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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. [ABSTRACT FROM AUTHOR]

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). [ABSTRACT FROM AUTHOR]

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.

The gene encoding the GTPase KRAS is frequently mutated in pancreatic, lung, and colorectal cancers. The KRAS fraction in the plasma membrane (PM) correlates with activation of the mitogen-activated protein kinase (MAPK) pathway and subsequent cellular proliferation. Understanding KRAS's interaction with the PM is challenging given the complexity of the cellular environment. To gain insight into key components necessary for KRAS signal transduction at the PM, we used synthetic membranes such as liposomes and giant unilamellar vesicles. Using surface plasmon resonance (SPR) spectroscopy, we demonstrated that KRAS and Raf-1 proto-oncogene Ser/Thr kinase (RAF1) domains interact with these membranes primarily through electrostatic interactions with negatively charged lipids reinforced by additional interactions involving phosphatidyl ethanolamine and cholesterol. We found that the RAF1 region spanning RBD through CRD (RBDCRD) interacts with the membrane significantly more strongly than the isolated RBD or CRD domains and synergizes KRAS partitioning to the membrane. We also found that calmodulin and phosphodiesterase 6 delta (PDE6δ), but not galectin3 previously proposed to directly interact with KRAS, passively sequester KRAS and prevent it from partitioning into the PM. RAF1 RBDCRD interacted with membranes preferentially at nonraft lipid domains. Moreover, a C-terminal O-methylation was crucial for KRAS membrane localization. These results contribute to a better understanding of how the KRAS-membrane interaction is tuned by multiple factors whose identification could inform drug discovery efforts to disrupt this critical interaction in diseases such as cancer.

The Kirsten rat sarcoma viral (KRAS) oncogene is the most frequently mutated isoform of RAS, associated with 85 % of RAS-driven cancers. KRAS functions as a signaling hub, participating in various cellular signaling pathways and regulating a wide range of important activities, including cell proliferation, differentiation, growth, metabolism, and migration. Despite being the most frequently altered oncogenic protein in solid tumors, over the past four decades, KRAS has historically been considered "undruggable" owing to a lack of pharmacologically targetable pockets within the mutant isoforms. However, improvements in drug design and development have culminated in the development of selective inhibitors for KRAS mutants. Recent developments have led to the successful targeting of the KRASG12C mutant through covalent inhibitors that exploit the unique cysteine residue introduced by the mutation at 12th position. These inhibitors bind covalently to C12, locking KRAS in its inactive GDP-bound state and preventing downstream signaling. Some of these inhibitors have shown encouraging results in KRASG12C mutant cancer patients but suffer from drug resistance, toxicity, and low therapeutic efficacy. Recently, there have been great advancements in the discovery of drugs that directly target the switch I (S-I), switch-II (S-II) and S-I/II interface sites of KRAS mutant proteins. These include KRASG12C inhibitors like AMG510 (Sotorasib) and MRTX849 (Adagrasib), which have got FDA approval for non-small cell lung cancer harboring the KRASG12C mutation. There is no approved drug for cancers harboring other KRAS mutations, although efforts have expanded to target other KRAS mutations and the Switch I/II interface, aiming to disrupt KRAS-driven oncogenic signaling. Structure-activity relationship (SAR) studies have been instrumental in optimizing the binding affinity, selectivity, and pharmacokinetic properties of these inhibitors, leading to the development of promising therapeutic agents like Sotorasib and Adagrasib. This review provides an overview of the KRAS pathway, KRAS binding sites, strategies for direct and indirect inhibition using small molecules, and SAR based on the co-crystal structures of inhibitors with KRAS mutants which is expected to offer new hope for patients with KRAS-driven cancers through the development of new KRAS-targeted drugs. [Display omitted] • Cancer remains the leading cause of global mortality, accounting for one in every six fatalities. • KRAS, a GTPase, is frequently mutated in cancers. • The development of KRAS inhibitors is a significant advancement in targeted cancer therapy. • There is a need of new drug discovery and optimization for KRAS mutants. • Analysis of the resolved co-crystal structures of KRAS inhibitors have provided insights to guide new drug discovery. [ABSTRACT FROM AUTHOR]

