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2.
Cancer Res ; 2020 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-31911550

RESUMO

Pancreatic cancer is a disease with limited therapeutic options. Resistance to chemotherapies poses a significant clinical challenge for pancreatic cancer patients and contributes to a high rate of recurrence. Here we showed that oncogenic KRAS, a critical driver of pancreatic cancer, promotes metabolic reprogramming and upregulates NRF2, a master regulator of the antioxidant network. NRF2 contributed to chemoresistance and was associated with a poor prognosis in pancreatic cancer patients. NRF2 activation metabolically rewired and elevated pathways involved in glutamine metabolism. This curbed chemoresistance in KRAS-mutant pancreatic cancers. Additionally, manipulating glutamine metabolism restrained the assembly of stress granules, an indicator of chemoresistance. Glutaminase inhibitors sensitized chemoresistant pancreatic cancer cells to gemcitabine, thereby improving the effectiveness of chemotherapy. This therapeutic approach holds promise as a novel therapy for pancreatic cancer patients harboring KRAS mutation.

3.
Elife ; 92020 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-31958057

RESUMO

The RAS proteins are GTP-dependent switches that regulate signaling pathways and are frequently mutated in cancer. RAS proteins concentrate in the plasma membrane via lipid-tethers and hypervariable side-chain interactions in distinct nano-domains. However, little is known about RAS membrane dynamics and the details of RAS activation of downstream signaling. Here we characterize RAS in live human and mouse cells using single molecule tracking methods and estimate RAS mobility parameters. KRAS4b exhibits confined mobility with three diffusive states distinct from the other RAS isoforms (KRAS4a, NRAS, and HRAS); and although most of the amino acid differences between RAS isoforms lie within the hypervariable region, the additional confinement of KRAS4b is largely determined by the protein's globular domain. To understand the altered mobility of an oncogenic KRAS4b we used complementary experimental and molecular dynamic simulation approaches to reveal a detailed mechanism.

4.
J Biol Chem ; 295(4): 1105-1119, 2020 Jan 24.
Artigo em Inglês | MEDLINE | ID: mdl-31836666

RESUMO

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.

6.
Life Sci Alliance ; 2(4)2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31296567

RESUMO

Plasma membrane (PM) curvature defines cell shape and intracellular organelle morphologies and is a fundamental cell property. Growth/proliferation is more stimulated in flatter cells than the same cells in elongated shapes. PM-anchored K-Ras small GTPase regulates cell growth/proliferation and plays key roles in cancer. The lipid-anchored K-Ras form nanoclusters selectively enriched with specific phospholipids, such as phosphatidylserine (PS), for efficient effector recruitment and activation. K-Ras function may, thus, be sensitive to changing lipid distribution at membranes with different curvatures. Here, we used complementary methods to manipulate membrane curvature of intact/live cells, native PM blebs, and synthetic liposomes. We show that the spatiotemporal organization and signaling of an oncogenic mutant K-Ras G12V favor flatter membranes with low curvature. Our findings are consistent with the more stimulated growth/proliferation in flatter cells. Depletion of endogenous PS abolishes K-Ras G12V PM curvature sensing. In cells and synthetic bilayers, only mixed-chain PS species, but not other PS species tested, mediate K-Ras G12V membrane curvature sensing. Thus, K-Ras nanoclusters act as relay stations to convert mechanical perturbations to mitogenic signaling.


Assuntos
Membrana Celular/enzimologia , Membrana Celular/ultraestrutura , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Linhagem Celular Tumoral , Membrana Celular/química , Células Epiteliais/metabolismo , Humanos , Lipossomos/metabolismo , Fosfatidilserinas/metabolismo , Isoformas de Proteínas/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética , Transdução de Sinais/genética , Análise Espacial , Análise Espaço-Temporal
7.
Sci Rep ; 9(1): 10512, 2019 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-31324887

