization to different organelle membrane and lipid rafts 24,[34][35][36][37] . Even with these insights, a link between EV loading, cellular trafficking, and raft association for lipidated, peripheral membrane proteins has not yet been established. ...
Naturally generated lipid nanoparticles termed extracellular vesicles (EVs) hold significant promise as engineerable therapeutic delivery vehicles. However, active loading of protein cargo into EVs in a manner that is useful for delivery remains a challenge. Here, we demonstrate that by rationally designing proteins to traffic to the plasma membrane and associate with lipid rafts, we can enhance loading of protein cargo into EVs for a set of structurally diverse transmembrane and peripheral membrane proteins. We then demonstrate the capacity of select lipid tags to mediate increased EV loading and functional delivery of an engineered transcription factor to modulate gene expression in target cells. We envision that this technology could be leveraged to develop new EV-based therapeutics that deliver a wide array of macromolecular cargo.
... SmgGDS is a chaperone protein that regulates the prenylation and trafficking of small GTPases in the Ras and Rho families (15)(16)(17)(18) and promotes breast, lung, prostate, and pancreatic cancer (15)(16)(17)(19)(20)(21)(22)(23)(24)(25). Two SmgGDS splice variants are expressed in most cell types (24). ...
... SmgGDS is a chaperone protein that regulates the prenylation and trafficking of small GTPases in the Ras and Rho families (15)(16)(17)(18) and promotes breast, lung, prostate, and pancreatic cancer (15)(16)(17)(19)(20)(21)(22)(23)(24)(25). Two SmgGDS splice variants are expressed in most cell types (24). ...
... Two SmgGDS splice variants are expressed in most cell types (24). The longer splice variant SmgGDS-607 escorts pre-prenylated small GTPases to prenyltransferases, while the shorter splice variant SmgGDS-558 ( Fig. 1) escorts prenylated small GTPases throughout the cell (15)(16)(17)(18). Even though malignant cells often express much more SmgGDS-607 than SmgGDS-558 (24), a certain amount of SmgGDS-558 must be expressed to maintain the malignant phenotype. ...
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 to 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.
... A group of proteins called GDP-dissociation inhibitors (GDIs) are known to help Rab and Rho GTPases cycle between membrane and cytosol (8,9). In addition, there are GDI-like factors, including the chaperones SmgGDS and PDEdelta, which can deliver J o u r n a l P r e -p r o o f as well as extract small GTPases (Ras and Rap) to and from the membrane (10,11). To date, no GDIs or GDI-like factors have been reported for ARFs and ARLs (12). ...
ARL5B, an ARF-like small GTPase localized to the trans-Golgi, is known for regulating endosome-Golgi trafficking and promoting the migration and invasion of breast cancer cells. Although a few interacting partners have been identified, the mechanism of the shuttling of ARL5B between the Golgi membrane and the cytosol is still obscure. Here, using GFP-binding protein (GBP) pull-down followed by mass spectrometry, we identified heat shock cognate protein (HSC70) as an additional interacting partner of ARL5B. Our pull-down and isothermal titration calorimetry (ITC) based studies suggested that HSC70 binds to ARL5B in an ADP-dependent manner. Additionally, we showed that the N-terminal helix and the nucleotide status of ARL5B contribute to its recognition by HSC70. The confocal microscopy and cell fractionation studies in MDA-MB-231 breast cancer cells revealed that the depletion of HSC70 reduces the localization of ARL5B to the Golgi. Using in vitro reconstitution approach, we provide evidence that HSC70 fine-tunes the association of ARL5B with Golgi membrane. Finally, we demonstrated that the interaction between ARL5B and HSC70 is important for the localization of cation independent mannose-6-phosphate receptor (CIMPR) at Golgi. Collectively, we propose a mechanism by which HSC70, a constitutively expressed chaperone, modulates the Golgi association of ARL5B, which in turn has implications for the Golgi-associated functions of this GTPase.
... The best characterized mechanism that controls the prenylation of Ras and Rho family members involves the interaction of these small GTPases with SmgGDS (pronounced "smidge-G-D-S", gene name RAP1GDS1). SmgGDS has emerged as a major regulator of both the prenylation and intracellular trafficking of many GTPases in the Ras and Rho families (Berg et al., 2010;Ntantie et al., 2013;Williams, 2013;Schuld N. J. et al., 2014;Jennings et al., 2018;Garcia-Torres and Fierke, 2019;Nissim et al., 2019;Brandt et al., 2020;Liao et al., 2020). This review describes how these events are regulated by the two splice variants of SmgGDS, named SmgGDS-607 and SmgGDS-558, and compares SmgGDS to the proteins that regulate the prenylation and trafficking of Rab family members. ...
