Megaviruses contain various genes encoding for eukaryotic vesicle trafficking factors.

Many intracellular pathogens, such as bacteria and large viruses, enter eukaryotic cells via phagocytosis, then replicate and proliferate inside the host. To avoid degradation in the phagosomes, they have developed strategies to modify vesicle trafficking. Although several strategies of bacteria have been characterized, it is not clear whether viruses also interfere with the vesicle trafficking of the host. Recently, we came across SNARE proteins encoded in the genomes of several bacteria of the order Legionellales. These pathogenic bacteria may use SNAREs to interfere with vesicle trafficking, since SNARE proteins are the core machinery for vesicle fusion during transport. They assemble into membrane-bridging SNARE complexes that bring membranes together. We now have also discovered SNARE proteins in the genomes of diverse giant viruses. Our biochemical experiments showed that these proteins are able to form SNARE complexes. We also found other key trafficking factors that work together with SNAREs such as NSF, SM, and Rab proteins encoded in the genomes of giant viruses, suggesting that viruses can make use of a large genetic repertoire of trafficking factors. Most giant viruses possess different collections, suggesting that these factors entered the viral genome multiple times. In the future, the molecular role of these factors during viral infection need to be studied.

Mapping the KRAS proteoform landscape in colorectal cancer identifies truncated KRAS4B that decreases MAPK signaling.

The KRAS gene is one of the most frequently mutated oncogenes in human cancer and gives rise to two isoforms, KRAS4A and KRAS4B. KRAS post-translational modifications (PTMs) have the potential to influence downstream signaling. However, the relationship between KRAS PTMs and oncogenic mutations remains unclear, and the extent of isoform-specific modification is unknown. Here, we present the first top-down proteomics study evaluating both KRAS4A and KRAS4B, resulting in 39 completely characterized proteoforms across colorectal cancer cell lines and primary tumor samples. We determined which KRAS PTMs are present, along with their relative abundance, and that proteoforms of KRAS4A versus KRAS4B are differentially modified. Moreover, we identified a subset of KRAS4B proteoforms lacking the C185 residue and associated C-terminal PTMs. By confocal microscopy, we confirmed that this truncated GFP-KRAS4BC185∗ proteoform is unable to associate with the plasma membrane, resulting in a decrease in mitogen-activated protein kinase signaling pathway activation. Collectively, our study provides a reference set of functionally distinct KRAS proteoforms and the colorectal cancer contexts in which they are present.

Biophysical and Biochemical Characterization of Structurally Diverse Small Molecule Hits for KRAS Inhibition.

We describe six compounds as early hits for the development of direct inhibitors of KRAS, an important anticancer drug target. We show that these compounds bind to KRAS with affinities in the low micromolar range and exert different effects on its interactions with binding partners. Some of the compounds exhibit selective binding to the activated form of KRAS and inhibit signal transduction through both the MAPK or the phosphatidylinositide 3-kinase PI3K-protein kinase B (AKT) pathway in cells expressing mutant KRAS. Most inhibit intrinsic and/or SOS-mediated KRAS activation while others inhibit RAS-effector interaction. We propose these compounds as starting points for the development of non-covalent allosteric KRAS inhibitors.

High Pressure Promotes Binding of the Allosteric Inhibitor Zn2+-Cyclen in Crystals of Activated H-Ras.

In this work, we experimentally investigate the potency of high pressure to drive a protein toward an excited state where an inhibitor targeted for this state can bind. Ras proteins are small GTPases cycling between active GTP-bound and inactive GDP-bound states. Various states of GTP-bound Ras in active conformation coexist in solution, amongst them, state 2 which binds to effectors, and state 1, weakly populated at ambient conditions, which has a low affinity for effectors. Zn2+-cyclen is an allosteric inhibitor of Ras protein, designed to bind specifically to the state 1. In H-Ras(wt).Mg2+.GppNHp crystals soaked with Zn2+-cyclen, no binding could be observed, as expected in the state 2 conformation which is the dominant state at ambient pressure. Interestingly, Zn2+-cyclen binding is observed at 500 MPa pressure, close to the nucleotide, in Ras protein that is driven by pressure to a state 1 conformer. The unknown binding mode of Zn2+-cyclen to H-Ras can thus be fully characterized in atomic details. As a more general conjunction from our study, high pressure x-ray crystallography turns out to be a powerful method to induce transitions allowing drug binding in proteins that are in low-populated conformations at ambient conditions, enabling the design of specific inhibitors.