RESUMO

Although post-translational modification of the C-terminus of RAS has been studied extensively, little is known about N-terminal processing. Mass spectrometric characterization of KRAS expressed in mammalian cells showed cleavage of the initiator methionine (iMet) and N-acetylation of the nascent N-terminus. Interestingly, structural studies on GDP- and GMPPNP-bound KRAS lacking the iMet and N-acetylation resulted in Mg2+-free structures of KRAS with flexible N-termini. In the Mg2+-free KRAS-GDP structure, the flexible N-terminus causes conformational changes in the interswitch region resulting in a fully open conformation of switch I. In the Mg2+-free KRAS-GMPPNP structure, the flexible N-terminus causes conformational changes around residue A59 resulting in the loss of Mg2+ and switch I in the inactive state 1 conformation. Structural studies on N-acetylated KRAS-GDP lacking the iMet revealed the presence of Mg2+ and a conformation of switch regions also observed in the structure of GDP-bound unprocessed KRAS with the iMet. In the absence of the iMet, the N-acetyl group interacts with the central beta-sheet and stabilizes the N-terminus and the switch regions. These results suggest there is crosstalk between the N-terminus and the Mg2+ binding site, and that N-acetylation plays an important role by stabilizing the N-terminus of RAS upon excision of the iMet.

8.
Cancers (Basel) ; 11(7)2019 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-31247990

RESUMO

Mutated forms of the RAS oncogene drive 30% of all cancers, but they cannot be targeted therapeutically using currently available drugs. The molecular and cellular mechanisms that create a heterogenous tumor environment harboring both mutant and wild-type RAS have not been elucidated. In this study, we examined horizontal transfer of mutant KRAS (Kirsten Rat Sarcoma Virus) between colorectal cancer (CRC) cells via a direct form of cell-to-cell communication called tunneling nanotubes (TNTs). TNT formation was significantly higher in CRC cell lines expressing mutant KRAS than CRC cell lines expressing wild-type RAS; this effect was most pronounced in metastatic CRC cell lines with both mutant KRAS and deficiency in mismatch repair proteins. Using inverted and confocal fluorescence time-lapse and fluorescence recovery after photobleaching (FRAP)-based microscopy, we observed GFP-tagged mutant KRASG12D protein trafficking between CRC cells through TNTs within a span of seconds to several minutes. Notably, acquisition of mutant KRAS increased Extracellular Signal-regulated Kinase (ERK) phosphorylation and upregulated tunneling nanotube formation in recipient wildtype CRC cells. In conclusion, these findings suggest that intercellular horizontal transfer of RAS can occur by TNTs. We propose that intercellular transfer of mutant RAS can potentially induce intratumoral heterogeneity and result in a more invasive phenotype in recipient cells.

9.
Biophys J ; 116(6): 1049-1063, 2019 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-30846362

RESUMO

Deregulation of KRAS4b signaling pathway has been implicated in 30% of all cancers. Membrane localization of KRAS4b is an essential step for the initiation of the downstream signaling cascades that guide various cellular mechanisms. KRAS4b plasma membrane (PM) binding is mediated by the insertion of a prenylated moiety that is attached to the terminal carboxy-methylated cysteine, in addition to electrostatic interactions of its positively charged hypervariable region with anionic lipids. Calmodulin (CaM) has been suggested to selectively bind KRAS4b to act as a negative regulator of the RAS/mitogen-activated protein kinase (MAPK) signaling pathway by displacing KRAS4b from the membrane. However, the mechanism by which CaM can recognize and displace KRAS4b from the membrane is not well understood. In this study, we employed biophysical and structural techniques to characterize this mechanism in detail. We show that KRAS4b prenylation is required for binding to CaM and that the hydrophobic pockets of CaM can accommodate the prenylated region of KRAS4b, which might represent a novel CaM-binding motif. Remarkably, prenylated KRAS4b forms a 2:1 stoichiometric complex with CaM in a nucleotide-independent manner. The interaction between prenylated KRAS4b and CaM is enthalpically driven, and electrostatic interactions also contribute to the formation of the complex. The prenylated KRAS4b terminal KSKTKC-farnesylation and carboxy-methylation is sufficient for binding and defines the minimal CaM-binding motif. This is the same region implicated in membrane and phosphodiesterase6-δ binding. Finally, we provide a structure-based docking model by which CaM binds to prenylated KRAS4b. Our data provide new insights into the KRAS4b-CaM interaction and suggest a possible mechanism whereby CaM can regulate KRAS4b membrane localization.

