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Loss of USP32 disrupts cargo trafficking and lysosomal proteolysis. a–e Effect of USP32 depletion on ligand-mediated trafficking and degradation of epidermal growth factor (EGF) receptor (EGFR). a Representative confocal z-projections of fixed HeLa cells transfected as indicated, starved and stimulated with 100 ng/mL EGF-555 (white) for 120 min. Perinuclear (PN) and peripheral (PP) insets show overlays of EGF (green) with immunostained CD63 (magenta). b EGF-positive pixel distribution expressed as fractional distance along a straight line from center of nucleus (0) to the PM (1.0). Red lines: mean, n = 2 independent experiments. c Colocalization of EGF with CD63 in PN (left), PP (middle), and overall (right) in control cells (siCtrl, white bars) vs. those depleted of USP32 (siUSP32_2, gray bars), n = 2 independent experiments. d Representative confocal images of fixed HeLa cells transfected as indicated, starved and stimulated with EGF-555 (white) for 120 min. PN and PP insets show overlays of EGF (magenta) with immunostained cathepsin D (green). e Colocalization of EGF with cathepsin D in control cells (siCtrl, white bars) vs. those depleted of USP32 (siUSP32_2, gray bars), n = 3 independent experiments. All colocalization plots report Mander’s overlap quantified from multicell images (black circles). Cell and nuclear boundaries are depicted in dashed magenta and white lines, respectively. Scale bars = 10 μm. f, g Effect of USP32 depletion on ligand-induced degradation of EGFR. f Lysates from HeLa cells transfected as indicated, serum starved, and stimulated with EGF (25 ng/mL) for 0, 30, 60, or 120 min were analyzed by immunoblot against total EGFR (rabbit anti-EGFR) and phosphorylated (pY) EGFR (mouse anti-phosphotyrosine 4G10), with actin as a loading control. g Total (left graph, relative to t = 0) and activated (right graph, pY relative to t = 30) EGFR remaining at 120 min following stimulation in control cells (siCtrl) vs. those depleted of USP32 using different siRNA oligos (siUSP32_2 and siUSP32_3 + 4), n = 3 independent experiments. Bar graphs report mean of independent measurements (black circles), error bars reflect ±s.d. Total number of cells analyzed per condition appears above each bar/scatter. All significance calculated using Student’s t test: *p < 0.05, **p < 0.01, and ***p < 0.001. See also Supplementary Fig. 2
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The endosomal system is a highly dynamic multifunctional organelle, whose complexity is regulated in part by reversible ubiquitylation. Despite the wide-ranging influence of ubiquitin in endosomal processes, relatively few enzymes utilizing ubiquitin have been described to control endosome integrity and function. Here we reveal the deubiquitylating...
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Citations
... Rab7 can undergo ubiquitination in cells by mammalian E3 ligases, including Parkin, primarily at the K38, K191, and K194 residues [50]. Importantly, a recent study reported that Rab7 K191/194R binds RILP more efficiently than the WT, suggesting a ubiquitination-dependent mechanism to regulate late endosome transport [68]. K194 is also a preferred ubiquitination site of Rab7 mediated by SidC/SdcA. ...
Protein ubiquitination is one of the most important posttranslational modifications (PTMs) in eukaryotes and is involved in the regulation of almost all cellular signaling pathways. The intracellular bacterial pathogen Legionella pneumophila translocates at least 26 effectors to hijack host ubiquitination signaling via distinct mechanisms. Among these effectors, SidC/SdcA are novel E3 ubiquitin ligases with the adoption of a Cys-His-Asp catalytic triad. SidC/SdcA are critical for the recruitment of endoplasmic reticulum (ER)-derived vesicles to the Legionella-containing vacuole (LCV). However, the ubiquitination targets of SidC/SdcA are largely unknown, which restricts our understanding of the mechanisms used by these effectors to hijack the vesicle trafficking pathway. Here, we demonstrated that multiple Rab small GTPases and target soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE) proteins are bona fide ubiquitination substrates of SidC/SdcA. SidC/SdcA-mediated ubiquitination of syntaxin 3 and syntaxin 4 promotes their unconventional pairing with the vesicle-SNARE protein Sec22b, thereby contributing to the membrane fusion of ER-derived vesicles with the phagosome. In addition, our data reveal that ubiquitination of Rab7 by SidC/SdcA is critical for its association with the LCV membrane. Rab7 ubiquitination could impair its binding with the downstream effector Rab-interacting lysosomal protein (RILP), which partially explains why LCVs avoid fusion with lysosomes despite the acquisition of Rab7. Taken together, our study reveals the biological mechanisms employed by SidC/SdcA to promote the maturation of the LCVs.