Targeted genetic and small molecule disruption of N-Ras CaaX cleavage alters its localization and oncogenic potential.

Ras GTPases and other CaaX proteins undergo multiple post-translational modifications at their carboxyl-terminus. These events initiate with prenylation of a cysteine and are followed by endoproteolytic removal of the 'aaX' tripeptide and carboxylmethylation. Some CaaX proteins are only subject to prenylation, however, due to the presence of an uncleavable sequence. In this study, uncleavable sequences were used to stage Ras isoforms in a farnesylated and uncleaved state to address the impact of CaaX proteolysis on protein localization and function. This targeted strategy is more specific than those that chemically inhibit the Rce1 CaaX protease or delete the RCE1 gene because global abrogation of CaaX proteolysis impacts the entire CaaX protein proteome and effects cannot be attributed to any specific CaaX protein of the many concurrently affected. With this targeted strategy, clear mislocalization and reduced activity of farnesylated and uncleaved Ras isoforms was observed. In addition, new peptidomimetics based on cleavable Ras CaaX sequences and the uncleavable CAHQ sequence were synthesized and tested as Rce1 inhibitors using in vitro and cell-based assays. Consistently, these non-hydrolyzable peptidomimetic Rce1 inhibitors recapitulate Ras mislocalization effects when modeled on cleavable but not uncleavable CaaX sequences. These findings indicate that a prenylated and uncleavable CaaX sequence, which can be easily applied to a wide range of mammalian CaaX proteins, can be used to probe the specific impact of CaaX proteolysis on CaaX protein properties under conditions of an otherwise normally processed CaaX protein proteome.

GTP-Bound N-Ras Conformational States and Substates Are Modulated by Membrane and Point Mutation.

Oncogenic Ras proteins are known to present multiple conformational states, as reported by the great variety of crystallographic structures. The GTP-bound states are grouped into two main states: the "inactive" state 1 and the "active" state 2. Recent reports on H-Ras have shown that state 2 exhibits two substates, directly related to the orientation of Tyr32: toward the GTP-bound pocket and outwards. In this paper, we show that N-Ras exhibits another substate of state 2, related to a third orientation of Tyr32, toward Ala18 and parallel to the GTP-bound pocket. We also show that this substate is highly sampled in the G12V mutation of N-Ras and barely present in its wild-type form, and that the G12V mutation prohibits the sampling of the GTPase-activating protein (GAP) binding substate, rendering this mutation oncogenic. Furthermore, using molecular dynamics simulations, we explore the importance of the membrane on N-Ras' conformational state dynamics and its strong influence on Ras protein stability. Moreover, the membrane has a significant influence on the conformational (sub)states sampling of Ras. This, in turn, is of crucial importance in the activation/deactivation cycle of Ras, due to the binding of guanine nucleotide exchange factor proteins (GEFs)/GTPase-activating proteins (GAPs).

A novel antiproliferative PKCα-Ras-ERK signaling axis in intestinal epithelial cells.

