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A unifying feature of the RAS superfamily is a conserved GTPase cycle by which these proteins transition between active and inactive states. We demonstrate that autophosphorylation of some GTPases is an intrinsic regulatory mechanism that reduces nucleotide hydrolysis and enhances nucleotide exchange, altering the on/off switch that forms the basis for their signaling functions. Using X-ray crystallography, nuclear magnetic resonance spectroscopy, binding assays, and molecular dynamics on autophosphorylated mutants of H-RAS and K-RAS, we show that phosphoryl transfer from GTP requires dynamic movement of the switch II region and that autophosphorylation promotes nucleotide exchange by opening the active site and extracting the stabilizing Mg2+. Finally, we demonstrate that autophosphorylated K-RAS exhibits altered effector interactions, including a reduced affinity for RAF proteins in mammalian cells. Thus, autophosphorylation leads to altered active site dynamics and effector interaction properties, creating a pool of GTPases that are functionally distinct from their non-phosphorylated counterparts.
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GTP Fosfo-Hidrolases , Transdução de Sinais , Animais , Cristalografia por Raios X , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Guanosina Trifosfato/metabolismo , Mamíferos/metabolismo , Nucleotídeos , ProteínasRESUMO
Mycobacterium ulcerans causes Buruli ulcer, the third most frequent mycobacterial disease after tuberculosis and leprosy. Transient clinical deteriorations, known as paradoxical reactions (PRs), occur in some patients during or after antibiotic treatment. We investigated the clinical and biological features of PRs in a prospective cohort of 41 patients with Buruli ulcer from Benin. Neutrophil counts decreased from baseline to day 90, and interleukin 6 (IL-6), granulocyte colony-stimulating factor, and vascular endothelial growth factor were the cytokines displaying a significant monthly decrease relative to baseline. PRs occurred in 10 (24%) patients. The baseline biological and clinical characteristics of the patients presenting with PRs did not differ significantly from those of the other patients. However, the patients with PRs had significantly higher IL-6 and tumor necrosis factor alpha (TNF-α) concentrations on days 30, 60, and 90 after the start of antibiotic treatment. The absence of a decrease in IL-6 and TNF-α levels during treatment should alert clinicians to the possibility of PR onset.
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Úlcera de Buruli , Humanos , Úlcera de Buruli/tratamento farmacológico , Estudos Prospectivos , Fator de Necrose Tumoral alfa , Interleucina-6 , Fator A de Crescimento do Endotélio Vascular , Antibacterianos/uso terapêuticoRESUMO
Regenerative medicine is emerging as a novel field in organ transplantation. In September 2019, the European Cell Therapy and Organ Regeneration Section (ECTORS) of the European Society for Organ Transplantation (ESOT) held its first meeting to discuss the state-of-the-art of regenerative medicine in organ transplantation. The present article highlights the key areas of interest and major advances in this multidisciplinary field in organ regeneration and discusses its implications for the future of organ transplantation.
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Transplante de Órgãos , Medicina Regenerativa , Terapia Baseada em Transplante de Células e Tecidos , RegeneraçãoRESUMO
OBJECTIVE: The morbidity and mortality associated with the Marfan Syndrome (MFS) warrant timely diagnosis and intervention that can improve long-term prognosis. The aim of this study was to test the hypothesis that a distinct craniofacial morphology exists for patients with MFS that can be described quantitatively and qualitatively. METHODS: Subjects with a positive diagnosis of MFS were recruited for this study (N = 36). Craniofacial anthropometric measurements were made on each subject and compared to established norms of age- and sex-matched controls using z-scores calculated for measurements of MFS patients. Lateral and frontal photographs were obtained to make qualitative assessments and describe facial features of subjects, and a clinical examination was completed to document occlusal relationships. RESULTS: The subjects were primarily female (58%) ranging in age between 4 and 57 years (mean age 10.7 ± 6.0 years). Comparison of craniofacial measurements revealed that for 10 of the 12 measurements, ≥65% of the study population had a z-score of ± 2 and fell within the normal range for facial dimension. For 2 of the 12 measurements, over half of the subjects fell outside of the normal range (z-score < -2 or > 2) for facial dimension. Specifically, the majority of participants resided in the supernormal category for biocular width and the subnormal category for width of the face. Photographic assessment revealed retrognathia (54%) and down-slanting palpebral fissures (62%) were most prevalent in MFS patients. CONCLUSION: Our data suggest there are quantitative differences in the facial morphology of patients with MFS when compared to a control population.
