RESUMO
The characterization of ligand binding modes is a crucial step in the drug discovery process and is especially important in campaigns arising from phenotypic screening, where the protein target and binding mode are unknown at the outset. Elucidation of target binding regions is typically achieved by X-ray crystallography or photoaffinity labeling (PAL) approaches; yet, these methods present significant challenges. X-ray crystallography is a mainstay technique that has revolutionized drug discovery, but in many cases structural characterization is challenging or impossible. PAL has also enabled binding site mapping with peptide- and amino-acid-level resolution; however, the stoichiometric activation mode can lead to poor signal and coverage of the resident binding pocket. Additionally, each PAL probe can have its own fragmentation pattern, complicating the analysis by mass spectrometry. Here, we establish a robust and general photocatalytic approach toward the mapping of protein binding sites, which we define as identification of residues proximal to the ligand binding pocket. By utilizing a catalytic mode of activation, we obtain sets of labeled amino acids in the proximity of the target protein binding site. We use this methodology to map, in vitro, the binding sites of six protein targets, including several kinases and molecular glue targets, and furthermore to investigate the binding site of the STAT3 inhibitor MM-206, a ligand with no known crystal structure. Finally, we demonstrate the successful mapping of drug binding sites in live cells. These results establish µMap as a powerful method for the generation of amino-acid- and peptide-level target engagement data.
Assuntos
Peptídeos , Proteínas , Ligantes , Proteínas/química , Sítios de Ligação , Peptídeos/química , Ligação ProteicaRESUMO
Over half of new therapeutic approaches fail in clinical trials due to a lack of target validation. As such, the development of new methods to improve and accelerate the identification of cellular targets, broadly known as target ID, remains a fundamental goal in drug discovery. While advances in sequencing and mass spectrometry technologies have revolutionized drug target ID in recent decades, the corresponding chemical-based approaches have not changed in over 50 y. Consigned to outdated stoichiometric activation modes, modern target ID campaigns are regularly confounded by poor signal-to-noise resulting from limited receptor occupancy and low crosslinking yields, especially when targeting low abundance membrane proteins or multiple protein target engagement. Here, we describe a broadly general platform for photocatalytic small molecule target ID, which is founded upon the catalytic amplification of target-tag crosslinking through the continuous generation of high-energy carbene intermediates via visible light-mediated Dexter energy transfer. By decoupling the reactive warhead tag from the small molecule ligand, catalytic signal amplification results in unprecedented levels of target enrichment, enabling the quantitative target and off target ID of several drugs including (+)-JQ1, paclitaxel (Taxol), dasatinib (Sprycel), as well as two G-protein-coupled receptors-ADORA2A and GPR40.
Assuntos
Sistemas de Liberação de Medicamentos , Transferência de Energia , Proteômica , Descoberta de Drogas , Espectrometria de MassasRESUMO
Although current antiretroviral therapy can control HIV-1 replication and prevent disease progression, it is not curative. Identifying mechanisms that can lead to eradication of persistent viral reservoirs in people living with HIV-1 (PLWH) remains an outstanding challenge to achieving cure. Utilizing a phenotypic screen, we identified a novel chemical class capable of killing HIV-1 infected peripheral blood mononuclear cells. Tool compounds ICeD-1 and ICeD-2 ("inducer of cell death-1 and 2"), optimized for potency and selectivity from screening hits, were used to deconvolute the mechanism of action using a combination of chemoproteomic, biochemical, pharmacological, and genetic approaches. We determined that these compounds function by modulating dipeptidyl peptidase 9 (DPP9) and activating the caspase recruitment domain family member 8 (CARD8) inflammasome. Efficacy of ICeD-1 and ICeD-2 was dependent on HIV-1 protease activity and synergistic with efavirenz, which promotes premature activation of HIV-1 protease at high concentrations in infected cells. This in vitro synergy lowers the efficacious cell kill concentration of efavirenz to a clinically relevant dose at concentrations of ICeD-1 or ICeD-2 that do not result in complete DPP9 inhibition. These results suggest engagement of the pyroptotic pathway as a potential approach to eliminate HIV-1 infected cells.