We have previously shown that the serine/threonine kinase PKCα triggers MAPK/ERK kinase (MEK)-dependent G1→S cell cycle arrest in intestinal epithelial cells, characterized by downregulation of cyclin D1 and inhibitor of DNA-binding protein 1 (Id1) and upregulation of the cyclin-dependent kinase inhibitor p21Cip1. Here, we use pharmacological inhibitors, genetic approaches, siRNA-mediated knockdown, and immunoprecipitation to further characterize antiproliferative ERK signaling in intestinal cells. We show that PKCα signaling intersects the Ras-Raf-MEK-ERK kinase cascade at the level of Ras small GTPases and that antiproliferative effects of PKCα require active Ras, Raf, MEK, and ERK, core ERK pathway components that are also essential for pro-proliferative ERK signaling induced by epidermal growth factor (EGF). However, PKCα-induced antiproliferative signaling differs from EGF signaling in that it is independent of the Ras guanine nucleotide exchange factors (Ras-GEFs), SOS1/2, and involves prolonged rather than transient ERK activation. PKCα forms complexes with A-Raf, B-Raf, and C-Raf that dissociate upon pathway activation, and all three Raf isoforms can mediate PKCα-induced antiproliferative effects. At least two PKCα-ERK pathways that collaborate to promote growth arrest were identified: one pathway requiring the Ras-GEF, RasGRP3, and H-Ras, leads to p21Cip1 upregulation, while additional pathway(s) mediate PKCα-induced cyclin D1 and Id1 downregulation. PKCα also induces ERK-dependent SOS1 phosphorylation, indicating possible negative crosstalk between antiproliferative and growth-promoting ERK signaling. Importantly, the spatiotemporal activation of PKCα and ERK in the intestinal epithelium in vivo supports the physiological relevance of these pathways and highlights the importance of antiproliferative ERK signaling to tissue homeostasis in the intestine.

LY6D-induced macropinocytosis as a survival mechanism of senescent cells.

Although senescent cells display various morphological changes including vacuole formation, it is still unclear how these processes are regulated. We have recently identified the gene, lymphocyte antigen 6 complex, locus D (LY6D), to be upregulated specifically in senescent cells. LY6D is a glycosylphosphatidylinositol-anchored cell-surface protein whose function remains unknown. Here, we analyzed the functional relationship between LY6D and the senescence processes. We found that overexpression of LY6D induced vacuole formation and knockdown of LY6D suppressed the senescence-associated vacuole formation. The LY6D-induced vacuoles were derived from macropinocytosis, a distinct form of endocytosis. Furthermore, Src family kinases and Ras were found to be recruited to membrane lipid rafts in an LY6D-dependent manner, and inhibition of their activity impaired the LY6D-induced macropinocytosis. Finally, reduction of senescent-cell survival induced by glutamine deprivation was recovered by albumin supplementation to the culture media in an LY6D-dependent manner. Because macropinocytosis acts as an amino acid supply route, these results suggest that LY6D-mediated macropinocytosis contributes to senescent-cell survival through the incorporation of extracellular nutrients.

The KRAS-regulated kinome identifies WEE1 and ERK coinhibition as a potential therapeutic strategy in KRAS-mutant pancreatic cancer.

Oncogenic KRAS drives cancer growth by activating diverse signaling networks, not all of which have been fully delineated. We set out to establish a system-wide profile of the KRAS-regulated kinase signaling network (kinome) in KRAS-mutant pancreatic ductal adenocarcinoma (PDAC). We knocked down KRAS expression in a panel of six cell lines and then applied multiplexed inhibitor bead/MS to monitor changes in kinase activity and/or expression. We hypothesized that depletion of KRAS would result in downregulation of kinases required for KRAS-mediated transformation and in upregulation of other kinases that could potentially compensate for the deleterious consequences of the loss of KRAS. We identified 15 upregulated and 13 downregulated kinases in common across the panel of cell lines. In agreement with our hypothesis, all 15 of the upregulated kinases have established roles as cancer drivers (e.g., SRC, TGF-ß1, ILK), and pharmacological inhibition of one of these upregulated kinases, DDR1, suppressed PDAC growth. Interestingly, 11 of the 13 downregulated kinases have established driver roles in cell cycle progression, particularly in mitosis (e.g., WEE1, Aurora A, PLK1). Consistent with a crucial role for the downregulated kinases in promoting KRAS-driven proliferation, we found that pharmacological inhibition of WEE1 also suppressed PDAC growth. The unexpected paradoxical activation of ERK upon WEE1 inhibition led us to inhibit both WEE1 and ERK concurrently, which caused further potent growth suppression and enhanced apoptotic death compared with WEE1 inhibition alone. We conclude that system-wide delineation of the KRAS-regulated kinome can identify potential therapeutic targets for KRAS-mutant pancreatic cancer.