... The participation of REP1 in the prenylation and trafficking of newly synthesized Rab family members suggests that proteins with functions similar to REP1 might participate in the prenylation and trafficking of newly synthesized Ras and Rho family members. Such proteins that might assist Ras and Rho family members during prenylation were not known prior to the discovery of the two major splice variants of SmgGDS (Berg et al., 2010). The discovery of these SmgGDS splice variants has led to an increasing understanding of how cells can suppress or promote the prenylation of Ras and Rho family members, and has stimulated a growing exploration of how Ras and Rho family members can actively signal both before and after they are prenylated. ...
... An explanation for these conflicting reports that only prenylated GTPases bind SmgGDS was provided in 2010, when the Williams group reported the identification of two splice variants of SmgGDS that differ in their ability to bind prenylated GTPases (Berg et al., 2010). A long form of SmgGDS that has 607 amino acids was identified and named SmgGDS-607, and the shorter form of SmgGDS that has 558 amino acids was named SmgGDS-558 (Berg et al., 2010). ...
Newly synthesized small GTPases in the Ras and Rho families are prenylated by cytosolic prenyltransferases and then escorted by chaperones to membranes, the nucleus, and other sites where the GTPases participate in a variety of signaling cascades. Understanding how prenylation and trafficking are regulated will help define new therapeutic strategies for cancer and other disorders involving abnormal signaling by these small GTPases. A growing body of evidence indicates that splice variants of SmgGDS (gene name RAP1GDS1) are major regulators of the prenylation, post-prenylation processing, and trafficking of Ras and Rho family members. SmgGDS-607 binds pre-prenylated small GTPases, while SmgGDS-558 binds prenylated small GTPases. This review discusses the history of SmgGDS research and explains our current understanding of how SmgGDS splice variants regulate the prenylation and trafficking of small GTPases. We discuss recent evidence that mutant forms of RabL3 and Rab22a control the release of small GTPases from SmgGDS, and review the inhibitory actions of DiRas1, which competitively blocks the binding of other small GTPases to SmgGDS. We conclude with a discussion of current strategies for therapeutic targeting of SmgGDS in cancer involving splice-switching oligonucleotides and peptide inhibitors.
... Some small GTPases are regulated and trafficked through GGTase-Icatalyzed S-prenylation by the splice variants SmgGDS607 and SmgGDS558 chaperone proteins, with SmgGDS607 bearing an extra exon (Ex5). 423,424 SmgGDS607 delivers the protein substrate to the prenyltransferase, while SmgGDS shuttles the prenylated protein to the ER for maturation ( Figure 15A). Since a high SmgGDS607:SmgGDS558 ratio is implicated in some cancers, a splice-switch oligonucleotide (SSO) therapeutic strategy was developed to reprogram the ratio of the SmgGDS isoforms. ...
Protein lipid modification involves the attachment of hydrophobic groups to proteins via ester, thioester, amide, or thioether linkages. In this review, the specific click chemical reactions that have been employed to study protein lipid modification and their use for specific labeling applications are first described. This is followed by an introduction to the different types of protein lipid modifications that occur in biology. Next, the roles of click chemistry in elucidating specific biological features including the identification of lipid-modified proteins, studies of their regulation, and their role in diseases are presented. A description of the use of protein-lipid modifying enzymes for specific labeling applications including protein immobilization, fluorescent labeling, nanostructure assembly, and the construction of protein-drug conjugates is presented next. Concluding remarks and future directions are presented in the final section.
... When the CAAX motif ends with leucine, GGTase1 adds a20-carbon polyisoprene lipid to the cysteine residue [24,25]. Although CAAX prenylation is considered to be immediate and unregulated, small GTP-binding protein GDP-dissociation stimulator (SmgGDS), classified as a guanine nucleotide exchange factor, can regulate the farnesylation of multiple small GTPases [26]. Moreover, SmgGDS-607 was discovered to promote the farnesylation of HRAS by accelerating protein release from FTase [27]. ...
Aberrant activation of the RAS superfamily is one of the critical factors in carcinogenesis. Among them, KRAS is the most frequently mutated one which has inspired extensive studies for developing approaches to intervention. Although the cognition toward KRAS remains far from complete, mounting evidence suggests that a variety of post-translational modifications regulate its activation and localization. In this review, we summarize the regulatory mode of post-translational modifications on KRAS including prenylation, post-prenylation, palmitoylation, ubiquitination, phosphorylation, SUMOylation, acetylation, nitrosylation, etc. We also highlight the recent studies targeting these modifications having exhibited potent anti-tumor activities.