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Síndrome de Marfan , Retrognatismo , Adolescente , Adulto , Criança , Pré-Escolar , Face , Feminino , Humanos , Pessoa de Meia-Idade , Prognóstico , Adulto JovemRESUMO
H-Ras, K-Ras, and N-Ras are small GTPases that are important in the control of cell proliferation, differentiation, and survival, and their mutants occur frequently in human cancers. The G-domain, which catalyzes GTP hydrolysis and mediates downstream signaling, is 95% conserved between the Ras isoforms. Because of their very high sequence identity, biochemical studies done on H-Ras have been considered representative of all three Ras proteins. We show here that this is not a valid assumption. Using enzyme kinetic assays under identical conditions, we observed clear differences between the three isoforms in intrinsic catalysis of GTP by Ras in the absence and presence of the Ras-binding domain (RBD) of the c-Raf kinase protein (Raf-RBD). Given their identical active sites, isoform G-domain differences must be allosteric in origin, due to remote isoform-specific residues that affect conformational states. We present the crystal structure of N-Ras bound to a GTP analogue and interpret the kinetic data in terms of structural features specific for H-, K-, and N-Ras.
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GTP Fosfo-Hidrolases/metabolismo , Guanosina Trifosfato/metabolismo , Proteínas de Membrana/metabolismo , Modelos Moleculares , Proteínas Proto-Oncogênicas c-raf/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Regulação Alostérica , Sítio Alostérico , Substituição de Aminoácidos , Biocatálise , Domínio Catalítico , Cristalografia por Raios X , Fosfatos de Dinucleosídeos/química , Fosfatos de Dinucleosídeos/metabolismo , Estabilidade Enzimática , GTP Fosfo-Hidrolases/química , GTP Fosfo-Hidrolases/genética , Guanosina Trifosfato/análogos & derivados , Guanosina Trifosfato/química , Humanos , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Ligantes , Proteínas de Membrana/química , Proteínas de Membrana/genética , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Mutação Puntual , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas Proto-Oncogênicas c-raf/química , Proteínas Proto-Oncogênicas c-raf/genética , Proteínas Proto-Oncogênicas p21(ras)/química , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMO
This study examines the available data used to surveil state-level drug overdoses and characterizes them based on their timeliness, drug involvement, and specification of nonfatal outcomes.
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Analgésicos Opioides , Revelação , Overdose de Drogas , Transtornos Relacionados ao Uso de Opioides , Vigilância em Saúde Pública , Humanos , Analgésicos Opioides/efeitos adversos , Overdose de Drogas/epidemiologia , Overdose de Drogas/etiologia , Transtornos Relacionados ao Uso de Opioides/complicações , Transtornos Relacionados ao Uso de Opioides/epidemiologia , Estados Unidos/epidemiologia , Vigilância em Saúde Pública/métodosRESUMO
RAS proteins are signal transduction gatekeepers that mediate cell growth, survival, and differentiation through interactions with multiple effector proteins. The RAS effector RAS- and RAB-interacting protein 1 (RIN1) activates its own downstream effectors, the small GTPase RAB5 and the tyrosine kinase Abelson tyrosine-protein kinase (ABL), to modulate endocytosis and cytoskeleton remodeling. To identify ABL substrates downstream of RAS-to-RIN1 signaling, we examined human HEK293T cells overexpressing components of this pathway. Proteomic analysis revealed several novel phosphotyrosine peptides, including Harvey rat sarcoma oncogene (HRAS)-pTyr(137). Here we report that ABL phosphorylates tyrosine 137 of H-, K-, and NRAS. Increased RIN1 levels enhanced HRAS-Tyr(137) phosphorylation by nearly 5-fold, suggesting that RAS-stimulated RIN1 can drive ABL-mediated RAS modification in a feedback circuit. Tyr(137) is well conserved among RAS orthologs and is part of a transprotein H-bond network. Crystal structures of HRAS(Y137F) and HRAS(Y137E) revealed conformation changes radiating from the mutated residue. Although consistent with Tyr(137) participation in allosteric control of HRAS function, the mutations did not alter intrinsic GTP hydrolysis rates in vitro. HRAS-Tyr(137) phosphorylation enhanced HRAS signaling capacity in cells, however, as reflected by a 4-fold increase in the association of phosphorylated HRAS(G12V) with its effector protein RAF proto-oncogene serine/threonine protein kinase 1 (RAF1). These data suggest that RAS phosphorylation at Tyr(137) allosterically alters protein conformation and effector binding, providing a mechanism for effector-initiated modulation of RAS signaling.