... Indeed Rab7C205,207S mutant fails to bind to endosomal membranes and is almost completely cytosolic (Modica et al., 2017). In addition to prenylation, other modifications such as ubiquitination (Sapmaz et al., 2019;Song et al., 2016), phosphorylation (Francavilla et al., 2016;Heo et al., 2018;Malik et al., 2021;Ritter et al., 2020;Shinde and Maddika, 2016) and palmitoylation (Modica et al., 2017) have been shown to play a role in regulating Rab7A function. ...
... Image analysis was performed using Fiji (Schindelin et al., 2012) and the coloc2 plugins for the co-localization analysis. Statistical analysis was performed using GraphPad Prism Version 8.2.1 (GraphPad Software, San Diego, California USA, www.graphpad.com) ...
The small GTPase Rab7A has a key role in regulating membrane trafficking at late endosomes. By interacting with several different effectors, this small GTPase controls late endosome mobility, orchestrates fusion events between late endosomes and lysosomes, and participates in the formation of and regulates the fusion between autophagosomes and lysosomes. Rab7A is also responsible for the spatiotemporal recruitment of retromer, which is required for the endosome-to-TGN retrieval of cargo-receptors such as sortilin and CI-MPR. Recently several post-translational modifications have been shown to modulate Rab7A functions, including palmitoylation, ubiquitination and phosphorylation. Here we show that phosphorylation of Rab7A at serine 72 is important to modulate its interaction with retromer, as the non-phosphorylatable Rab7AS72A mutant is not able to interact with and recruit retromer to late endosomes. We have previously shown that Rab7A palmitoylation is also required for efficient retromer recruitment. We found that palmitoylation of Rab7AS72A is reduced compared to the wild-type protein, suggesting an interplay between S72 phosphorylation and palmitoylation in regulating the Rab7A/retromer interaction. Finally, we identify NEK7 as the kinase required to phosphorylate Rab7A to promote retromer binding and recruitment.
... Research has demonstrated that USP32 is expressed in the cytoplasm as well as the cell membrane. It is also one of the few DUBs connected to the endocytosis pathway that can regulate the function of a number of factors [15,16]. In the meantime, USP32 participates in physiological processes like cell cycle, invasion, migration, multiplication of cells, and repair of DNA damage [11]. ...
The regulatory significance of ubiquitin-specific peptidase 32 (USP32) in tumor is significant, nevertheless, the biological roles and regulatory mechanisms of USP32 in non-small cell lung cancer (NSCLC) remain unclear. According to our research, USP32 was strongly expressed in NSCLC cell lines and tissues and was linked to a bad prognosis for NSCLC patients. Interference with USP32 resulted in a significant inhibition of NSCLC cell proliferation, migration potential, and EMT development; on the other hand, USP32 overexpression had the opposite effect. To further elucidate the mechanism of action of USP32 in NSCLC, we screened H1299 cells for interacting proteins and found that USP32 interacts with BAG3 (Bcl2-associated athanogene 3) and deubiquitinates and stabilizes BAG3 in a deubiquitinating activity-dependent manner. Functionally, restoration of BAG3 expression abrogated the antitumor effects of USP32 silencing. Furthermore, USP32 increased the phosphorylation level of the RAF/MEK/ERK signaling pathway in NSCLC cells by stabilizing BAG3. In summary, these findings imply that USP32 is critical to the development of NSCLC and could offer a theoretical framework for the clinical diagnosis and management of NSCLC patients in the future.
... Many of these ubiquitination sites were imputed, suggesting that the ubiquitination of these proteins may result from L.p. effector activity (Supplemental Figure S6). While several of the small GTPases ubiquitinated during infection are known to be regulated by ubiquitin outside of the context of infection, these ubiquitination events are often transient and hard to detect (Lachance et al., 2013;Shin et al., 2017;Sapmaz et al., 2019;Duncan et al., 2022), suggesting that L.p. infection may ubiquitinate small GTPases at a higher frequency or with a greater stability than observed in uninfected cells. In contrast to WT infection, ΔdotA infection induced ubiquitination of few small GTPases, consistent with cross-family small GTPase ubiquitination being a process induced by secreted effectors. ...
