KRAS, a small GTPase involved in cell proliferation and differentiation, frequently gains activating mutations in human cancers. For KRAS to function, it must bind the plasma membrane (PM) via interactions between its membrane anchor and phosphatidylserine (PtdSer). Therefore, depleting PM PtdSer abrogates KRAS PM binding and activity. From a genome-wide siRNA screen to identify genes regulating KRAS PM localization, we identified a set of phosphatidylinositol (PI) 3-phosphatases: myotubularin-related proteins (MTMR) 2, 3, 4, and 7. Here, we show that silencing MTMR 2/3/4/7 disrupts KRAS PM interactions by reducing PM PI 4-phosphate (PI4P) levels, thereby disrupting the localization and operation of ORP5, a lipid transfer protein maintaining PM PtdSer enrichment. Concomitantly, silencing MTMR 2/3/4/7 elevates PM PI3P levels while reducing PM and total PtdSer levels. We also observed MTMR 2/3/4/7 expression is interdependent. We propose that the PI 3-phosphatase activity of MTMR is required for generating PM PI, necessary for PM PI4P synthesis, promoting the PM localization of PtdSer and KRAS.

KRAS, a small GTPase involved in cell proliferation and differentiation, frequently gains activating mutations in human cancers. For KRAS to function, it must bind the plasma membrane (PM) via interactions between its membrane anchor and phosphatidylserine (PtdSer). Therefore, depleting PM PtdSer abrogates KRAS PM binding and activity. From a genome-wide siRNA screen to identify genes regulating KRAS PM localization, we identified a set of phosphatidylinositol (PI) 3-phosphatases: myotubularin-related proteins (MTMR) 2, 3, 4, and 7. Here, we show that silencing MTMR 2/3/4/7 disrupts KRAS PM interactions by reducing PM PI 4-phosphate (PI4P) levels, thereby disrupting the localization and operation of ORP5, a lipid transfer protein maintaining PM PtdSer enrichment. Concomitantly, silencing MTMR 2/3/4/7 elevates PM PI3P levels while reducing PM and total PtdSer levels. We also observed MTMR 2/3/4/7 expression is interdependent. We propose that the PI 3-phosphatase activity of MTMR is required for generating PM PI, necessary for PM PI4P synthesis, promoting the PM localization of PtdSer and KRAS.

We discovered that silencing the phosphatidylinositol (PI) 3-phosphatase, MTMR , disrupts the PM localization of PtdSer and KRAS. We propose a model, where MTMR loss depletes PM PI needed for PM PI4P synthesis, an essential phospholipid for PM PtdSer enrichment, thereby impairing KRAS PM localization.

The KRAS mutation stands out as one of the most influential oncogenic mutations, which directly regulates the hallmark features of cancer and interacts with other cancer-causing driver mutations. However, there remains a lack of precise information on their cooccurrence with mutated variants of KRAS and any correlations between KRAS and other driver mutations. To enquire about this issue, we delved into cBioPortal, TCGA, UALCAN, and Uniport studies. We aimed to unravel the complexity of KRAS and its relationships with other driver mutations. We noticed that G12D and G12V are the prevalent mutated variants of KRAS and coexist with the TP53 mutation in PAAD and CRAD, while G12C and G12V coexist with LUAD. We also noticed similar observations in the case of PIK3CA and APC mutations in CRAD. At the transcript level, a positive correlation exists between KRAS and PIK3CA and between APC and KRAS in CRAD. The existence of the co-mutation of KRAS and other driver mutations could influence the signaling pathway in the neoplastic transformation. Moreover, it has immense prognostic and predictive implications, which could help in better therapeutic management to treat cancer.

Cellular membranes serve as an epicentre combining extracellular and cytosolic components with membranous effectors, which together support numerous fundamental cellular signalling pathways that mediate biological responses. To execute their functions, membrane proteins, lipids and carbohydrates arrange, in a highly coordinated manner, into well-defined assemblies displaying diverse biological and biophysical characteristics that modulate several signalling events. The loss of membrane homeostasis can trigger oncogenic signalling. More recently, it has been documented that select membrane active dietaries (MADs) can reshape biological membranes and subsequently decrease cancer risk. In this review, we emphasize the significance of membrane domain structure, organization and their signalling functionalities as well as how loss of membrane homeostasis can steer aberrant signalling. Moreover, we describe in detail the complexities associated with the examination of these membrane domains and their association with cancer. Finally, we summarize the current literature on MADs and their effects on cellular membranes, including various mechanisms of dietary chemoprevention/interception and the functional links between nutritional bioactives, membrane homeostasis and cancer biology.

The development of effective drugs targeting the K-Ras oncogene product is a significant focus in anticancer drug development. Despite the lack of successful Ras signaling inhibitors, recent research has identified PDEδ, a KRAS transporter, as a potential target for inhibiting the oncogenic KRAS signaling pathway. This study aims to investigate the interactions between eight K-Ras inhibitors (deltarazine, deltaflexin 1 and 2, and its analogues) and PDEδ to understand their binding modes. The research will utilize computational techniques such as density functional theory (DFT) and molecular electrostatic surface potential (MESP), molecular docking, binding site analyses, molecular dynamic (MD) simulations, electronic structure computations, and predictions of the binding free energy. Molecular dynamic simulations (MD) will be used to predict the binding conformations and pharmacophoric features in the active site of PDEδ for the examined structures. The binding free energies determined using the MMPB(GB)SA method will be compared with the observed potency values of the tested compounds. This computational approach aims to enhance understanding of the PDEδ selective mechanism, which could contribute to the development of novel selective inhibitors for K-Ras signaling.