10.
J Biol Chem ; 294(6): 2193-2207, 2019 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-30559287

RESUMO

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.


Assuntos
Membrana Celular/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Calmodulina/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 6/metabolismo , Humanos , Sistema de Sinalização das MAP Quinases , Proteínas de Membrana/metabolismo , Membranas Artificiais , Domínios Proteicos , Proteínas Proto-Oncogênicas c-raf , Transdução de Sinais , Eletricidade Estática
11.
Oncotarget ; 9(41): 26431-26452, 2018 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-29899869

RESUMO

The most widely used cancer animal model is the human-murine tumor xenograft. Unbiased molecular dissection of tumor parenchyma versus stroma in human-murine xenografts is critical for elucidating dysregulated protein networks/pathways and developing therapeutics that may target these two functionally codependent compartments. Although antibody-reliant technologies (e.g., immunohistochemistry, imaging mass cytometry) are capable of distinguishing tumor-proper versus stromal proteins, the breadth or extent of targets is limited. Here, we report an antibody-free targeted cross-species glycoproteomic (TCSG) approach that enables direct dissection of human tumor parenchyma from murine tumor stroma at the molecular/protein level in tumor xenografts at a selectivity rate presently unattainable by other means. This approach was used to segment/dissect and obtain the protein complement phenotype of the tumor stroma and parenchyma of the metastatic human lung adenocarcinoma A549 xenograft, with no need for tissue microdissection prior to mass-spectrometry analysis. An extensive molecular map of the tumor proper and the associated microenvironment was generated along with the top functional N-glycosylated protein networks enriched in each compartment. Importantly, immunohistochemistry-based cross-validation of selected parenchymal and stromal targets applied on human tissue samples of lung adenocarcinoma and normal adjacent tissue is indicative of a noteworthy translational capacity for this unique approach that may facilitate identifications of novel targets for next generation antibody therapies and development of real time preclinical tumor models.

12.
Cell ; 170(1): 17-33, 2017 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-28666118

RESUMO

RAS proteins are binary switches, cycling between ON and OFF states during signal transduction. These switches are normally tightly controlled, but in RAS-related diseases, such as cancer, RASopathies, and many psychiatric disorders, mutations in the RAS genes or their regulators render RAS proteins persistently active. The structural basis of the switch and many of the pathways that RAS controls are well known, but the precise mechanisms by which RAS proteins function are less clear. All RAS biology occurs in membranes: a precise understanding of RAS' interaction with membranes is essential to understand RAS action and to intervene in RAS-driven diseases.


Assuntos
Proteínas ras/metabolismo , Animais , Membrana Celular/metabolismo , Anormalidades Congênitas/metabolismo , Humanos , Transtornos Mentais/metabolismo , Mutação , Neoplasias/metabolismo , Filogenia , Transdução de Sinais , Leveduras , Proteínas ras/química , Proteínas ras/genética
13.
Cancer Inform ; 16: 1176935117711944, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28634423

RESUMO

The 3 human RAS genes play pivotal roles regulating proliferation, differentiation, and survival in normal cells and become mutated in 15% to 20% of all human tumors and amplified in many others. In this report, we examined data from The Cancer Genome Atlas to investigate the relationship between RAS gene mutational status and messenger RNA expression. We show that all 3 RAS genes exhibit increased expression when they are mutated in a context-dependent manner. In the case of KRAS, this increase is manifested by a larger proportional increase in KRAS4A than KRAS4B, although both increase significantly. In addition, the mutational status of RAS genes can be associated with expression changes in other RAS genes, with most of these cases showing decreased expression. The mutational status associations with expression are recapitulated in cancer cell lines. Increases in expression are mediated by both copy number variation and contextual differences, including mutational status of epidermal growth factor receptor (EGFR) and BRAF. These findings potentially reveal an adaptive response during tumor evolution that is dependent on the mutational status of proximal genes in the RAS pathway and cellular context. Cell contextual differences in these adaptations may influence therapeutic responsiveness and alternative resistance mechanisms.