Assuntos
Infecções por HIV , HIV-1 , Alcinos , Benzoxazinas , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Ciclopropanos , Dipeptidil Peptidases e Tripeptidil Peptidases/metabolismo , Infecções por HIV/tratamento farmacológico , HIV-1/metabolismo , Humanos , Inflamassomos/metabolismo , Leucócitos Mononucleares , Proteínas de Neoplasias/metabolismoRESUMO
Inhibiting the NLRP3 inflammasome mediates inflammation in an extensive number of preclinical models. As excitement in this field has grown, several companies have recently initiated testing of direct NLRP3 inhibitors in the clinic. At the same time, the NLRP3 inflammasome is part of a larger pro-inflammatory pathway, whose modulation is also being explored. Multiple targets in this pathway are already impinged upon by molecules that have been through clinical trials. These data, informed by the growing mechanistic understanding of the NLRP3 inflammasome in the preclinical space, provide a rich backdrop to assess the current state of the field. Here we explore attempts to inhibit the NLRP3 inflammasome in light of clinical and preclinical data around efficacy and safety.
Assuntos
Sistemas de Liberação de Medicamentos , Inflamassomos/efeitos dos fármacos , Proteína 3 que Contém Domínio de Pirina da Família NLR/antagonistas & inibidores , Animais , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Caspase 1/efeitos dos fármacos , Ensaios Clínicos como Assunto , Humanos , Inflamação/tratamento farmacológico , Interleucina-1beta/antagonistas & inibidores , Camundongos , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/fisiologia , Ligação Proteica , Receptores de Interleucina-1/antagonistas & inibidores , Transdução de SinaisRESUMO
Lysosomal acid lipase (LAL) is a serine hydrolase that hydrolyzes cholesteryl ester (CE) and TGs delivered to the lysosomes into free cholesterol and fatty acids. LAL deficiency due to mutations in the LAL gene (LIPA) results in accumulation of TGs and cholesterol esters in various tissues of the body leading to pathological conditions such as Wolman's disease and CE storage disease (CESD). Here, we present the first crystal structure of recombinant human LAL (HLAL) to 2.6 Å resolution in its closed form. The crystal structure was enabled by mutating three of the six potential glycosylation sites. The overall structure of HLAL closely resembles that of the evolutionarily related human gastric lipase (HGL). It consists of a core domain belonging to the classical α/ß hydrolase-fold family with a classical catalytic triad (Ser-153, His-353, Asp-324), an oxyanion hole, and a "cap" domain, which regulates substrate entry to the catalytic site. Most significant structural differences between HLAL and HGL exist at the lid region. Deletion of the short helix, 238NLCFLLC244, at the lid region implied a possible role in regulating the highly hydrophobic substrate binding site from self-oligomerization during interfacial activation. We also performed molecular dynamic simulations of dog gastric lipase (lid-open form) and HLAL to gain insights and speculated a possible role of the human mutant, H274Y, leading to CESD.
Assuntos
Doença do Armazenamento de Colesterol Éster/enzimologia , Esterol Esterase/química , Esterol Esterase/metabolismo , Doença do Armazenamento de Colesterol Éster/genética , Cristalografia por Raios X , Glicosilação , Humanos , Modelos Moleculares , Mutação , Domínios Proteicos , Esterol Esterase/genéticaRESUMO
The development of new antimicrobial drugs is a priority to combat the increasing spread of multidrug-resistant bacteria. This development is especially problematic in gram-negative bacteria due to the outer membrane (OM) permeability barrier and multidrug efflux pumps. Therefore, we screened for compounds that target essential, nonredundant, surface-exposed processes in gram-negative bacteria. We identified a compound, MRL-494, that inhibits assembly of OM proteins (OMPs) by the ß-barrel assembly machine (BAM complex). The BAM complex contains one essential surface-exposed protein, BamA. We constructed a bamA mutagenesis library, screened for resistance to MRL-494, and identified the mutation bamAE470K BamAE470K restores OMP biogenesis in the presence of MRL-494. The mutant protein has both altered conformation and activity, suggesting it could either inhibit MRL-494 binding or allow BamA to function in the presence of MRL-494. By cellular thermal shift assay (CETSA), we determined that MRL-494 stabilizes BamA and BamAE470K from thermally induced aggregation, indicating direct or proximal binding to both BamA and BamAE470K Thus, it is the altered activity of BamAE470K responsible for resistance to MRL-494. Strikingly, MRL-494 possesses a second mechanism of action that kills gram-positive organisms. In microbes lacking an OM, MRL-494 lethally disrupts the cytoplasmic membrane. We suggest that the compound cannot disrupt the cytoplasmic membrane of gram-negative bacteria because it cannot penetrate the OM. Instead, MRL-494 inhibits OMP biogenesis from outside the OM by targeting BamA. The identification of a small molecule that inhibits OMP biogenesis at the cell surface represents a distinct class of antibacterial agents.