Prenylation is an irreversible post-translational modification that supports membrane interactions of proteins involved in various cellular processes, including migration, proliferation, and survival. Dysregulation of prenylation contributes to multiple disorders, including cancers and vascular and neurodegenerative diseases. Prenyltransferases tether isoprenoid lipids to proteins via a thioether linkage during prenylation. Pharmacological inhibition of the lipid synthesis pathway by statins is a therapeutic approach to control hyperlipidemia. Building on our previous finding that statins inhibit membrane association of G protein γ (Gγ) in a subtype-dependent manner, we investigated the molecular reasoning for this differential inhibition. We examined the prenylation of carboxy-terminus (Ct) mutated Gγ in cells exposed to Fluvastatin and prenyl transferase inhibitors and monitored the subcellular localization of fluorescently tagged Gγ subunits and their mutants using live-cell confocal imaging. Reversible optogenetic unmasking-masking of Ct residues was used to probe their contribution to prenylation and membrane interactions of the prenylated proteins. Our findings suggest that specific Ct residues regulate membrane interactions of the Gγ polypeptide, statin sensitivity, and extent of prenylation. Our results also show a few hydrophobic and charged residues at the Ct are crucial determinants of a protein's prenylation ability, especially under suboptimal conditions. Given the cell and tissue-specific expression of different Gγ subtypes, our findings indicate a plausible mechanism allowing for statins to differentially perturb heterotrimeric G protein signaling in cells depending on their Gγ-subtype composition. Our results may also provide molecular reasoning for repurposing statins as Ras oncogene inhibitors and the failure of using prenyltransferase inhibitors in cancer treatment.

Ras proteins are membrane-bound GTPases that regulate essential cellular processes at the plasma membrane (PM). Constitutively active mutations of K-Ras, one of the three Ras isoforms in mammalian cells, are frequently found in human cancers. Ferrocene derivatives, which elevate cellular reactive oxygen species (ROS), have shown to block the growth of non-small cell lung cancers harboring oncogenic mutant K-Ras. Here, we tested a novel ferrocene derivative on the growth of pancreatic ductal adenocarcinoma and non-small cell lung cancer. Our compound, which elevated cellular ROS levels, inhibited the growth of K-Ras-driven cancers, and abrogated the PM binding and signaling of K-Ras in an isoform-specific manner. These effects were reversed upon antioxidant supplementation, suggesting a ROS-mediated mechanism. We further identified that K-Ras His95 residue plays an important role in this process, and it is putatively oxidized by cellular ROS. Together, our study demonstrates that the redox system directly regulates K-Ras/PM binding and signaling via oxidative modification at the His95, and proposes a role of oncogenic mutant K-Ras in the recently described antioxidant-induced growth and metastasis of K-Ras-driven cancers.

Prenylation is a universal and irreversible post-translational modification that supports membrane interactions of proteins involved in various cellular processes, including migration, proliferation, and survival. Thus, dysregulation of prenylation contributes to multiple disorders, including cancers, vascular diseases, and neurodegenerative diseases. During prenylation, prenyltransferase enzymes tether metabolically produced isoprenoid lipids to proteins via a thioether linkage. Pharmacological inhibition of the lipid synthesis pathway by statins has long been a therapeutic approach to control hyperlipidemia. Building on our previous finding that statins inhibit membrane association of G protein γ (Gγ) in a subtype-dependent manner, we investigated the molecular reasoning for this differential. We examined the prenylation efficacy of carboxy terminus (Ct) mutated Gγ in cells exposed to Fluvastatin and prenyl transferase inhibitors and monitored the subcellular localization of fluorescently tagged Gγ subunits and their mutants using live-cell confocal imaging. Reversible optogenetic unmasking-masking of Ct residues was used to probe their contribution to the prenylation process and membrane interactions of the prenylated proteins. Our findings suggest that specific Ct residues regulate membrane interactions of the Gγ polypeptide statin sensitivity, and prenylation efficacy. Our results also show that a few hydrophobic and charged residues at the Ct are crucial determinants of a protein's prenylation ability, especially under suboptimal conditions. Given the cell and tissue-specific expression of different Gγ subtypes, our findings explain how and why statins differentially perturb heterotrimeric G protein signaling in specific cells and tissues. Our results may provide molecular reasoning for repurposing statins as Ras oncogene inhibitors and the failure of using prenyltransferase inhibitors in cancer treatment.

Ras proteins are membrane-bound GTPases that regulate essential cellular processes at the plasma membrane (PM). Constitutively active mutations of K-Ras, one of the three Ras isoforms in mammalian cells, are frequently found in human cancers. Ferrocene derivatives, which elevate cellular reactive oxygen species (ROS), have shown to block the growth of non-small cell lung cancers (NSCLCs) harboring oncogenic mutant K-Ras. Here, we developed and tested a novel ferrocene derivative on the growth of human pancreatic ductal adenocarcinoma (PDAC) and NSCLC. Our compound inhibited the growth of K-Ras-dependent PDAC and NSCLC and abrogated the PM binding and signaling of K-Ras, but not other Ras isoforms. These effects were reversed upon antioxidant supplementation, suggesting a ROS-mediated mechanism. We further identified K-Ras His95 residue in the G-domain as being involved in the ferrocene-induced K-Ras PM dissociation via oxidative modification. Together, our studies demonstrate that the redox system directly regulates K-Ras PM binding and signaling via oxidative modification at the His95, and proposes a role of oncogenic mutant K-Ras in the recently described antioxidant-induced metastasis in K-Ras-driven lung cancers.

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