14.
Proc Natl Acad Sci U S A ; 113(44): E6766-E6775, 2016 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-27791178

RESUMO

Farnesylation and carboxymethylation of KRAS4b (Kirsten rat sarcoma isoform 4b) are essential for its interaction with the plasma membrane where KRAS-mediated signaling events occur. Phosphodiesterase-δ (PDEδ) binds to KRAS4b and plays an important role in targeting it to cellular membranes. We solved structures of human farnesylated-methylated KRAS4b in complex with PDEδ in two different crystal forms. In these structures, the interaction is driven by the C-terminal amino acids together with the farnesylated and methylated C185 of KRAS4b that binds tightly in the central hydrophobic pocket present in PDEδ. In crystal form II, we see the full-length structure of farnesylated-methylated KRAS4b, including the hypervariable region. Crystal form I reveals structural details of farnesylated-methylated KRAS4b binding to PDEδ, and crystal form II suggests the potential binding mode of geranylgeranylated-methylated KRAS4b to PDEδ. We identified a 5-aa-long sequence motif (Lys-Ser-Lys-Thr-Lys) in KRAS4b that may enable PDEδ to bind both forms of prenylated KRAS4b. Structure and sequence analysis of various prenylated proteins that have been previously tested for binding to PDEδ provides a rationale for why some prenylated proteins, such as KRAS4a, RalA, RalB, and Rac1, do not bind to PDEδ. Comparison of all four available structures of PDEδ complexed with various prenylated proteins/peptides shows the presence of additional interactions due to a larger protein-protein interaction interface in KRAS4b-PDEδ complex. This interface might be exploited for designing an inhibitor with minimal off-target effects.


Assuntos
3',5'-GMP Cíclico Fosfodiesterases/química , 3',5'-GMP Cíclico Fosfodiesterases/metabolismo , Domínios e Motivos de Interação entre Proteínas , Prenilação de Proteína/fisiologia , Proteínas Proto-Oncogênicas p21(ras)/química , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , 3',5'-GMP Cíclico Fosfodiesterases/genética , Sequência de Aminoácidos , Sítios de Ligação , Membrana Celular/metabolismo , Cristalografia por Raios X , Genes ras , Humanos , Metilação , Modelos Moleculares , Conformação Molecular , Mutação , Ligação Proteica/fisiologia , Proteínas Proto-Oncogênicas p21(ras)/genética , Análise de Sequência , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteínas ral de Ligação ao GTP/metabolismo
15.
Oncotarget ; 7(52): 86948-86971, 2016 Dec 27.
Artigo em Inglês | MEDLINE | ID: mdl-27894102

RESUMO

Oncogenic Ras mutants play a major role in the etiology of most aggressive and deadly carcinomas in humans. In spite of continuous efforts, effective pharmacological treatments targeting oncogenic Ras isoforms have not been developed. Cell-surface proteins represent top therapeutic targets primarily due to their accessibility and susceptibility to different modes of cancer therapy. To expand the treatment options of cancers driven by oncogenic Ras, new targets need to be identified and characterized at the surface of cancer cells expressing oncogenic Ras mutants. Here, we describe a mass spectrometry-based method for molecular profiling of the cell surface using KRasG12V transfected MCF10A (MCF10A-KRasG12V) as a model cell line of constitutively activated KRas and native MCF10A cells transduced with an empty vector (EV) as control. An extensive molecular map of the KRas surface was achieved by applying, in parallel, targeted hydrazide-based cell-surface capturing technology and global shotgun membrane proteomics to identify the proteins on the KRasG12V surface. This method allowed for integrated proteomic analysis that identified more than 500 cell-surface proteins found unique or upregulated on the surface of MCF10A-KRasG12V cells. Multistep bioinformatic processing was employed to elucidate and prioritize targets for cross-validation. Scanning electron microscopy and phenotypic cancer cell assays revealed changes at the cell surface consistent with malignant epithelial-to-mesenchymal transformation secondary to KRasG12V activation. Taken together, this dataset significantly expands the map of the KRasG12V surface and uncovers potential targets involved primarily in cell motility, cellular protrusion formation, and metastasis.