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Proteínas Oncogênicas v-abl/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Transdução de Sinais/fisiologia , Substituição de Aminoácidos , Animais , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Mutação de Sentido Incorreto , Proteínas Oncogênicas v-abl/química , Proteínas Oncogênicas v-abl/genética , Fosforilação/genética , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas p21(ras)/química , Proteínas Proto-Oncogênicas p21(ras)/genética , Ratos , Tirosina/química , Tirosina/genética , Tirosina/metabolismo , Proteínas rab5 de Ligação ao GTP/química , Proteínas rab5 de Ligação ao GTP/genética , Proteínas rab5 de Ligação ao GTP/metabolismo , Quinases raf/química , Quinases raf/genética , Quinases raf/metabolismoRESUMO
PURPOSE OF REVIEW: The current review presents an update on the existing preclinical and human experience of mesenchymal stromal cell (MSC) therapies for post-transplant immunomodulation. RECENT FINDINGS: Although results from early clinical studies have demonstrated that the application of autologous and allogeneic MSC to be both safe and feasible in a solid organ transplantation setting, for example in liver, the efficacy of MSC immunotherapy demonstrated in preclinical models has yet to be replicated in human clinical trials. SUMMARY: Eagerly awaited results from the second generation of solid organ transplantation clinical trials, many of which are nearing completion, will perhaps establish the effectiveness of combining MSCs and low-dose pharmacological immunosuppression in promoting graft acceptance. At present, the question of whether infusional cell products based on MSCs will have a significant clinical impact in the field of liver transplantation remains open.
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Imunomodulação , Transplante de Fígado , Transplante de Células-Tronco Mesenquimais , Pesquisa Biomédica , HumanosRESUMO
In the past decade, therapeutic use of mesenchymal stem cells (MSCs) has increased dramatically. The weight of existing evidence supports that the short-term application of MSCs is safe and feasible; however, concerns remain over the possibility of unwanted long-term effects. One fundamental difference between MSCs and pharmacotherapy is that, once applied, the effects of cell products cannot be easily reversed. Therefore, a carefully considered decision process is indispensable before cell infusion. In addition to unwanted interactions of MSCs with the host immune system, there are concerns that MSCs may promote tumor progression or even give rise to cancer themselves. As animal models and first-in-man clinical studies have provided conflicting results, it is challenging to estimate the long-term risk of individual patients. In addition, most animal models, especially rodents, are ill-suited to adequately address questions over long-term side effects. Based on the available evidence, we address the potential pitfalls for the use of MSCs as a therapeutic agent to control alloimmune effects. The aim of this review was not to discourage investigators from clinical studies, but to raise awareness of the intrinsic risks of MSC therapy.