Assuntos
Antibacterianos/farmacologia , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Escherichia coli/antagonistas & inibidores , Escherichia coli/efeitos dos fármacos , Multimerização Proteica/efeitos dos fármacos , Triazinas/farmacologia , Proteínas da Membrana Bacteriana Externa/antagonistas & inibidores , Proteínas da Membrana Bacteriana Externa/genética , Transporte Biológico/fisiologia , Membrana Celular/efeitos dos fármacos , Permeabilidade da Membrana Celular/fisiologia , Avaliação Pré-Clínica de Medicamentos , Farmacorresistência Bacteriana/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Testes de Sensibilidade MicrobianaRESUMO
Dorsal root ganglion (DRG) are a key tissue in the nervous system that have a role in neurological disease, particularly pain. Despite the importance of this tissue, the proteome of DRG is poorly understood, and it is unknown whether the proteome varies between organisms or different DRG along the spine. Therefore, we profiled the proteome of human and rat DRG. We identified 5,245 proteins in human DRG and 4959 proteins in rat DRG. Across species the proteome is largely conserved with some notable differences. While the most abundant proteins in both rat and human DRG played a role in extracellular functions and myelin sheeth, proteins detected only in humans mapped to roles in immune function whereas those detected only in rat mapped to roles in localization and transport. The DRG proteome between human T11 and L2 vertebrae was nearly identical indicating DRG from different vertebrae are representative of one another. Finally, we asked if this data could be used to enhance translatability by identifying mechanisms that modulate cellular phenotypes representative of pain in different species. Based on our data we tested and discovered that MAP4K4 inhibitor treatment increased neurite outgrowth in rat DRG as in human SH-SY5Y cells.
Assuntos
Gânglios Espinais/citologia , Neurônios/metabolismo , Proteoma/química , Animais , Linhagem Celular Tumoral , Células Cultivadas , Humanos , Proteoma/metabolismo , Ratos , Especificidade da EspécieRESUMO
Compound potency is a key metric that is often used to drive medicinal chemistry programs. Compound potency is also taken into account when identifying the mechanism of action of compounds whose pharmacological target is unknown, particularly when these compounds are identified in phenotypic screens. Often compound potency is determined from assays using recombinantly generated, purified protein. It is well understood in the medicinal chemistry community that potency measured with recombinant enzyme and potency measured in cell may not entirely coincide. Decreases in cellular vs recombinant potency are often anticipated or explainable. What is less often realized is that compound potency can increase in a cellular environment due to several factors including cellular metabolism of compounds, protein-protein interactions, post-translational modifications, and asymmetric intracellular localization of compound. Here we discuss these factors and highlight examples where increases in cellular compound potency were critical to the development of probes or drugs.
Assuntos
Química Farmacêutica/métodos , Composição de Medicamentos , Equivalência Terapêutica , Animais , Humanos , Preparações FarmacêuticasRESUMO
Bone metastasis is a deadly consequence of cancers, in which osteoclast forms a vicious cycle with tumor cells. Bone metastasis attenuation by clinical usage of osteoclast inhibitors and in our osteopetrotic mouse genetic models with ß-catenin constitutive activation or peroxisome proliferator-activated receptor γ deficiency fully support the important role of osteoclast in driving the bone metastatic niche. However, the mechanisms for this "partnership in crime" are underexplored. Here we show that osteoclasts reprogram their lipid secretion to support cancer cells. Metabolomic profiling reveals elevated prometastatic arachidonic acid (AA) but reduced antimetastatic lysophosphatidylcholines (LPCs). This shift in lipid osteoclastokines synergistically stimulates tumor cell proliferation, migration, survival, and expression of prometastatic genes. Pharmacologically, combined treatment with LPCs and BW-755C, an inhibitor of AA signaling via blocking lipoxygenase and cyclooxygenase, impedes breast cancer bone metastasis. Our findings elucidate key paracrine mechanisms for the osteoclast-cancer vicious cycle and uncover important therapeutic targets for bone metastasis.
Assuntos
Neoplasias Ósseas/secundário , Neoplasias da Mama/patologia , Citocinas/metabolismo , Metabolismo dos Lipídeos , Osteoclastos/fisiologia , Animais , Ácido Araquidônico/metabolismo , Movimento Celular , Feminino , Lisofosfatidilcolinas , Metabolômica , Camundongos Knockout , Camundongos Nus , Metástase Neoplásica , Transplante de Neoplasias , Osteogênese , Comunicação ParácrinaRESUMO
Loss of LIPA activity leads to diseases such as Wolman's Disease and Cholesterol Ester Storage Disease. While it is possible to measure defects in LIPA protein levels, it is difficult to directly measure LIPA activity in cells. In order to measure LIPA activity directly we developed a LIPA specific activity based probe. LIPA is heavily glycosylated although it is unclear how glycosylation affects LIPA activity or function. Our probe is specific for a glycosylated form of LIPA in cells, although it labels purified LIPA regardless of glycosylation.