Assuntos
Proteínas de Membrana/análise , Proteínas Mutantes/análise , Proteômica/métodos , Proteínas Proto-Oncogênicas p21(ras)/análise , Antígenos CD/análise , Basigina/análise , Moléculas de Adesão Celular/análise , Linhagem Celular Tumoral , Movimento Celular , Biologia Computacional , Transição Epitelial-Mesenquimal , Glicoproteínas/classificação , Glicoproteínas/fisiologia , Humanos , Espectrometria de Massas , Microscopia Eletrônica de Varredura , Proteínas de Neoplasias/análise
16.
Sci Rep ; 5: 15916, 2015 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-26522388

RESUMO

Prenylated proteins play key roles in several human diseases including cancer, atherosclerosis and Alzheimer's disease. KRAS4b, which is frequently mutated in pancreatic, colon and lung cancers, is processed by farnesylation, proteolytic cleavage and carboxymethylation at the C-terminus. Plasma membrane localization of KRAS4b requires this processing as does KRAS4b-dependent RAF kinase activation. Previous attempts to produce modified KRAS have relied on protein engineering approaches or in vitro farnesylation of bacterially expressed KRAS protein. The proteins produced by these methods do not accurately replicate the mature KRAS protein found in mammalian cells and the protein yield is typically low. We describe a protocol that yields 5-10 mg/L highly purified, farnesylated, and methylated KRAS4b from insect cells. Farnesylated and methylated KRAS4b is fully active in hydrolyzing GTP, binds RAF-RBD on lipid Nanodiscs and interacts with the known farnesyl-binding protein PDEδ.


Assuntos
Lipídeos/fisiologia , Prenilação de Proteína/fisiologia , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Animais , Biofísica/métodos , Membrana Celular/metabolismo , Células Cultivadas , Guanosina Trifosfato/metabolismo , Humanos , Insetos/metabolismo , Metilação , Ligação Proteica/fisiologia , Quinases raf/metabolismo
17.
Biomark Med ; 8(2): 269-86, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24521024

RESUMO

The discovery of clinically relevant cancer biomarkers using mass spectrometry (MS)-based proteomics has proven difficult, primarily because of the enormous dynamic range of blood-derived protein concentrations and the fact that the 22 most abundant blood-derived proteins constitute approximately 99% of the total plasma protein mass. Immunodepletion of clinical body fluid specimens (e.g., serum/plasma) for the removal of highly abundant proteins is a reasonable and reproducible solution. Often overlooked, clinical tissue specimens also contain a formidable amount of highly abundant blood-derived proteins present in tissue-embedded networks of blood/lymph capillaries and interstitial fluid. Hence, the dynamic range impediment to biomarker discovery remains a formidable obstacle, regardless of clinical sample type (solid tissue and/or body fluid). Thus, we optimized and applied simultaneous immunodepletion of blood-derived proteins from solid tissue and peripheral blood, using clear cell renal cell carcinoma as a model disease. Integrative analysis of data from this approach and genomic data obtained from the same type of tumor revealed concordant key pathways and protein targets germane to clear cell renal cell carcinoma. This includes the activation of the lipogenic pathway characterized by increased expression of adipophilin (PLIN2) along with 'cadherin switching', a phenomenon indicative of transcriptional reprogramming linked to renal epithelial dedifferentiation. We also applied immunodepletion of abundant blood-derived proteins to various tissue types (e.g., adipose tissue and breast tissue) showing unambiguously that the removal of abundant blood-derived proteins represents a powerful tool for the reproducible profiling of tissue proteomes. Herein, we show that the removal of abundant blood-derived proteins from solid tissue specimens is of equal importance to depletion of body fluids and recommend its routine use in the context of biological discovery and/or cancer biomarker research. Finally, this perspective presents the background, rationale and strategy for using tissue-directed high-resolution/accuracy MS-based shotgun proteomics to detect genuine tumor proteins in the peripheral blood of a patient diagnosed with nonmetastatic cancer, employing concurrent liquid chromatography-MS analysis of immunodepleted clinical tissue and blood specimens.