The sheer amplitude of biological actions of insulin-like growth factor I (IGF-1) affecting all type of cells in all tissues suggests a vast signaling landscape for this ubiquitous humoral signal. While the canonical signaling pathways primarily involve the Ras/MAPK and PI3K/AKT cascades, the evolutionary conservation of insulin-like peptides (ILPs) and their pathways hints at the potential for novel functions to emerge over time. Indeed, the evolutionary trajectory of ILPs open the possibility of either novel functions for these two pathways, novel downstream routes, or both. Evidence supporting this notion includes observations of neofunctionalization in bony fishes or crustaceans, and the involvement of ILPs pathways in invertebrate eusociality or in vertebrate bone physiology, respectively. Such evolutionary processes likely contribute to the rich diversity of ILPs signaling observed today. Moreover, the interplay between conserved signaling pathways, such as those implicated in aging (predominantly involving the PI3K-AKT route), and lesser-known pathways, such as those mediated by biased G-protein coupled receptors and others even less known, may underpin the context-dependent actions characteristic of ILPs signaling. While canonical IGF-1 signaling is often assumed to account for the intracellular pathways utilized by this growth factor, a comprehensive analysis of all the pathways mediated by the IGF-1 receptor (IGF-1R) remains lacking. This review aims to explore both canonical and non-canonical routes of IGF-1R action across various cell types, offering a detailed examination of the mechanisms underlying IGF-1 signaling and highlighting the significant gaps in our current understanding., Competing Interests: Conflict of interests The authors declare that they have no conflicts of interest with the contents of this article, (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

In the modern pharmaceutical industry, monoclonal antibodies are often used as therapeutic agents. However, they are restricted to cell surface antigens due to their inability to penetrate the outer cell membrane and maintain normal function in the reducing environment. Additionally, it can lead to cytotoxicity since it attacks cancerous cells by mimicking the human immune system. As an alternative, this study modifies the hyperstable single-chain fragment variable(scFv) antibody to eliminate cancer using its linear shape. The scFv(F8) antibody model was modified to recognize human Ras protein by altering residues in the antigen-binding site. Furthermore, a cell-penetrating peptide (CPP) was attached to the scFv(Ras) antibody model to allow entrance to the cell, creating CPP-scFv(Ras). Sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE), western blotting, and the binding assay were performed to prove its effectiveness. As a result, CPP-scFv(Ras) was successfully engineered and bound to the antigen, HRas(G12V).

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. [ABSTRACT FROM AUTHOR]

Mitochondrial metabolism and dynamics (fission and fusion) critically regulate cell survival and proliferation, and abnormalities in these pathways are implicated in both neurodegenerative disorders and cancer. Mitochondrial fission is necessary for the growth of mutant Ras-dependent tumors. Here, we investigated whether loss of PTEN-induced kinase 1 (PINK1) - a mitochondrial kinase linked to recessive familial Parkinsonism - affects the growth of oncogenic Ras-induced tumor growth in vitro and in vivo. We show that RasG12D-transformed embryonic fibroblasts (MEFs) from PINK1-deficient mice display reduced growth in soft agar and in nude mice, as well as increased necrosis and decreased cell cycle progression, compared to RasG12D-transformed MEFs derived from wildtype mice. PINK1 re-expression (overexpression) at least partially rescues these phenotypes. Neither PINK1 deletion nor PINK1 overexpression altered Ras expression levels. Intriguingly, PINK1-deficient Ras-transformed MEFs exhibited elongated mitochondria and altered DRP1 phosphorylation, a key event in regulating mitochondrial fission. Inhibition of DRP1 diminished PINK1-regulated mitochondria morphological changes and tumor growth suggesting that PINK1 deficiency primarily inhibits Ras-driven tumor growth through disturbances in mitochondrial fission and associated cell necrosis and cell cycle defects. Moreover, we substantiate the requirement of PINK1 for optimal growth of Ras-transformed cells by showing that human HCT116 colon carcinoma cells (carrying an endogenous RasG13D mutation) with CRISPR/Cas9-introduced PINK1 gene deletions also show reduced mitochondrial fission and decreased growth. Our results support the importance of mitochondrial function and dynamics in regulating the growth of Ras-dependent tumor cells and provide insight into possible mechanisms underlying the lower incidence of cancers in Parkinson's disease and other neurodegenerative disorders. [ABSTRACT FROM AUTHOR]

Mitochondrial metabolism and dynamics (fission and fusion) critically regulate cell survival and proliferation, and abnormalities in these pathways are implicated in both neurodegenerative disorders and cancer. Mitochondrial fission is necessary for the growth of mutant Ras-dependent tumors. Here, we investigated whether loss of PTEN-induced kinase 1 (PINK1) - a mitochondrial kinase linked to recessive familial Parkinsonism - affects the growth of oncogenic Ras-induced tumor growth in vitro and in vivo. We show that RasG12D-transformed embryonic fibroblasts (MEFs) from PINK1-deficient mice display reduced growth in soft agar and in nude mice, as well as increased necrosis and decreased cell cycle progression, compared to RasG12D-transformed MEFs derived from wildtype mice. PINK1 re-expression (overexpression) at least partially rescues these phenotypes. Neither PINK1 deletion nor PINK1 overexpression altered Ras expression levels. Intriguingly, PINK1-deficient Ras-transformed MEFs exhibited elongated mitochondria and altered DRP1 phosphorylation, a key event in regulating mitochondrial fission. Inhibition of DRP1 diminished PINK1-regulated mitochondria morphological changes and tumor growth suggesting that PINK1 deficiency primarily inhibits Ras-driven tumor growth through disturbances in mitochondrial fission and associated cell necrosis and cell cycle defects. Moreover, we substantiate the requirement of PINK1 for optimal growth of Ras-transformed cells by showing that human HCT116 colon carcinoma cells (carrying an endogenous RasG13D mutation) with CRISPR/Cas9-introduced PINK1 gene deletions also show reduced mitochondrial fission and decreased growth. Our results support the importance of mitochondrial function and dynamics in regulating the growth of Ras-dependent tumor cells and provide insight into possible mechanisms underlying the lower incidence of cancers in Parkinson’s disease and other neurodegenerative disorders.