... Of the many detected GTPases, almost all fell below both the adjusted p value and the Log2FC significance cutoffs, suggesting that GTPases do not significantly change in abundance during infection. This result is consistent with past work demonstrating that L.p.-induced Rab1 ubiquitination is removed at later time points during infection in a proteasome-independent process (Horenkamp et al., 2014), and with past work on nondegradative small GTPase monoubiquitination (Sapmaz et al., 2019;Kholmanskikh et al., 2022). Consistent with this insight from our proteomics analysis, we do not see a decrease in small GTPase abundance across the time course of infection by Western blot analysis for all small GTPases tested (Figures 2, B−E and 5; Supplemental Figure S7). ...
... Monoubiquitination of RhoC, Rab11a, and KRas on either the G5 SAK motif or the preceding α4 helix appears to be activating (Sasaki et al., 2011;Baker et al., 2013;Lachance et al., 2013;Kholmanskikh et al., 2022), while ubiquitination of Rab5 in the same region appears to impair activity (Shin et al., 2017). Equally paradoxical, ubiquitination of Rab7 in the HVD appears to maintain it in the membrane (Sapmaz et al., 2019), while ubiquitination of H/N/KRas in this region prevents membrane association (Steklov et al., 2018). ...
The intracellular bacterial pathogen Legionella pneumophila ( L.p.) manipulates eukaryotic host ubiquitination machinery to form its replicative vacuole. While nearly 10% of L.p.’s ∼330 secreted effector proteins are ubiquitin ligases or deubiquitinases, a comprehensive measure of temporally resolved changes in the endogenous host ubiquitinome during infection has not been undertaken. To elucidate how L.p hijacks host cell ubiquitin signaling, we generated a proteome-wide analysis of changes in protein ubiquitination during infection. We discover that L.p. infection increases ubiquitination of host regulators of subcellular trafficking and membrane dynamics, most notably ∼40% of mammalian Ras superfamily small GTPases. We determine that these small GTPases undergo non-degradative ubiquitination at the Legionella-containing vacuole membrane. Finally, we find that the bacterial effectors SidC/SdcA play a central role in cross-family small GTPase ubiquitination, and that these effectors function upstream of SidE-family ligases in the poly-ubiquitination and retention of GTPases in the LCV membrane. This work highlights the extensive reconfiguration of host ubiquitin signaling by bacterial effectors during infection and establishes simultaneous ubiquitination of small GTPases across the Ras superfamily as a novel consequence of L.p. infection. Our findings position L.p. as a tool to better understand how small GTPases can be regulated by ubiquitination in uninfected contexts.
... [20] For example, the ubiquitination of the small GTPase RAB7 at K194 (Figure 1d) has been shown to induce its binding to vacuolar protein sorting 35 (VPS35), thereby maintaining spatiotemporal order in the endosomal system. [21] The ubiquitination of ADP-Ribosylation Factor 6 (ARF6) at K69 (Figure 1d), has been suggested to reduce its affinity with the downstream effector multidomain ARF GAP protein (AMAP1), potentially affecting endothelial cell migration and tubulogenesis. [22] In contrast to these examples, the role of shear stress-induced ubiquitination on the function of the RAP1A and RAP1B GTPases ( Figure 1d) remains unexplored, even though these two highly homologous RAP1 isoforms are key regulators of endothelial function. ...