... 11 RAP1GDS1 consists of two main splice variants, SmgGDS-558 (NCBI accession # NP_001093899) and SmgGDS-607 (NCBI accession # NP_001093897), which are reported to have different physiological roles in posttranslational modification of RhoA. 12,13 However, the understanding of biological functions of the RAP1GDS1 gene and the potential mechanisms involved are sparse. ...
Background
RAP1GDS1 (RAP1, GTP‐GDP dissociation stimulator 1), also known as SmgGDS, is a guanine nucleotide exchange factor (GEF) that regulates small GTPases, including, RHOA, RAC1, and KRAS. RAP1GDS1 was shown to be highly expressed in different tissue types including the brain. However, mutations in the RAP1GDS1 gene associated with human diseases have not previously been reported.
Methods
We report on four affected individuals, presenting intellectual disability, global developmental delay (GDD), and hypotonia. The probands’ DNA was subjected to whole‐genome sequencing, revealing a homozygous splice acceptor site mutation in the RAP1GDS1 gene (1444‐1G > A). Sanger sequencing was performed to confirm the segregation of the variant in two Saudi families. The possible aberrant splicing in the patients’ RNA was investigated using RT‐PCR and changes in mRNA expression of the patients were confirmed using qRT‐PCR.
Results
The identified splice variant was found to segregate within the two families. RT‐PCR showed that the mutation affected RAP1GDS1 gene splicing, resulting in the production of aberrant transcripts in the affected individuals. Quantitative gene expression analysis demonstrated that the RAP1GDS1 mRNA expression in all the probands was significantly decreased compared to that of the control, and Sanger sequencing of the probands’ cDNA revealed skipping of exon 13, further strengthening the pathogenicity of this variant.
Conclusion
We are the first to report the mutation of the RAP1GDS1 gene as a potential cause of GDD and hypotonia. However, further investigations into the molecular mechanisms involved are required to confirm the role of RAP1GDS1 gene in causing GDD and hypotonia.
... RAP1GDS1, or SmgGDS, is less well characterized but has serves as GEF for RHOA and RHOC (36). RAP1GDS1 also binds other small GTPases including KRAS (37). RAP1GDS1 differs structurally from canonical RHO and RAS GEFs (38) and may also act as a chaperone for these small GTPases (Berg et al, 2010; was not certified by peer review) is the author/funder. ...
... RAP1GDS1 also binds other small GTPases including KRAS (37). RAP1GDS1 differs structurally from canonical RHO and RAS GEFs (38) and may also act as a chaperone for these small GTPases (Berg et al, 2010; was not certified by peer review) is the author/funder. All rights reserved. ...
... RAP1GDS1 is a non-canonical GEF for several RHO family members. It promotes prenylation and membrane trafficking of RHO and RAS proteins, which are critical for their signaling (37,53). We identified RAP1GDS1 in the PPI data with all of the RAS and RAP GTPases as baits, making it a highly central member of this signaling network ( Figure 1F, 3B, S1B, S2A). ...
Activating mutations in RAS GTPases drive one fifth of cancers, but poor understanding of many RAS effectors and regulators, and of the roles of their different paralogs, continues to impede drug development. We developed a multi-stage discovery and screening process to understand RAS function and identify RAS-related susceptibilities in lung adenocarcinoma. Using affinity purification mass spectrometry (AP/MS), we generated a protein-protein interaction map of the RAS pathway containing thousands of interactions. From this network we constructed a CRISPR dual knockout library targeting 119 RAS-related genes that we screened for genetic interactions (GIs). We found important new effectors of RAS-driven cellular functions, RADIL and the GEF RIN1, and over 250 synthetic lethal GIs, including a potent KRAS -dependent interaction between RAP1GDS1 and RHOA . Many GIs link specific paralogs within and between gene families. These findings illustrate the power of the multiomic approach to identify synthetic lethal combinations for hitherto undruggable cancers.
STATEMENT OF SIGNIFICANCE
We present a thorough survey of protein-protein and genetic interactions in the Ras pathway. These interactions suggested new discoveries that we validate here, and demonstrate important new paralog specificities and redundancies. By comparing synthetic lethal interactions across KRAS -dependent and -independent tumors, we identify new combination therapy targets against Ras-driven cancers.