Abstract Background Aberrant expression of ubiquitin-specific peptide 22 (USP22) has been detected in various cancers. This study aimed to investigate the role of USP22 and the underlying mechanism in human gastric cancer. Methods The expression pattern of USP22 in human gastric cancer was detected in a tissue microarray containing 88 pairs of gastric cancer tissue and adjacent normal tissue samples from patients with primary gastric cancer using immunohistochemical staining. The correlation of USP22 expression with clinical characteristics of patients, as well as their prognostic values in the overall survival of patients, were evaluated. USP22-overexpressing SGC7901 and USP22-silencing AGS cells were used to explore the role of USP22 in gastric cancer cell behavior in vitro and in vivo. Chromatin immunoprecipitation was performed to identify differentially expressed genes induced by USP22 overexpression. Western blot analysis was conducted to detect the activation of RAS/ERK and PI3K/AKT signaling in USP22-overexpressing SGC7901 cells and xenograft tumor tissues. Knockdown of RAS activator son of sevenless 1 (SOS1) was performed to investigate the role of SOS1 in USP22-regulated gastric cancer cell behavior and RAS signaling both in vitro and in vivo. Results USP22 protein expression was significantly increased in human gastric cancer tissues, compared with adjacent normal tissues, and was positively correlated with local tumor stage. Gain- and loss-of-function assays showed that USP22 promoted gastric cancer cell growth and cell cycle transition while suppressing apoptosis in vitro. Consistent results were observed in a xenograft mouse model. Chromatin immunoprecipitation revealed that the overexpression of USP22 induced the upregulation of RAS activator son of sevenless 1 (SOS1) in SGC7901 cells. Western blot analysis showed that USP22 overexpression also induced activation of the RAS/ERK and PI3K/AKT pathways in SGC7901 cells and xenograft tumor tissues. Furthermore, SOS1 silencing could reverse the effects of USP22 on gastric cancer cell behavior and RAS signaling both in vitro and in vivo. Conclusions Our results suggest that USP22 acts as an oncogene in gastric cancer in a SOS1-dependent manner, identifying the USP22/SOS1/RAS axis as a potential therapeutic target in gastric cancer.

As one of the world’s five terminally ills, tumours can cause important genetic dysfunction. However, some current medicines for tumours usually have strong toxic side effects and are prone to drug resistance. Studies have found that farnesyltransferase inhibitors (FTIs) extracted from natural materials have a good inhibiting ability on tumours with fewer side effects. This article describes several FTIs extracted from natural materials and clarifies the current research progress, which provides a new choice for the treatment of tumours. Executive summary Farnesyltransferase (FTase) When Ras gene is activated, it becomes an oncogene with oncogenic activity. Ras protein plays a crucial role in cancer cells. FTase is the first step to activate Ras protein. FTIs not only have anti-tumour effects, but also makes great contribution to the treatment of plasmodium falciparum, parasitic diseases and progeria. There are still some problems with FTIs, but natural products FTIs are worthy of further research as a new class of low-toxic, safe and effective anticancer drugs. Summarised natural product-derived FTase inhibitors This review summarised several FTIs extracted from natural materials and clarifies their anti-tumour activity (IC50 value) and structure, providing a reference for further research on tumour therapy.

[Display omitted] • N -Ras is mislocalized when its CaaX cleavage is disrupted. • N -Ras that is staged as a farnesylated and uncleaved intermediate has less activity. • Farnesylated substrate mimetics of the Rce1 protease are cell permeable. 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. [ABSTRACT FROM AUTHOR]

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. Zn 2+ -cyclen is an allosteric inhibitor of Ras protein, designed to bind specifically to the state 1. In H-Ras(wt).Mg 2+ .GppNHp crystals soaked with Zn 2+ -cyclen, no binding could be observed, as expected in the state 2 conformation which is the dominant state at ambient pressure. Interestingly, Zn 2+ -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 Zn 2+ -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., (© 2024 Wiley-VCH GmbH.)