Blood flow produces shear stress exerted on the endothelial layer of the vessels. Spatial characterization of the endothelial proteome is required to uncover the mechanisms of endothelial activation by shear stress, as blood flow varies in the vasculature. An integrative ubiquitinome and proteome analysis of shear‐stressed endothelial cells demonstrated that the non‐degradative ubiquitination of several GTPases is regulated by mechano‐signaling. Spatial analysis reveals increased ubiquitination of the small GTPase RAP1 in the descending aorta, a region exposed to laminar shear stress. The ubiquitin ligase WWP2 is identified as a novel regulator of RAP1 ubiquitination during shear stress response. Non‐degradative ubiquitination fine‐tunes the function of GTPases by modifying their interacting network. Specifically, WWP2‐mediated RAP1 ubiquitination at lysine 31 switches the balance from the RAP1/ Talin 1 (TLN1) toward RAP1/ Afadin (AFDN) or RAP1/ RAS Interacting Protein 1 (RASIP1) complex formation, which is essential to suppress shear stress‐induced reactive oxygen species (ROS) production and maintain endothelial barrier integrity. Increased ROS production in endothelial cells in the descending aorta of endothelial‐specific Wwp2‐knockout mice leads to increased levels of oxidized lipids and inflammation. These results highlight the importance of the spatially regulated non‐degradative ubiquitination of GTPases in endothelial mechano‐activation.
... RILP has been widely studied due to its unique role in various biological processes, such as autophagosome biogenesis, transport, and degradation Sapmaz et al. 2019;. Recently, an increasing number of researches have also associated the regulation of autophagy by RILP in tumors (Qi et al. 2022;Luca et al. 2021;Steffan et al. 2010). ...
Background
Rab-interacting lysosomal protein (RILP) contains an alpha-helical coil with an unexplored biological function in osteosarcoma. This study investigated the expression of RILP in osteosarcoma cells and tissues to determine the effect of RILP on the biological behaviors of osteosarcoma cells and the underlying mechanism.
Methods
Tumor Immune Estimation Resource (TIMER) database, The Cancer Genome Atlas (TCGA) database and Gene Expression Omnibus (GEO) database were used for bioinformatic analysis. Co-immunoprecipitation experiment was used to determine whether the two proteins were interacting. In functional tests, cell counting kit-8 (CCK-8) assay, colony formation assay, wound healing assay, transwell invasion assay, Immunofluorescence (IF) assay and immunohistochemical (IHC) assay were performed.
Results
Overexpression of RILP significantly inhibited proliferation and impaired metastasis ability of osteosarcoma cells, while silencing of RILP showed the opposite trend. RNA-seq data analysis was applied in 143B cells and pathway enrichment analysis revealed that differentially expressed genes were mainly enriched in the PI3K/AKT pathway. We further verified that overexpression of RILP restrained the PI3K/AKT/mTOR signaling pathway and induced autophagy in osteosarcoma cells, while the opposite trend was observed when PI3K pathway activator 740Y-P was used. 3-Methyladenine (3-MA), a selective autophagy inhibitor, partially attenuated the inhibitory effect of RILP on the migration and invasion ability of osteosarcoma cells, suggesting the involvement of autophagy in epithelial–mesenchymal transition regulation in osteosarcoma cells. Growth factor receptor binding protein-10 (Grb10), an adaptor protein, was confirmed as a potential target of RILP to restrain the PI3K/AKT signaling pathway. We subcutaneously injected stably overexpressing 143B osteosarcoma cells into nude mice and observed that overexpression of RILP inhibited tumor growth by inhibiting the PI3K/AKT/mTOR pathway.
Conclusion
Our study revealed that the expression of RILP was associated with favorable prognosis of osteosarcoma and RILP inhibits proliferation, migration, and invasion and promotes autophagy in osteosarcoma cells via Grb10-mediated inhibition of the PI3K/AKT/mTOR signaling pathway. In the future, targeting RILP may be a potential strategy for osteosarcoma treatment.
... In the investigation of drug resistance in tumor cells, USP32, a membrane protein, can result in resistance to the anticancer medication YM155 by interfering with the steady expression of SLC35F2 [28]. Subcellular localization studies also show that USP32 may be co-located with Golgi [23], and some studies have found that USP32 can regulate the participation of small GTPase Rab7 in Golgi endosome selection [35]. The absence of USP32 will also affect the structure and function of intracellular lysosomal vesicles and the transport of nuclear endosomes, thus affecting the occurrence of some diseases [35]. ...
... Subcellular localization studies also show that USP32 may be co-located with Golgi [23], and some studies have found that USP32 can regulate the participation of small GTPase Rab7 in Golgi endosome selection [35]. The absence of USP32 will also affect the structure and function of intracellular lysosomal vesicles and the transport of nuclear endosomes, thus affecting the occurrence of some diseases [35]. ...