Development of a versatile HPLC-based method to evaluate the activation status of small GTPases.

Small GTPases cycle between an inactive GDP-bound and an active GTP-bound state to control various cellular events, such as cell proliferation, cytoskeleton organization, and membrane trafficking. Clarifying the guanine nucleotide-bound states of small GTPases is vital for understanding the regulation of small GTPase functions and the subsequent cellular responses. Although several methods have been developed to analyze small GTPase activities, our knowledge of the activities for many small GTPases is limited, partly because of the lack of versatile methods to estimate small GTPase activity without unique probes and specialized equipment. In the present study, we developed a versatile and straightforward HPLC-based assay to analyze the activation status of small GTPases by directly quantifying the amounts of guanine nucleotides bound to them. This assay was validated by analyzing the RAS-subfamily GTPases, including HRAS, which showed that the ratios of GTP-bound forms were comparable with those obtained in previous studies. Furthermore, we applied this assay to the investigation of psychiatric disorder-associated mutations of RHEB (RHEB/P37L and RHEB/S68P), revealing that both mutations cause an increase in the ratio of the GTP-bound form in cells. Mechanistically, loss of sensitivity to TSC2 (a GTPase-activating protein for RHEB) for RHEB/P37L, as well as both decreased sensitivity to TSC2 and accelerated guanine-nucleotide exchange for RHEB/S68P, is involved in the increase of their GTP-bound forms, respectively. In summary, the HPLC-based assay developed in this study provides a valuable tool for analyzing small GTPases for which the activities and regulatory mechanisms are less well understood.

Optogenetic control of small GTPases reveals RhoA mediates intracellular calcium signaling.

Rho/Ras family small GTPases are known to regulate numerous cellular processes, including cytoskeletal reorganization, cell proliferation, and cell differentiation. These processes are also controlled by Ca2+, and consequently, cross talk between these signals is considered likely. However, systematic quantitative evaluation has not yet been reported. To fill this gap, we constructed optogenetic tools to control the activity of small GTPases (RhoA, Rac1, Cdc42, Ras, Rap, and Ral) using an improved light-inducible dimer system (iLID). We characterized these optogenetic tools with genetically encoded red fluorescence intensity-based small GTPase biosensors and confirmed these optogenetic tools' specificities. Using these optogenetic tools, we investigated calcium mobilization immediately after small GTPase activation. Unexpectedly, we found that a transient intracellular calcium elevation was specifically induced by RhoA activation in RPE1 and HeLa cells. RhoA activation also induced transient intracellular calcium elevation in MDCK and HEK293T cells, suggesting that generally RhoA induces calcium signaling. Interestingly, the molecular mechanisms linking RhoA activation to calcium increases were shown to be different among the different cell types: In RPE1 and HeLa cells, RhoA activated phospholipase C epsilon (PLCε) at the plasma membrane, which in turn induced Ca2+ release from the endoplasmic reticulum (ER). The RhoA-PLCε axis induced calcium-dependent nuclear factor of activated T cells nuclear translocation, suggesting that it does activate intracellular calcium signaling. Conversely, in MDCK and HEK293T cells, RhoA-ROCK-myosin II axis induced the calcium transients. These data suggest universal coordination of RhoA and calcium signaling in cellular processes, such as cellular contraction and gene expression.

Biochemical and structural analyses reveal that the tumor suppressor neurofibromin (NF1) forms a high-affinity dimer.