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Transplante de Células-Tronco Mesenquimais/efeitos adversos , Células-Tronco Mesenquimais/citologia , Transplante de Órgãos/métodos , Animais , Diferenciação Celular , Ensaios Clínicos como Assunto , Progressão da Doença , Transplante de Células-Tronco Hematopoéticas , Humanos , Sistema Imunitário , Imunossupressores/uso terapêutico , Camundongos , Neoplasias/cirurgia , Neoplasias/terapia , Neovascularização PatológicaRESUMO
PURPOSE OF REVIEW: Despite their potential to supplement the donor organ pool, expanded donor criteria grafts are associated with an elevated risk of graft failure and increased early mortality. Likewise, attempts to promote operational graft tolerance through conventional immunosuppressive therapy have demonstrated significant safety-related drawbacks. Because of their potent regenerative and immunomodulative potential, adjunct mesenchymal stem cell (MSC) therapy represents an innovative approach to both of these clinical problems. RECENT FINDINGS: Recent studies have begun to delineate the benefit and mechanisms of short-term therapy combining MSCs and low-dose immunosuppressive drugs in promoting graft acceptance and potentially regeneration. SUMMARY: The current review presents our rationale for the first-in-man clinical trial in liver transplantation utilizing a mesenchymal cell product (MultiStem, Athersys, Cleveland, Ohio, USA). The long-term objective of this program is to safely minimize the dose of complementary immunosuppressive drugs while achieving long-term allograft survival and operational tolerance. The use of adjunct cellular therapy as a means of reducing long-term pharmacotherapy would represent a major advancement in the field of liver transplantation.
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Transplante de Fígado/métodos , Transplante de Células-Tronco Mesenquimais/métodos , Células-Tronco Mesenquimais/citologia , Humanos , Terapia de Imunossupressão/métodos , Imunossupressores/administração & dosagem , Tolerância ao TransplanteRESUMO
BACKGROUND: Prescription drug monitoring programs (PDMPs) have been widely adopted as a tool to address the prescription opioid epidemic in the United States. PDMP integration and mandatory use policies are 2 approaches states have implemented to increase use of PDMPs by prescribers. While the effectiveness of these approaches is mixed, it is unclear what factors motivated states to implement them. This study examines whether opioid dispensing, adverse health outcomes, or other non-health-related factors motivated implementation of these PDMP approaches. METHODS: Time-to-event analysis was performed using lagged state-year covariates to reflect values from the year prior. Extended Cox regression estimated the association of states' rates of opioid dispensing, prescription opioid overdose deaths, and neonatal opioid withdrawal syndrome with implementation of PDMP integration and mandatory use policies from 2009 to 2020, controlling for demographic and economic factors, state government and political factors, and prior opioid policies. RESULTS: In our main model, prior opioid dispensing (HR 2.31, 95% CI 1.17, 4.57), neonatal opioid withdrawal syndrome hospitalizations (HR 1.55, 95% CI 1.09, 2.19), and number of prior opioid policies (HR 2.13, 95% CI 1.13, 4.00) were associated with mandatory use policies. Prior prescription opioid overdose deaths (HR 1.21, 95% CI 1.08, 1.35) were also associated with mandatory use policies in a model that did not include opioid dispensing or neonatal opioid withdrawal syndrome. No study variables were associated with implementation of PDMP integration. CONCLUSION: Understanding state-level factors associated with implementing PDMP approaches can provide insights into factors that motivate the adoption of future public health interventions.
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OBJECTIVE: Previous nonfatal overdose is a key risk factor for drug overdose death; however, current nonfatal overdose surveillance is limited to people who receive medical attention. We identified states that tracked nonfatal overdoses treated in prehospital and hospital care settings, assessed the effects of different surveillance methods on the magnitude of nonfatal overdose estimates, and estimated states' nonfatal-to-fatal overdose ratio. METHODS: Two analysts independently reviewed state websites to characterize states' methods of capturing nonfatal overdose events from December 2022 through February 2023. We collected information on surveillance methods in 5 states that met the inclusion criteria, including data source, measure specification, drug(s) involved, and whether states performed deduplication or published mutually exclusive measure specifications to capture unique events across care settings. We calculated nonfatal-to-fatal overdose ratios to assess the effects of different data sources on estimates of nonfatal overdoses. RESULTS: Illinois, Maine, North Carolina, and West Virginia used syndromic surveillance data and New Jersey used hospital discharge data to track nonfatal overdose-related emergency department visits. Illinois and West Virginia tracked nonfatal overdose-related encounters with emergency medical services. Other states tracked opioid overdoses reversed following naloxone administration by emergency medical services, law enforcement, and community members. Maine, New Jersey, and West Virginia published nonfatal overdose information by using mutually exclusive measure specifications; the number of nonfatal overdoses per fatal overdose in these states ranged from approximately 5:1 to 14:1. PRACTICE IMPLICATIONS: Establishing a standard framework to combine data from existing national surveillance systems in prehospital and hospital care settings can improve nonfatal overdose estimates and enable comparisons between jurisdictions to help decision makers identify areas most in need of essential services.