... All human DUBs have the two distinct structures of the N-terminal calcium-binding EF hand and the C-terminal prenylation site (CAAX box), the latter of which is present only in the USP32 structure and is associated with USP32doped membrane structures [34]. Researchers discovered that the USP domain in USP32, which has substrates for both mono-and double-ubiquitin cleavage, does not often tend to break one of the eight ubiquitin connections, M1, K6, K11, K27, K29, K33, K48, and K63 [35]. In addition, a ubiquitin C-terminal hydrolase (UBP12) exists in the 518-1316 amino acid position of the USP32 structure, which can undergo post-translational modification and protein turnover, while the ubiquitin carboxyl terminal hydrolase has the 1231-1564 amino acid position of the USP32 structure (Fig. 4A). ...
An essential protein regulatory system in cells is the ubiquitin-proteasome pathway. The substrate is modified by the ubiquitin ligase system (E1-E2-E3) in this pathway, which is a dynamic protein bidirectional modification regulation system. Deubiquitinating enzymes (DUBs) are tasked with specifically hydrolyzing ubiquitin molecules from ubiquitin-linked proteins or precursor proteins and inversely regulating protein degradation, which in turn affects protein function. The ubiquitin-specific peptidase 32 (USP32) protein level is associated with cell cycle progression, proliferation, migration, invasion, and other cellular biological processes. It is an important member of the ubiquitin-specific protease family. It is thought that USP32, a unique enzyme that controls the ubiquitin process, is closely linked to the onset and progression of many cancers, including small cell lung cancer, gastric cancer, breast cancer, epithelial ovarian cancer, glioblastoma, gastrointestinal stromal tumor, acute myeloid leukemia, and pancreatic adenocarcinoma. In this review, we focus on the multiple mechanisms of USP32 in various tumor types and show that USP32 controls the stability of many distinct proteins. Therefore, USP32 is a key and promising therapeutic target for tumor therapy, which could provide important new insights and avenues for antitumor drug development. The therapeutic importance of USP32 in cancer treatment remains to be further proven. In conclusion, there are many options for the future direction of USP32 research.
... Surprisingly, many of these ubiquitination sites were imputed, suggesting that the ubiquitination of these proteins may be catalyzed by L.p. effectors (Fig 2-S2). While several of the small GTPases ubiquitinated during infection are known to be regulated by ubiquitin outside of the context of infection, these ubiquitination events are often transient and hard to detect (Lachance et al., 2013;Shin et al., 2017;Sapmaz et al., 2019;Duncan et al., 2022), suggesting that L.p. infection may ubiquitinate small GTPases at a higher frequency or with a greater stability than observed in uninfected cells. In contrast to WT infection, ΔdotA infection induced ubiquitination of few small GTPases, consistent with mass small GTPase ubiquitination being an effector-induced process. ...
... p-value and the Log2FC significance cutoffs, suggesting that GTPases do not significantly change in abundance during infection. This result is consistent with past work demonstrating that L.p.-induced Rab1 ubiquitination is removed at later time points during infection in a proteasome-independent process (Horenkamp et al., 2014), and with past work on non-degradative small GTPase monoubiquitination (Sapmaz et al., 2019;Kholmanskikh et al., 2022). Consistent with this insight from our proteomics analysis, we do not see a decrease in small GTPase abundance across the time course of infection by Western blot for all small GTPases tested (Fig 2B-E, Fig 2-S3, Fig 5). ...
... 1101/2023.08.03.551750 doi: bioRxiv preprint al., 2013Kholmanskikh et al., 2022), while ubiquitination of Rab5 in the same region appears to impair activity (Shin et al., 2017). Equally paradoxical, ubiquitination of Rab7 in the HVD appears to maintain it in the membrane (Sapmaz et al., 2019), while ubiquitination of H/N/KRas in this region prevents membrane association (Steklov et al., 2018). It is worth noting that GTPases can form protein-protein binding contacts outside of the Switch regions. ...