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., (© 2024 Wiley‐VCH GmbH.)

As one of the world's five terminally ills, tumours can cause important genetic dysfunction. However, some current medicines for tumours usually have strong toxic side effects and are prone to drug resistance. Studies have found that farnesyltransferase inhibitors (FTIs) extracted from natural materials have a good inhibiting ability on tumours with fewer side effects. This article describes several FTIs extracted from natural materials and clarifies the current research progress, which provides a new choice for the treatment of tumours. Executive summary Farnesyltransferase (FTase) When Ras gene is activated, it becomes an oncogene with oncogenic activity. Ras protein plays a crucial role in cancer cells. FTase is the first step to activate Ras protein. FTIs not only have anti-tumour effects, but also makes great contribution to the treatment of plasmodium falciparum, parasitic diseases and progeria. There are still some problems with FTIs, but natural products FTIs are worthy of further research as a new class of low-toxic, safe and effective anticancer drugs. Summarised natural product-derived FTase inhibitors This review summarised several FTIs extracted from natural materials and clarifies their anti-tumour activity (IC50 value) and structure, providing a reference for further research on tumour therapy. [ABSTRACT FROM AUTHOR]

Ras proteins are among the most recurrently mutated drivers in human cancer and still an indefinable pharmaceutical target. Novel approaches of cancer therapeutic based on the inhibition of Ras-mediated signaling, including inhibition of Ras protein synthesis, Ras processing and blockage of downstream Ras effectors. In 2013, researchers successfully selected a high-affinity ssDNAaptamer recognizing the Ras protein called Ra1 aptamer. Ra1 aptamer may serve as preventive and/or remedial agents for cancer in the future by inhibiting Ras function. This study aimed to determine the therapeutic effect of Ra1 aptamer on breast cancer cell lines. The results showed that 100 µM Ra1 aptamer could be inhibited the proliferation of the MCF-7 breast cancer cell line in vitro after 72h by 90.5%. The IC50 of Ra1 aptamer on the MCF-7 cell line was 55μM at 72h. Also, the Ra1 aptamer caused to inhibit the NF-κB transcriptional factor activity in this cell line. The results showed that the inactivation of RAS protein function by Ra1 aptamer and the effect of this ssDNA on NF-kB transcription factor activity represents a viable approach to prevent the growth of MCF-7 cancer cell lines and therapeutic agents for breast cancer in the future. [ABSTRACT FROM AUTHOR]

Background: Aberrant expression of ubiquitin-specific peptide 22 (USP22) has been detected in various cancers. This study aimed to investigate the role of USP22 and the underlying mechanism in human gastric cancer. Methods: The expression pattern of USP22 in human gastric cancer was detected in a tissue microarray containing 88 pairs of gastric cancer tissue and adjacent normal tissue samples from patients with primary gastric cancer using immunohistochemical staining. The correlation of USP22 expression with clinical characteristics of patients, as well as their prognostic values in the overall survival of patients, were evaluated. USP22-overexpressing SGC7901 and USP22-silencing AGS cells were used to explore the role of USP22 in gastric cancer cell behavior in vitro and in vivo. Chromatin immunoprecipitation was performed to identify differentially expressed genes induced by USP22 overexpression. Western blot analysis was conducted to detect the activation of RAS/ERK and PI3K/AKT signaling in USP22-overexpressing SGC7901 cells and xenograft tumor tissues. Knockdown of RAS activator son of sevenless 1 (SOS1) was performed to investigate the role of SOS1 in USP22-regulated gastric cancer cell behavior and RAS signaling both in vitro and in vivo. Results: USP22 protein expression was significantly increased in human gastric cancer tissues, compared with adjacent normal tissues, and was positively correlated with local tumor stage. Gain- and loss-of-function assays showed that USP22 promoted gastric cancer cell growth and cell cycle transition while suppressing apoptosis in vitro. Consistent results were observed in a xenograft mouse model. Chromatin immunoprecipitation revealed that the overexpression of USP22 induced the upregulation of RAS activator son of sevenless 1 (SOS1) in SGC7901 cells. Western blot analysis showed that USP22 overexpression also induced activation of the RAS/ERK and PI3K/AKT pathways in SGC7901 cells and xenograft tumor tissues. Furthermore, SOS1 silencing could reverse the effects of USP22 on gastric cancer cell behavior and RAS signaling both in vitro and in vivo. Conclusions: Our results suggest that USP22 acts as an oncogene in gastric cancer in a SOS1-dependent manner, identifying the USP22/SOS1/RAS axis as a potential therapeutic target in gastric cancer. [ABSTRACT FROM AUTHOR]