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BACKGROUND: Prescription drug monitoring programs (PDMPs) have been shown to reduce opioid prescribing for pain, but it is not well understood whether PDMPs influence utilization of medications for opioid use disorder. PDMP integration and mandatory use policies are two approaches implemented by states to increase use of PDMPs by prescribers. This study examined the effect of these approaches on distribution of methadone and buprenorphine from 2009 to 2021 for 50 states and DC. METHODS: The effect of PDMP integration and mandatory use policies on four outcomes (distribution of buprenorphine to opioid treatment programs, distribution of buprenorphine to pharmacies, distribution of methadone to opioid treatment programs, and the total combined distribution of methadone and buprenorphine) was estimated using a Callaway and Sant'Anna difference-in-differences model, controlling for co-occurring opioid-related state policies. RESULTS: Distribution of buprenorphine to pharmacies decreased 8 % (95 % CI -14 %, -1 %) following implementation of mandatory use policies. Distribution of methadone to opioid treatment programs increased 17 % (95 % CI 4 %, 34 %) and the total combined distribution of methadone and buprenorphine increased 6 % (95 % CI -0 %, 14 %) following the joint implementation of both approaches. CONCLUSION: Distribution of methadone and buprenorphine has increased since 2009, but less than a quarter of people with opioid use disorder currently receive these medications. We observed a small net benefit of PDMP integration and mandatory use policies on distribution of methadone and buprenorphine. Policymakers should continue to assess the impact of PDMPs on access to medications for opioid use disorder and consider additional approaches to increase access to treatment.
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The classical lineage of Mycobacterium ulcerans is the most prevalent clonal group associated with Buruli ulcer in humans. Its reservoir is strongly associated with the environment. We analyzed together 1,045 isolates collected from 13 countries on two continents to define the evolutionary history and population dynamics of this lineage. We confirm that this lineage spread over 7,000 years from Australia to Africa with the emergence of outbreaks in distinct waves in the 18th and 19th centuries. In sharp contrast with its global spread over the last century, transmission chains are now mostly local, with little or no dissemination between endemic areas. This study provides new insights into the phylogeography and population dynamics of M. ulcerans, highlighting the importance of comparative genomic analyses to improve our understanding of pathogen transmission. IMPORTANCE: Mycobacterium ulcerans is an environmental mycobacterial pathogen that can cause Buruli ulcer, a severe cutaneous infection, mostly spread in Africa and Australia. We conducted a large genomic study of M. ulcerans, combining genomic and evolutionary approaches to decipher its evolutionary history and pattern of spread at different geographic scales. At the scale of villages in an endemic area of Benin, the circulating genotypes have been introduced in recent decades and are not randomly distributed along the river. On a global scale, M. ulcerans has been spreading for much longer, resulting in distinct and compartmentalized endemic foci across Africa and Australia.
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Úlcera de Buruli , Mycobacterium ulcerans , Humanos , Mycobacterium ulcerans/genética , Úlcera de Buruli/epidemiologia , Úlcera de Buruli/microbiologia , Filogenia , Genômica , Evolução BiológicaRESUMO
Spores of Bacillus megaterium QM B1551 germinate rapidly when exposed to a number of single-trigger germinant compounds, including glucose, proline, leucine, and certain inorganic salts. However, spores of strain PV361, a plasmidless QM B1551 derivative that lacks the GerU germinant receptor (GR) responsible for mediating germination in response to single-trigger compounds, can germinate efficiently when incubated in nutritionally rich media, presumably via activation of additional germinant receptors. In this work, we have identified five chromosomally encoded GRs and attempted to characterize, by mutational analysis, germinant recognition profiles associated with the respective receptors in strain PV361. Of strains engineered with single GR insertion-deletions, only GerK-null spores displayed significant defective germination phenotypes when incubated in 5% (wt/vol) beef extract or plated on rich solid medium. Cumulative decreases in viability were observed in GerK-null spores that also lacked GerA or GerA2, indicating that these GRs, which exerted little effect on spore germination when disrupted individually, have a degree of functionality. Unexpectedly, an efficient germination response to combinations of germinants was restored in GerA(+) spores, which lack all other functional GRs, providing evidence for negative cooperativity between some GRs within the spore. Tetrazolium-based germinative assays conducted with purified spores indicated that these newly characterized B. megaterium GRs are cognate for a wide and chemically diverse range of germinant molecules, but unlike GerU, can only be induced to trigger germination when stimulated by at least two different germinants.