The intracellular bacterial pathogen Legionella pneumophila ( L.p. ) manipulates eukaryotic host ubiquitination machinery to form its replicative vacuole. While nearly 10% of L.p. ’s arsenal of ∼330 secreted effector proteins have been biochemically characterized as ubiquitin ligases or deubiquitinases, a comprehensive measure of temporally resolved changes in the endogenous host ubiquitinome during infection has not been undertaken. To elucidate how L.p hijacks ubiquitin signaling within the host cell, we undertook a proteome-wide analysis of changes in protein ubiquitination during infection. We discover that L.p. infection results in increased ubiquitination of host proteins regulating subcellular trafficking and membrane dynamics, most notably 63 of ∼160 mammalian Ras superfamily small GTPases. We determine that these small GTPases predominantly undergo non-degradative monoubiquitination, and link ubiquitination to recruitment to the Legionella -containing vacuole membrane. Finally, we find that the bacterial effectors SidC/SdcA play a central, but likely indirect, role in cross-family small GTPase ubiquitination. This work highlights the extensive reconfiguration of host ubiquitin signaling by bacterial effectors during infection and establishes simultaneous ubiquitination of small GTPases across the Ras superfamily as a novel consequence of L.p. infection. This work positions L.p. as a tool to better understand how small GTPases can be regulated by ubiquitination in uninfected contexts.
... She then focused on USP32, a critical DUB in the endolysosomal system and the high expression of which is connected to a poor survival rate in different cancer types. 48 A group of potent USP32 inhibitors were identified via high-throughput screening. Among them, one inhibitor showed the highest potency and best selectivity toward USP32 within and outside the target family. ...
... This molecule was later developed into a chemical probe to explore USP32 activity in vitro and in the living cell, as well as to determine its specificity toward USP32 in pull-down experiments. 48 Finally, the presentation moved to USP32 ubiquitome and proteome analysis between the inhibitor treated and untreated cells. The results further validated the deubiquitylating involvement of USP32 in the endolysosomal system and provided new insights into USP32 biology. ...
... The results further validated the deubiquitylating involvement of USP32 in the endolysosomal system and provided new insights into USP32 biology. 48 Matthew Robers (Promega, USA) described a live cell target engagement method for intractable protein complexes. The first part of the presentation highlighted that novel interactions with type II inhibitors are commonly observed in cellular settings compared with isolated target settings. ...
ICBS 2022 was a refreshing multi-day event where it was justified that the advancement of chemical biology did not halt due to the pandemic, but in contrast, amazing findings were discovered within the restrictions of the SARS-CoV-2 pandemic. All aspects of this annual gathering reinforced that interconnecting the branches of chemical biology through collaboration, the sharing of ideas and knowledge, and networking are enabling the discovery and diversification of applications that will arm scientists of this world in "uncovering solutions for diseases."
... 193,194 K38 in Rab7A is subjected to ubiquitination by PARKIN and deubiquitination by USP32, which alters interactions of Rab7A with its effector and regulates the Rab7A-dependent endosome pathway during PD progression. 195,196 An interesting interplay among β 2 -adrenergic receptor (β 2 -AR), HACE1 ubiquitination ligase, and Rab11A has been observed. HACE1 mediates recycling of β 2 -AR through ubiquitinating RAB11A at K145 whereas the β 2 -AR/HACE1 interactions are required for activation of HACE1. ...
Small GTPases including Ras, Rho, Rab, Arf, and Ran are omnipresent molecular switches in regulating key cellular functions. Their dysregulation is a therapeutic target for tumors, neurodegeneration, cardiomyopathies, and infection. However, small GTPases have been historically recognized as “undruggable”. Targeting KRAS, one of the most frequently mutated oncogenes, has only come into reality in the last decade due to the development of breakthrough strategies such as fragment-based screening, covalent ligands, macromolecule inhibitors, and PROTACs. Two KRAS G12C covalent inhibitors have obtained accelerated approval for treating KRAS G12C mutant lung cancer, and allele-specific hotspot mutations on G12D/S/R have been demonstrated as viable targets. New methods of targeting KRAS are quickly evolving, including transcription, immunogenic neoepitopes, and combinatory targeting with immunotherapy. Nevertheless, the vast majority of small GTPases and hotspot mutations remain elusive, and clinical resistance to G12C inhibitors poses new challenges. In this article, we summarize diversified biological functions, shared structural properties, and complex regulatory mechanisms of small GTPases and their relationships with human diseases. Furthermore, we review the status of drug discovery for targeting small GTPases and the most recent strategic progress focused on targeting KRAS. The discovery of new regulatory mechanisms and development of targeting approaches will together promote drug discovery for small GTPases.