Summary: Post‐translational farnesylation can regulate subcellular localization and protein–protein interaction in eukaryotes. The function of farnesylation is not well identified in plant pathogenic fungi, particularly during the process of fungal infection. Here, through functional analyses of the farnesyltransferase β‐subunit gene, RAM1, we examine the importance of protein farnesylation in the rice blast fungus Magnaporthe oryzae. Targeted disruption of RAM1 resulted in the reduction of hyphal growth and sporulation, and an increase in the sensitivity to various stresses. Importantly, loss of RAM1 also led to the attenuation of virulence on the plant host, characterized by decreased appressorium formation and invasive growth. Interestingly, the defect in appressoria formation of the Δram1 mutant can be recovered by adding exogenous cAMP and IBMX, suggesting that RAM1 functions upstream of the cAMP signalling pathway. We found that two Ras GTPases, RAS1 and RAS2, can interact with Ram1, and their plasma membrane localization was regulated by Ram1 through their C‐terminal farnesylation sites. Adding a farnesyltransferase inhibitor Tipifarnib can result in similar defects as in Δram1 mutant, including decreased appressorium formation and invasive growth, as well as mislocalized RAS proteins. Our findings indicate that protein farnesylation regulates the RAS protein‐mediated signaling pathways required for appressorium formation and host infection, and suggest that abolishing farnesyltransferase could be an effective strategy for disease control. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Palmitoylation, which is mediated by protein acyltransferase (PAT) and performs important biological functions, is the only reversible lipid modification in organism. To study the effect of protein palmitoylation on hypopharyngeal squamous cell carcinoma (HPSCC), the expression levels of 23 PATs in tumor tissues of 8 HPSCC patients were determined, and high mRNA and protein levels of DHHC9 and DHHC15 were found. Subsequently, we investigated the effect of 2-bromopalmitate (2BP), a small-molecular inhibitor of protein palmitoylation, on the behavior of Fadu cells in vitro (50 μM) and in nude mouse xenograft models (50 μmol/kg), and found that 2BP suppressed the proliferation, invasion, and migration of Fadu cells without increasing cell apoptosis. Mechanistically, the effect of 2BP on the transduction of BMP, Wnt, Shh, and FGF signaling pathways was tested with qRT-PCR, and its drug target was explored with western blotting and acyl-biotinyl exchange assay. Our results showed that 2BP inhibited the transduction of the FGF/ERK signaling pathway. The palmitoylation level of Ras protein decreased after 2BP treatment, and its distribution in the cell membrane structure was reduced significantly. The findings of this work reveal that protein palmitoylation mediated by DHHC9 and DHHC15 may play important roles in the occurrence and development of HPSCC. 2BP is able to inhibit the malignant biological behaviors of HPSCC cells, possibly via hindering the palmitoylation and membrane location of Ras protein, which might, in turn, offer a low-toxicity anti-cancer drug for targeting the treatment of HPSCC., Competing Interests: Declaration of conflicting interestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Protein prenylation such as farnesylation and geranylgeranylation is associated with various diseases. Thus, many inhibitors of prenyltransferase have been developed. We report novel inhibitors of farnesyltransferase with a zinc-site recognition moiety and a farnesyl/dodecyl group. Molecular docking analysis showed that both parts of the inhibitor fit well into the catalytic domain of farnesyltransferase. The synthesized inhibitors showed activity against farnesyltransferase in vitro and inhibited proliferation of the pancreatic cell line AsPC-1. Among the compounds with farnesyl and dodecyl groups, the inhibitor with a farnesyl group was found to have stronger and more selective activity. [ABSTRACT FROM AUTHOR]

Prenylation pathway is responsible for post translational modification of various signal proteins, including proteins of Ras superfamily. CAAX prenyl proteases are known to be key players in prenylation pathway. In the current study, we have evaluated CAAX prenyl protease II as a possible drug target against Leishmania donovani parasite, the causative agent of visceral leishmaniasis. Gene knockout strategy was employed to target CAAX prenyl protease II and subsequent effects were studied. CAAX prenyl protease II knockout resulted in significant decrease in growth and infectivity. [ABSTRACT FROM AUTHOR]

Small-molecule inhibition of the interaction between the KRas oncoprotein and the chaperone PDE6δ impairs KRas spatial organization and signaling in cells. However, despite potent binding in vitro ( KD<10 n m), interference with Ras signaling and growth inhibition require 5-20 μ m compound concentrations. We demonstrate that these findings can be explained by fast release of high-affinity inhibitors from PDE6δ by the release factor Arl2. This limitation is overcome by novel highly selective inhibitors that bind to PDE6δ with up to 7 hydrogen bonds, resulting in picomolar affinity. Their release by Arl2 is greatly decreased, and representative compounds selectively inhibit growth of KRas mutated and -dependent cells with the highest activity recorded yet. Our findings indicate that very potent inhibitors of the KRas-PDE6δ interaction may impair the growth of tumors driven by oncogenic KRas. [ABSTRACT FROM AUTHOR]