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Bacillus megaterium/metabolismo , Bacillus megaterium/fisiologia , Proteínas de Bactérias/metabolismo , Esporos Bacterianos/metabolismo , Esporos Bacterianos/fisiologia , Bacillus megaterium/genética , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica/genética , Regulação Bacteriana da Expressão Gênica/fisiologia , Esporos Bacterianos/genéticaRESUMO
OBJECTIVES: To (1) track the integration of telehealth- and COVID-19-related apps with electronic health records (EHRs) over time, (2) identify the primary functionality of apps designed to support the COVID-19 response, and (3) examine whether apps available prior to the pandemic added new telehealth- or COVID-19-related functionalities during the pandemic. STUDY DESIGN: Data were collected from public EHR app galleries on a monthly basis from December 31, 2019, through June 1, 2021. METHODS: Apps were identified as relating to COVID-19 or telehealth using text analysis of the app marketing materials. Descriptive analyses were conducted to characterize telehealth- and COVID-19-related apps discovered through the app galleries, identify their primary functionality, and examine whether any apps added new telehealth- or COVID-19-related functionalities during the pandemic. RESULTS: The number of COVID-19-related apps increased from 0 in March 2020 to 19 a month later and continued to grow to 62 as of June 2021. The number of telehealth-related apps more than doubled from prepandemic levels (n = 41) to a total of 87 apps by June 2021. These apps were 2 times more likely to contain specialized capabilities used to support COVID-19 response efforts, such as secure messaging, vaccine administration, and laboratory testing, compared with all apps listed in the EHR app galleries. CONCLUSIONS: These findings demonstrate the potential of integrating third-party apps into EHRs to expand the range of tools that health care providers can use to diagnose, treat, and communicate with patients.
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COVID-19 , Aplicativos Móveis , Telemedicina , Envio de Mensagens de Texto , Humanos , COVID-19/epidemiologia , Registros Eletrônicos de SaúdeRESUMO
OBJECTIVE: The aim of this study was to identify racial and ethnic disparities in patient portal offers, access, and use and to examine the role of providers in facilitating access to electronic health information (EHI) by offering patient portals and encouraging their use. MATERIALS AND METHODS: Using nationally representative survey data from 2019 and 2020 (N = 8028), we examined disparities in patients being offered access to a portal by their provider and differences in subsequent access and use. Using multivariable models, we estimated the effect of race and ethnicity on the likelihood of being offered, accessing or using a portal. Among those offered, we examined the relationship between provider encouragement and portal access; and for those who did not access their portal, we explored reasons for nonuse. RESULTS: Black and Hispanic individuals were offered and accessed patient portals at significantly lower rates than White individuals. Compared to Whites, Black and Hispanic individuals were 5.2 percentage-points less likely to be offered a portal (P < .05) and, among those offered, 7.9 percentage-points less likely to access their portal (P < .05). Black and Hispanic individuals who were offered and accessed a portal were 12 percentage-points more likely than Whites to use it to download or transmit information (P < .01). Individuals who were offered a portal and encouraged to use it were 21 percentage-points more likely to access it. DISCUSSION: Differences in patient portal access and use are likely driven by disparities in which groups of patients reported being offered a portal. CONCLUSIONS: Providers play an important role in increasing access to EHI by facilitating access to patient portals.