Neurofibromin is a tumor suppressor encoded by the NF1 gene, which is mutated in Rasopathy disease neurofibromatosis type I. Defects in NF1 lead to aberrant signaling through the RAS-mitogen-activated protein kinase pathway due to disruption of the neurofibromin GTPase-activating function on RAS family small GTPases. Very little is known about the function of most of the neurofibromin protein; to date, biochemical and structural data exist only for its GAP domain and a region containing a Sec-PH motif. To better understand the role of this large protein, here we carried out a series of biochemical and biophysical experiments, including size-exclusion chromatography-multiangle light scattering (SEC-MALS), small-angle X-ray and neutron scattering, and analytical ultracentrifugation, indicating that full-length neurofibromin forms a high-affinity dimer. We observed that neurofibromin dimerization also occurs in human cells and likely has biological and clinical implications. Analysis of purified full-length and truncated neurofibromin variants by negative-stain EM revealed the overall architecture of the dimer and predicted the potential interactions that contribute to the dimer interface. We could reconstitute structures resembling high-affinity full-length dimers by mixing N- and C-terminal protein domains in vitro The reconstituted neurofibromin was capable of GTPase activation in vitro, and co-expression of the two domains in human cells effectively recapitulated the activity of full-length neurofibromin. Taken together, these results suggest how neurofibromin dimers might form and be stabilized within the cell.

Alternative splicing reverses the cell-intrinsic and cell-extrinsic pro-oncogenic potentials of YAP1.

In addition to acting as a transcriptional co-activator, YAP1 directly mediates translocalization of the pro-oncogenic phosphatase SHP2 from the cytoplasm to nucleus. In the cytoplasm, SHP2 potentiates RAS-ERK signaling, which promotes cell proliferation and cell motility, whereas in the nucleus, it mediates gene regulation. As a result, elucidating the details of SHP2 trafficking is important for understanding its biological roles, including in cancer. YAP1 comprises multiple splicing isoforms defined in part by the presence (as in YAP1-2γ) or absence (as in YAP1-2α) of a γ-segment encoded by exon 6 that disrupts a critical leucine zipper. Although the disruptive segment is known to reduce co-activator function, it is unclear how this element impacts the physical and functional relationships between YAP1 and SHP2. To explore this question, we first demonstrated that YAP1-2γ cannot bind SHP2. Nevertheless, YAP1-2γ exhibits stronger mitogenic and motogenic activities than does YAP1-2α because the YAP1-2α-mediated delivery of SHP2 to the nucleus weakens cytoplasmic RAS-ERK signaling. However, YAP1-2γ confers less in vivo tumorigenicity than does YA1-2α by recruiting tumor-inhibitory macrophages. Mechanistically, YAP1-2γ transactivates and the YAP1-2α-SHP2 complex transrepresses the monocyte/macrophage chemoattractant CCL2 Thus, cell-intrinsic and cell-extrinsic pro-oncogenic YAP1 activities are inversely regulated by alternative splicing of exon 6. Notably, oncogenic KRAS down-regulates the SRSF3 splicing factor that prevents exon 6 skipping, thereby creating a YAP1-2α-dominant situation that supports a "cold" immune microenvironment.

RAS internal tandem duplication disrupts GTPase-activating protein (GAP) binding to activate oncogenic signaling.

The oncogene RAS is one of the most widely studied proteins in cancer biology, and mutant active RAS is a driver in many types of solid tumors and hematological malignancies. Yet the biological effects of different RAS mutations and the tissue-specific clinical implications are complex and nuanced. Here, we identified an internal tandem duplication (ITD) in the switch II domain of NRAS from a patient with extremely aggressive colorectal carcinoma. Results of whole-exome DNA sequencing of primary and metastatic tumors indicated that this mutation was present in all analyzed metastases and excluded the presence of any other clear oncogenic driver mutations. Biochemical analysis revealed increased interaction of the RAS ITD with Raf proto-oncogene Ser/Thr kinase (RAF), leading to increased phosphorylation of downstream MAPK/ERK kinase (MEK)/extracellular signal-regulated kinase (ERK). The ITD prevented interaction with neurofibromin 1 (NF1)-GTPase-activating protein (GAP), providing a mechanism for sustained activity of the RAS ITD protein. We present the first crystal structures of NRAS and KRAS ITD at 1.65-1.75 Å resolution, respectively, providing insight into the physical interactions of this class of RAS variants with its regulatory and effector proteins. Our in-depth bedside-to-bench analysis uncovers the molecular mechanism underlying a case of highly aggressive colorectal cancer and illustrates the importance of robust biochemical and biophysical approaches in the implementation of individualized medicine.