In this study, we investigated the influence of elevated RAS expression on the growth of meningioma in vivo and in vitro. The IOMM-LEE cells, representing a cell line derived from malignant meningioma, were divided into blank control group (cells without any drug treatment), negative control group (cells treated with an equal volume of normal saline to replace drug), and farnesyl thiosalicylic acid (FTS)-treated group (cells treated with FTS). Methyl-thiazole-tetrazolium bromide (MTT) assay and flow cytometer (with cells after FTS (75 μmol/L) treatment for 48 h) were utilized to determine the proliferation and apoptosis, respectively, of IOMM-LEE cells after RAS inhibition. Western blot analysis was used for semi-quantitative analysis of p-ERK and p-AKT levels. Animal model of human meningioma was established with sub-renal capsule transplantation, and mice were divided into two groups: experimental group (50 mg/kg group, 75 mg/kg group, and 100 mg/kg, hypodermic injection with FTS) and control group. Proliferating cell nuclear antigen (PCNA) was detected by immunohistochemistry (IHC). Western blot analysis was used for detecting ERK and AKT signal pathway. The proliferation of IOMM-LEE cells decreased dramatically and apoptosis rate increased significantly in FTS-treated group compared to blank control group and negative control group (all P < 0.05). At FTS concentration of 75 μmol/L, the apoptosis rate of IOMM-LEE cells reduced significantly over time ( P < 0.05). Cell cycle analysis showed that IOMM-LEE cells exhibited G1-arrest in the FTS-treated group, compared to no cell-cycle arrest in blank control group and the negative control group ( P < 0.05). Further, significantly decreased ERK and AKT phosphorylation levels were detected in IOMM-Lee cells after FTS (75 μmol/L) treatment for 48 h, compared to blank control group and negative control group ( P < 0.05). The results in vivo experiments showed that after FTS treatment, tumor volume, PCNA LI, and the levels of p-ERK and p-Akt decreased significantly in 75 mg/kg group and 100 mg/kg group when compared with the control group and 50 mg/kg group (all P < 0.05). Our findings provide strong evidence that RAS protein is highly expressed in meningioma cells, and the RAS activity is inhibited by downregulating ERK and AKT signal pathway, which may further inhibit the growth of meningioma. [ABSTRACT FROM AUTHOR]

Ras pathway signaling is a critical virulence determinant for pathogenic fungi. Localization of Ras to the plasma membrane (PM) is required for Ras network interactions supporting fungal growth and virulence. For example, loss of A. fumigatus RasA signaling at the PM via inhibition of palmitoylation leads to decreased growth, altered hyphal morphogenesis, decreased cell wall integrity and loss of virulence. In order to be properly localized and activated, Ras proteins must transit a series of post-translational modification (PTM) steps. These steps include farnesylation, proteolytic cleavage of terminal amino acids, carboxymethylation, and palmitoylation. Because Ras activation drives tumor development, Ras pathways have been extensively studied in mammalian cells as a potential target for anti-cancer therapy. Inhibitors of mammalian Ras interactions and PTM components have been, or are actively being, developed. This review will focus on the potential for building upon existing scaffolds to exploit fungal Ras proteins for therapy, synthesizing data from studies employing both mammalian and fungal systems.

Introduction: In Poland, colorectal cancer is the second leading cause of death. The incidence of colorectal cancer increases with age and early onset indicates and increased likelihood for genetic predisposition for this disease. The somatic genetics of tumor development in relation to patients age, gender, sex and morphological factors are unknown in Podlaskie region, Poland. Materials and methods: We investigated seventy five patients (43 men and 32 women) who underwent surgery for cancer of the colorectal in the II Department of General and Gastroenterological Surgery, Medical University of Białystok in 2002-2007. The average age of patients was 64.8 years (the average age of women 66.7, men 63.1). All patients for the study of molecular research (absence or presence of K-RAS mutations) had histopathology confirmed adenocarcinoma. Results: There was no correlation presence or absence of mutations in K-RAS of the following clinical and morphological factors: gender, age, location, degree of tumor differentiation, tumor size and metastases to lymph nodes and other organs The gene encoding the K-Ras protein is mutated in 20-50% of cases of colorectal cancer. Such a difference of results is influenced by several factors: differences of the techniques used for detecting mutations, differences in codon of the gene that is considered codon 12 and /or 13 and / or 61 and differences in the selection and study population. Conclusions: These data suggest the clinical and morphological factors in patients with colorectal cancer have no effect on the presence of K-RAS. mutation. [ABSTRACT FROM AUTHOR]