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Disparidades em Assistência à Saúde , Portais do Paciente , Papel do Médico , Humanos , Registros Eletrônicos de Saúde , Hispânico ou Latino , Portais do Paciente/estatística & dados numéricos , População Branca , Disparidades em Assistência à Saúde/etnologia , Negro ou Afro-AmericanoRESUMO
The development of direct inhibitors of KRASG12C represents a monumental step forward in the field of oncology. Nevertheless, there is considerable opportunity to enhance response rates to KRASG12C inhibitors. In this issue of Cancer Research, three investigative teams explore the modulation of KRASG12C inhibitor activity in lung, colorectal, and pancreatic cancers using CRISPR-based knockout screens. While each group identified and validated a variety of genes and pathways conferring resistance to KRASG12C inhibition, all three groups converged upon activation of YAP/TAZ as a common means of resistance. While coinhibition of KRASG12C and YAP/TAZ did not cause complete tumor regression in xenograft models, combining YAP/TAZ inhibition was capable of significantly extending the response of tumors to KRASG12C inhibition. See related articles by Mukhopadhyay et al., p. 4095, Edwards et al., p. 4112, and Prahallad et al., p. 4130.
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Proteínas Adaptadoras de Transdução de Sinal , Neoplasias Pancreáticas , Humanos , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transativadores/metabolismo , Proteínas de Sinalização YAP , Proteínas com Motivo de Ligação a PDZ com Coativador Transcricional , Cidade de Roma , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismoRESUMO
RAS GTPases are proto-oncoproteins that regulate cell growth, proliferation, and differentiation in response to extracellular signals. The signaling functions of RAS, and other small GTPases, are dependent on their ability to cycle between GDP-bound and GTP-bound states. Structural analyses suggest that GTP hydrolysis catalyzed by HRAS can be regulated by an allosteric site located between helices 3, 4, and loop 7. Here we explore the relationship between intrinsic GTP hydrolysis on HRAS and the position of helix 3 and loop 7 through manipulation of the allosteric site, showing that the two sites are functionally connected. We generated several hydrophobic mutations in the allosteric site of HRAS to promote shifts in helix 3 relative to helix 4. By combining crystallography and enzymology to study these mutants, we show that closure of the allosteric site correlates with increased hydrolysis of GTP on HRAS in solution. Interestingly, binding to the RAS binding domain of RAF kinase (RAF-RBD) inhibits GTP hydrolysis in the mutants. This behavior may be representative of a cluster of mutations found in human tumors, which potentially cooperate with RAF complex formation to stabilize the GTP-bound state of RAS.
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Quinases raf , Proteínas ras , Humanos , Sítio Alostérico , Hidrólise , Quinases raf/química , Quinases raf/genética , Quinases raf/metabolismo , Proteínas ras/genética , Proteínas ras/metabolismo , Guanosina Trifosfato/metabolismoRESUMO
RAS proteins are GTPases that regulate a wide range of cellular processes. RAS activity is dependent on its nucleotide-binding status, which is modulated by guanine nucleotide exchange factors (GEF) and GTPase-activating proteins (GAP). KRAS can be acetylated at lysine 104 (K104), and an acetylation-mimetic mutation of K104 to glutamine (K104Q) attenuates the in vitro-transforming capacity of oncogenic KRAS by interrupting GEF-induced nucleotide exchange. To assess the effect of this mutation in vivo, we used CRISPR-Cas9 to generate mouse models carrying the K104Q point mutation in wild-type and conditional KrasLSL-G12D alleles. Homozygous animals for K104Q were viable, fertile, and arose at the expected Mendelian frequency, indicating that K104Q is not a complete loss-of-function mutation. Consistent with our previous findings from in vitro studies, however, the oncogenic activity of KRASG12D was significantly attenuated by mutation at K104. Biochemical and structural analysis indicated that the G12D and K104Q mutations cooperate to suppress GEF-mediated nucleotide exchange, explaining the preferential effect of K104Q on oncogenic KRAS. Furthermore, K104 functioned in an allosteric network with M72, R73, and G75 on the α2 helix of the switch-II region. Intriguingly, point mutation of glycine 75 to alanine (G75A) also showed a strong negative regulatory effect on KRASG12D. These data demonstrate that lysine at position 104 is critical for the full oncogenic activity of mutant KRAS and suggest that modulating the sites in its allosteric network may provide a unique therapeutic approach in cancers expressing mutant KRAS. SIGNIFICANCE: An allosteric network formed by interaction between lysine 104 and residues in the switch-II domain is required for KRAS oncogenicity, which could be exploited for developing inhibitors of the activated oncoprotein.