Development of Noonan syndrome by deregulation of allosteric SOS autoactivation.

Ras family proteins play an essential role in several cellular functions, including growth, differentiation, and survival. The mechanism of action of Ras mutants in Costello syndrome and cancers has been identified, but the contribution of Ras mutants to Noonan syndrome, a genetic disorder that prevents normal development in various parts of the body, is unknown. Son of Sevenless (SOS) is a Ras guanine nucleotide exchange factor. In response to Ras-activating cell signaling, SOS autoinhibition is released and is followed by accelerative allosteric feedback autoactivation. Here, using mutagenesis-based kinetic and pulldown analyses, we show that Noonan syndrome Ras mutants I24N, T50I, V152G, and D153V deregulate the autoactivation of SOS to populate their active form. This previously unknown process has been linked so far only to the development of Noonan syndrome. In contrast, other Noonan syndrome Ras mutants-V14I, T58I, and G60E-populate their active form by deregulation of the previously documented Ras GTPase activities. We propose a novel mechanism responsible for the deregulation of SOS autoactivation, where I24N, T50I, V152G, and D153V Ras mutants evade SOS autoinhibition. Consequently, they are capable of forming a complex with the SOS allosteric site, thus aberrantly promoting SOS autoactivation, resulting in the population of active Ras mutants in cells. The results of this study elucidate the molecular mechanism of the Ras mutant-mediated development of Noonan syndrome.

The molecular basis for immune dysregulation by the hyperactivated E62K mutant of the GTPase RAC2.

The RAS-related C3 botulinum toxin substrate 2 (RAC2) is a member of the RHO subclass of RAS superfamily GTPases required for proper immune function. An activating mutation in a key switch II region of RAC2 (RAC2E62K) involved in recognizing modulatory factors and effectors has been identified in patients with common variable immune deficiency. To better understand how the mutation dysregulates RAC2 function, we evaluated the structure and stability, guanine nucleotide exchange factor (GEF) and GTPase-activating protein (GAP) activity, and effector binding of RAC2E62K Our findings indicate the E62K mutation does not alter RAC2 structure or stability. However, it does alter GEF specificity, as RAC2E62K is activated by the DOCK GEF, DOCK2, but not by the Dbl homology GEF, TIAM1, both of which activate the parent protein. Our previous data further showed that the E62K mutation impairs GAP activity for RAC2E62K As this disease mutation is also found in RAS GTPases, we assessed GAP-stimulated GTP hydrolysis for KRAS and observed a similar impairment, suggesting that the mutation plays a conserved role in GAP activation. We also investigated whether the E62K mutation alters effector binding, as activated RAC2 binds effectors to transmit signaling through effector pathways. We find that RAC2E62K retains binding to an NADPH oxidase (NOX2) subunit, p67phox, and to the RAC-binding domain of p21-activated kinase, consistent with our earlier findings. Taken together, our findings indicate that the RAC2E62K mutation promotes immune dysfunction by promoting RAC2 hyperactivation, altering GEF specificity, and impairing GAP function yet retaining key effector interactions.

The effects of mutant Ras proteins on the cell signalome.