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-KRAS4B C185 ∗ 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., Competing Interests: Conflict of interest N. L. K. is involved in commercialization of proteomics software. All other authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Ras pathway signaling is a critical virulence determinant for pathogenic fungi. Localization of Ras to the plasma membrane (PM) is required for Ras network interactions supporting fungal growth and virulence. For example, loss of Aspergillus fumigatus RasA signaling at the PM via inhibition of palmitoylation leads to decreased growth, altered hyphal morphogenesis, decreased cell wall integrity and loss of virulence. In order to be properly localized and activated, Ras proteins must transit a series of post-translational modification (PTM) steps. These steps include farnesylation, proteolytic cleavage of terminal amino acids, carboxymethylation, and palmitoylation. Because Ras activation drives tumor development, Ras pathways have been extensively studied in mammalian cells as a potential target for anti-cancer therapy. Inhibitors of mammalian Ras interactions and PTM components have been, or are actively being, developed. This review will focus on the potential for building upon existing scaffolds to exploit fungal Ras proteins for therapy, synthesizing data from studies employing both mammalian and fungal systems. [ABSTRACT FROM AUTHOR]

The aim of this study was to develop high-affinity single-stranded DNA (ssDNA) aptamers that can selectively recognize the protein Ras and can be used as preventive and therapeutic agents for restenosis occurring after coronary surgery or angioplasty. For this purpose, we used the systematic evolution of ligands by exponential enrichment (SELEX) technique, also known as in vitro selection. Using this technique, ssDNA aptamers recognizing the Ras protein were obtained from a synthesized random ssDNA library in vitro. The binding rate and affinity of each aptamer pool, isolated in successive rounds of selection, were measured using ELISA, and the finally selected aptamer pool was cloned and sequenced. The binding affinities of each aptamer in this pool were measured. Their primary and secondary structures were analyzed using the DNAMAN 5.29 software, and the relationship between these structures and corresponding binding affinities was analyzed. The rate of aptamer pool binding to the Ras protein gradually increased from 2.4 to 34.5% along the selection process. Optical density (OD) and equilibrium dissociation constant (Kd) measurements showed that OD gradually increased from 0.220 to 1.080 and Kd decreased from 51.5 to 18.3 nM. The 11th pool of aptamers was selected based on these analyses, and cloning and sequencing of individual aptamers was performed. Secondary structure analysis revealed different conformations, but of a single type: stem-loop. The aptamer Ra1 showed the highest affinity, with a measured OD of 1.213 and an estimated Kd of 15.3 nM. The binding affinity of the aptamer Ra1 to Ras was dose-dependent. In conclusion, high-affinity ssDNA aptamers recognizing the Ras protein have been successfully selected. These aptamers may serve in the future as preventive and/or therapeutic agents for restenosis occurring after coronary surgery or angioplasty. [ABSTRACT FROM AUTHOR]

TNF-like weak inducer of apoptosis (TWEAK) is an inflammatory cytokine that activates the FGF-inducible 14 receptor. Both TWEAK and the FGF-inducible 14 receptor are constitutively expressed in the kidney. TWEAK has been shown to modulate several biological responses, such as inflammation, proliferation, differentiation, and apoptosis, that contribute to kidney injury. However, the role of TWEAK in fibrosis and TWEAKactivated intracellular signaling pathways remain poorly understood. We tested the hypothesis that TWEAK can be a potent inducer of renal fibrosis by increasing transforming growth factor (TGF)-β1 expression (a well-known switch in the fibrosis process) through PKG-I downregulation. We showed that in human mesangial cells, TWEAK increased TGF-β1 expression and activity, leading to higher levels of the extracellular matrix protein fibronectin and decreased PKG-I expression and activity via the Ras pathway. PKG-I activation with 8-bromo-cGMP, Ras inactivation with dominant negative Ras, or Ras pathway inhibition with the ERK1/2 inhibitor PD-98059 resulted in the prevention of TWEAK-induced TGF-β1 upregulation. In vivo, exogenous administration of TWEAK to wild-type mice downregulated kidney PKG-I and increased kidney TGF-β1 expression. These effects were blunted in H-Ras knockout mice. Together, these data demonstrate, for the first time, the key role of PKG-I in TGF-β1 induction by TWEAK in kidney cells. [ABSTRACT FROM AUTHOR]