Assuntos
Biomarcadores Tumorais/análise , Espectrometria de Massas , Anticorpos/imunologia , Biomarcadores Tumorais/sangue , Proteínas Sanguíneas/imunologia , Proteínas Sanguíneas/isolamento & purificação , Carcinoma de Células Renais/sangue , Carcinoma de Células Renais/metabolismo , Carcinoma de Células Renais/patologia , Cromatografia Líquida de Alta Pressão , Perfilação da Expressão Gênica , Humanos , Neoplasias Renais/sangue , Neoplasias Renais/metabolismo , Neoplasias Renais/patologia , Proteômica
18.
PLoS One ; 4(3): e4806, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19277127

RESUMO

OBJECTIVE: The use of antiretrovirals (ARV) during pregnancy has drastically reduced the rate of the human immunodeficiency virus perinatal transmission (MTCT). As a consequence of widespread ARV use, transmission of drug resistant strains from mothers to their babies is increasing. Ultra-sensitive PCR techniques have permitted the quantification of minority viral populations, but little is known about the transmission of drug-resistant HIV-1 minority population in the setting of MTCT. METHODOLOGY/PRINCIPAL FINDINGS: We describe the case of a female child born to an HIV-infected mother, which had not taken any ARV during the pregnancy. The child's first genotype demonstrated a minor non-nucleoside reverse transcriptase inhibitor (K101E), and during her treatment with reverse transcriptase and protease inhibitors full resistance to non-nucleoside reverse transcriptase inhibitors (NNRTI) emerged (G190A). Phenotypic/genotypic analysis of variant quasispecies through yeast TyHRT assay was conducted to characterize minority resistant viral strains circulating in both mother and child. Maximum likelihood and Bayesian MCMC phylogenetic analyses were performed with samples from the pair to assess genetic relatedness among minor viral strains. The analysis showed that the child received a minor NNRTI resistant variant, containing the mutation K101E that was present in less than 1% of the mother's quasispecies. Phylogenetic analyses have suggested common ancestry between the mother's virus strain carrying K101E with the viral sequences from the child. CONCLUSION: This is the first documentation of MTCT of a minority resistant strain of HIV-1. The transmission of minor resistant variants carries the threat of emergence of multi-drug primary mutations without identified specific selective pressures.


Assuntos
Terapia Antirretroviral de Alta Atividade , Farmacorresistência Viral Múltipla/genética , Infecções por HIV/virologia , HIV-1/genética , Transmissão Vertical de Doença Infecciosa , Adulto , Didanosina/administração & dosagem , Didanosina/farmacologia , Didanosina/uso terapêutico , Feminino , Infecções por HIV/congênito , Infecções por HIV/tratamento farmacológico , Infecções por HIV/transmissão , HIV-1/efeitos dos fármacos , HIV-1/isolamento & purificação , Humanos , Recém-Nascido , Lamivudina/administração & dosagem , Lamivudina/farmacologia , Lamivudina/uso terapêutico , Mutação de Sentido Incorreto , Nelfinavir/administração & dosagem , Nelfinavir/farmacologia , Nelfinavir/uso terapêutico , Filogenia , Mutação Puntual , Gravidez , Complicações Infecciosas na Gravidez/virologia , Seleção Genética , Zidovudina/administração & dosagem , Zidovudina/farmacologia , Zidovudina/uso terapêutico
19.
J Virol ; 83(8): 3826-33, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19193782