The genetic alterations in cancer cells are tightly linked to signaling pathway dysregulation. Ras is a key molecule that controls several tumorigenesis-related processes, and mutations in RAS genes often lead to unbiased intensification of signaling networks that fuel cancer progression. In this article, we review recent studies that describe mutant Ras-regulated signaling routes and their cross-talk. In addition to the two main Ras-driven signaling pathways, i.e., the RAF/MEK/ERK and PI3K/AKT/mTOR pathways, we have also collected emerging data showing the importance of Ras in other signaling pathways, including the RAC/PAK, RalGDS/Ral, and PKC/PLC signaling pathways. Moreover, microRNA-regulated Ras-associated signaling pathways are also discussed to highlight the importance of Ras regulation in cancer. Finally, emerging data show that the signal alterations in specific cell types, such as cancer stem cells, could promote cancer development. Therefore, we also cover the up-to-date findings related to Ras-regulated signal transduction in cancer stem cells.

Low Expression of Rasal2 Promotes Non-small Cell Lung Cancer Metastasis through Ras/ERK Pathway.

The RAS protein activator like 2 (Rasal2) has been reported to be a tumor suppressor in variety of cancers; while an oncogenic protein in ovarian cancer and triple negative breast cancer (TNBC). However, the exact role of Rasal2 in non-small cell lung cancer (NSCLC) is lacking. This study aimed to investigate the role of Rasal2 in NSCLC and the underlying mechanisms. Rasal2 expression level was measured in NSCLC tissue and cells by using quantitative (q)-PCR and immunoblotting analysis. The clinical implication of Rasal2 in NSCLC patients was also analyzed. The function role of Rasal2 in NSCLC cells were measured by small interfering RNA (si-RNA), immunostaining, transwell assay and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Low Rasal2 expression level was observed in human NSCLC tissue and cell lines and significantly related to tumor thickness, ulceration and TNM staging in NSCLC patients. Rasal2 knockdown significantly increased NSCLC cell invasion and migration. Mechanistically, we showed that Rasal2 knockdown significantly increased the phosphorylation level of extracellular signal-regulated kinase (ERK)/Raf1/mitogen-activated protein extracellular kinase (MEK) thus activated Ras/ERK signal pathway. Thus, our data showed that Rasal2 is downregulated in NSCLC cells and act as an epithelial-mesenchymal transition (EMT) and metastasis suppressor through the Ras/ERK pathway. Rasal2 may be a prognostic biomarker for NSCLC in the future.

Implications of farnesyltransferase and its inhibitors as a promising strategy for cancer therapy.

Ras proteins have been reported to play key role in oncologic diseases. Ras proteins are associated with cellular membranes for its carcinogenic activities through post-translational modifications, including farnesylation. Farnesyltransferase is responsible for a type of Ras membrane targeting, which leads to cancer origin and progression. Inhibitors of farnesyltransferase have been developed as novel anticancer agents. In this review, the role of farnesyltransferase in cancer progression and development has been discussed. Further, the current status of development of farnesyltransferase inhibitors for cancer prevention and treatment has also been reviewed.

Structural basis of the atypical activation mechanism of KRASV14I.

RAS regulation and signaling are largely accomplished by direct protein-protein interactions, making RAS protein dynamics a critical determinant of RAS function. Here, we report a crystal structure of GDP-bound KRASV14I, a mutated KRAS variant associated with the developmental RASopathy disorder Noonan syndrome (NS), at 1.5-1.6 Å resolution. The structure is notable for revealing a marked extension of switch 1 away from the G-domain and nucleotide-binding site of the KRAS protein. We found that this extension is associated with a loss of the magnesium ion and a tilt in the position of the guanine base because of the additional carbon introduced by the isoleucine substitution. Hydrogen-deuterium exchange MS analysis confirmed that this conformation occurs in solution, but also disclosed a difference in kinetics when compared with KRASA146T, another RAS mutant that displays a nearly identical conformation in previously reported crystal structures. This conformational change contributed to a high rate of guanine nucleotide-exchange factor (GEF)-dependent and -independent nucleotide exchange and to an increase in affinity for SOS Ras/Rac GEF 1 (SOS1), which appears to be the major mode of activation for this RAS variant. These results highlight a mechanistic connection between KRASA146T and KRASV14I that may have implications for the regulation of these variants and for the development of therapeutic strategies to manage KRAS variant-associated disorders.