Abstract: Mimetics of the C-terminal CAAX tetrapeptide of Ras protein were designed as farnesyltransferase (FTase) inhibitors (FTIs) by replacing AA with o-aryl or o-heteroaryl substituted p-hydroxy- or p-aminobenzoic acid, while maintaining the replacement of C with 1,4-benzodioxan-2-ylmethyl or 2-amino-4-thiazolylacetyl residue as in previous CAAX mimetics. Both FTase inhibition and antiproliferative effect were showed by two thiazole derivatives, namely those with 1-naphthyl (10 and 10a) or 3-furanyl (15 and 15a) in the central spacer, and by the benzodioxane derivative with 2-thienyl (6 and 6a) in the same position. Accumulation of unprenylated RAS was demonstrated in cells incubated with 15a. Consistently with FTIs literature, such results delineate the biaryl scaffold not only as a spacer but also as a sensible area of these mimetic molecules, where modifications at the branching aromatic ring are not indifferent and should be matter of further investigation. [Copyright &y& Elsevier]

A new type of allosteric inhibition by small molecules is proposed that should be applicable to all proteins involved intrinsically in protein–protein interactions. It is based on targeting their rare interaction states that can be detected by high‐pressure NMR spectroscopy (see picture). An example is the Ras‐protein where the protein–protein interaction of Ras with effectors can be modulated by small compounds that bind to the conformational states 1(T) or 1(0). [ABSTRACT FROM AUTHOR]

Abstract: Cellular communication is required for the life of pluricellular organisms. The informations exchanged between cells belong to six major types of order to be executed, opposite each other: proliferate or differentiate; remain attached or migrate; survive or die. The cancer cell is genetically unstable, able to explore all the functions encoded by the genome and to consider every proliferative or migratory advantage for selecting it and transmit it to its descent. All the signalling pathways involved in proliferation or differentiation, in adhesion and migration, in survival and death may be altered by oncogenic alterations. These alterations are precisely those which can be targeted for therapy: from this observation was forged the concept of targeted therapy. We present here some examples of therapeutic targeting at the level of a major proliferation pathway by showing how it was possible to identify and characterise relevant targets, invent original new therapeutic tools and decipher the mechanisms of resistance which occur and hinder the success of targeted therapies. This example is the proliferation signalling pathway which starts from the activation of tyrosine kinase receptors by cognate growth factors and ends by the activation of transcription factors which trigger the transcription of the genes required for DNA replication, after undergoing through numerous intermediate molecules constituting the MAP kinase pathway: RAS, RAF, MEK and ERK. [Copyright &y& Elsevier]

Abstract: The Ras family of proteins plays crucial roles in a variety of cell signaling networks where they have the function of a molecular switch. Their particular medical relevance arises from mutations in these proteins that are implicated in ~30% of human cancers. The various Ras proteins exhibit a high degree of homology in their soluble domains but extremely high variability in the membrane anchoring regions that are crucial for protein function and are the focus of this study. We have employed replica exchange molecular dynamics computer simulations to study a doubly lipidated heptapeptide, corresponding to the C-terminus of the human N-Ras protein, incorporated into a dimyristoylphosphatidylcholine lipid bilayer. This same system has previously been investigated experimentally utilizing a number of techniques, including neutron scattering. Here we present results of well converged simulations that describe the subtle changes in scattering density in terms of the location of the peptide and its lipid modifications and in terms of changes in phospholipid density arising from the incorporation of the peptide into the membrane bilayer. The detailed picture that emerges from the combination of experimental and computational data exemplifies the power of combining isotopic substitution neutron scattering with atomistic molecular dynamics simulation. This article is part of a Special Issue entitled: Membrane protein structure and function. [Copyright &y& Elsevier]

Structural, chemical, and mutational studies have shown that C-terminal cysteine residues on H-Ras could potentially be oxidized by nitrosylation. For investigating the effect of nitrosylation of Ras molecule on the adsorption of farnesylated H-Ras into lipid layer, experiments with optical waveguide lightmode spectroscopy were used. The analysis of association/dissociation kinetics to planar phospholipids under controlled hydrodynamic conditions has shown that preliminary treatment of protein by S-nitroso-cysteine decreased the adsorption of farnesylated H-Ras. The authors have found that compared with nitrosylated forms, farnesylated H-Ras has more compact configuration, because of the smaller area occupied by protein upon absorption at the membrane. The association rate coefficient for unmodified H-Ras was lower than similar parameter for farnesylated and nitrosylated forms. However, the desorbability, i.e., parameter, which reflects the rate of dissociation of protein from lipids is higher for farnesylated H-Ras. In addition, it was have found that farnesylation of cytoplasmic H-Ras, in contrast to membrane-derived forms, inhibits intrinsic GTPase activity of protein, and preliminary treatment of H-Ras by S-nitroso-cysteine restores the activity to the control level. These data suggest that nitrosylation of H-Ras rearranges the adsorptive potential and intrinsic GTPase activity of H-Ras through modification of C-terminal cysteines of molecule. [ABSTRACT FROM AUTHOR]