RESUMO

We previously identified a rare mutation in human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT), I132M, which confers high-level resistance to the nonnucleoside RT inhibitors (NNRTIs) nevirapine and delavirdine. In this study, we have further characterized the role of this mutation in viral replication capacity and in resistance to other RT inhibitors. Surprisingly, our data show that I132M confers marked hypersusceptibility to the nucleoside analogs lamivudine (3TC) and tenofovir at both the virus and enzyme levels. Subunit-selective mutagenesis studies revealed that the mutation in the p51 subunit of RT was responsible for the increased sensitivity to the drugs, and transient kinetic analyses showed that this hypersusceptibility was due to I132M decreasing the enzyme's affinity for the natural dCTP substrate but increasing its affinity for 3TC-triphosphate. Furthermore, the replication capacity of HIV-1 containing I132M is severely impaired. This decrease in viral replication capacity could be partially or completely compensated for by the A62V or L214I mutation, respectively. Taken together, these results help to explain the infrequent selection of I132M in patients for whom NNRTI regimens are failing and furthermore demonstrate that a single mutation outside of the polymerase active site and inside of the p51 subunit of RT can significantly influence nucleotide selectivity.


Assuntos
Fármacos Anti-HIV/farmacologia , Farmacorresistência Viral , Transcriptase Reversa do HIV/genética , HIV-1/efeitos dos fármacos , HIV-1/genética , Mutação de Sentido Incorreto , Inibidores da Transcriptase Reversa/farmacologia , Adenina/análogos & derivados , Adenina/farmacologia , Delavirdina/farmacologia , Humanos , Cinética , Lamivudina/farmacologia , Testes de Sensibilidade Microbiana , Nevirapina/farmacologia , Organofosfonatos/farmacologia , Ligação Proteica , Tenofovir
20.
Biochem J ; 404(1): 151-7, 2007 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-17286555

RESUMO

Several rare and novel NNRTI [non-nucleoside reverse transcriptase (RT) inhibitor] resistance mutations were recently detected at codons 132 and 135 in RTs from clinical isolates using the yeast-based chimaeric TyHRT (Ty1/HIV-1 RT) phenotypic assay. Ile132 and Ile135 form part of the beta7-beta8 loop of HIV-1 RT (residues 132-140). To elucidate the contribution of these residues in RT structure-function and drug resistance, we constructed twelve recombinant enzymes harbouring mutations at codons 132 and 135-140. Several of the mutant enzymes exhibited reduced DNA polymerase activities. Using the yeast two-hybrid assay for HIV-1 RT dimerization we show that in some instances this decrease in enzyme activity could be attributed to the mutations, in the context of the 51 kDa subunit of HIV-1 RT, disrupting the subunit-subunit interactions of the enzyme. Drug resistance analyses using purified RT, the TyHRT assay and antiviral assays demonstrated that the I132M mutation conferred high-level resistance (>10-fold) to nevirapine and delavirdine and low-level resistance (approximately 2-3-fold) to efavirenz. The I135A and I135M mutations also conferred low level NNRTI resistance (approximately 2-fold). Subunit selective mutagenesis studies again demonstrated that resistance was conferred via the p51 subunit of HIV-1 RT. Taken together, our results highlight a specific role of residues 132 and 135 in NNRTI resistance and a general role for residues in the beta7-beta8 loop in the stability of HIV-1 RT.


Assuntos
Transcriptase Reversa do HIV/metabolismo , Inibidores da Transcriptase Reversa/farmacologia , Dicroísmo Circular , Clonagem Molecular , DNA Viral/biossíntese , Transcriptase Reversa do HIV/química , Transcriptase Reversa do HIV/genética , HIV-1/enzimologia , Modelos Moleculares , Conformação Molecular , Conformação Proteica , Subunidades Proteicas/metabolismo , RNA Viral/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Inibidores da Transcriptase Reversa/química , Saccharomyces cerevisiae/genética
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