Assuntos
Sondas Moleculares/análise , Sondas Moleculares/metabolismo , Esterol Esterase/química , Esterol Esterase/metabolismo , Glicosilação , Humanos , Simulação de Acoplamento Molecular , Sondas Moleculares/síntese química , Sondas Moleculares/química , Estrutura MolecularRESUMO
Nuclear receptors (NRs) are key regulators of gene expression and physiology. Nearly half of all human NRs lack endogenous ligands including estrogen-related receptor α (ERRα). ERRα has important roles in cancer, metabolism, and skeletal homeostasis. Affinity chromatography of tissue lipidomes with the ERRα ligand-binding domain (LBD) and subsequent transcriptional assays identified cholesterol as an endogenous ERRα agonist. Perturbation of cholesterol biosynthesis or inhibition of ERRα revealed the interdependence of cholesterol and ERRα. In bone, the effects of cholesterol, statin, and bisphosphonate on osteoclastogenesis require ERRα; and consequently, cholesterol-induced bone loss or bisphosphonate osteoprotection is lost in ERRα knockout mice. Furthermore, statin induction of muscle toxicity and cholesterol suppression of macrophage cytokine secretion are impaired by loss or inhibition of ERRα. These findings reveal a key step in ERRα regulation and explain the actions of two highly prescribed drugs, statins and bisphosphonates.
Assuntos
Colesterol/fisiologia , Difosfonatos/farmacologia , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Receptores de Estrogênio/fisiologia , Sinvastatina/farmacologia , Animais , Sítios de Ligação , Reabsorção Óssea , Diferenciação Celular , Linhagem Celular , Feminino , Ligantes , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Músculo Esquelético/efeitos dos fármacos , Osteoclastos/efeitos dos fármacos , Osteoclastos/fisiologia , Ligação Proteica , Domínios Proteicos , Receptor ERRalfa Relacionado ao EstrogênioRESUMO
Mitogen-activated protein kinase 4 (MAP4K4) regulates the MEK kinase cascade and is implicated in cytoskeletal rearrangement and migration; however, identifying MAP4K4 substrates has remained a challenge. To ascertain MAP4K4-dependent phosphorylation events, we combined phosphoproteomic studies of MAP4K4 inhibition with in vitro assessment of its kinase specificity. We identified 235 phosphosites affected by MAP4K4 inhibition in cells and found that pTP and pSP motifs were predominant among them. In contrast, in vitro assessment of kinase specificity showed that MAP4K4 favors a pTL motif. We showed that MAP4K4 directly phosphorylates and coimmunoprecipitates with FERM, RhoGEF, and pleckstrin domain-containing protein 1 (FARP1). MAP4K4 inhibition in SH-SY5Y cells increases neurite outgrowth, a process known to involve FARP1. As FARP1 and MAP4K4 both contribute to cytoskeletal rearrangement, the results suggest that MAP4K4 exerts some of its effects on the cytoskeleton via phosphorylation of FARP1.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Bioensaio , Células Hep G2 , Humanos , Estrutura Molecular , Fosforilação , ProteômicaRESUMO
Adiponectin is an important adipokine. Increasing evidence suggests that altered adiponectin levels are linked with metabolic and inflammatory disorders. Here we report an important yet previously unrecognized function of adiponectin in lactation by which maternal adiponectin determines the inflammatory status in the nursing neonates. Surprisingly, both maternal adiponectin overexpression in the transgenic mice and maternal adiponectin deletion in the knockout mice lead to systemic inflammation in the pups, manifested as transient hair loss. However, distinct mechanisms are involved. Adiponectin deficiency triggers leukocyte infiltration and production of inflammatory cytokines in the lactating mammary gland. In contrast, adiponectin overabundance increases lipid accumulation in the lactating mammary gland, resulting in excessive long-chain saturated fatty acids in milk. Interestingly, in both cases, the inflammation and alopecia in the pups can be rescued by Toll-like receptor (TLR)-2/4 deletion because TLR2/4 double-knockout pups are resistant. Mechanistically, long-chain saturated fatty acid activation of inflammatory genes is TLR2/4 dependent and can be potentiated by proinflammatory cytokines, indicating that the inflammatory stimuli in both scenarios functionally converge by activating the TLR2/4 signaling. Therefore, our findings reveal adiponectin as a dosage-dependent regulator of lactation homeostasis and milk quality that critically controls inflammation in the nursing neonates. Furthermore, these results suggest that inflammatory infantile disorders may result from maternal adiponectin dysregulation that can be treated by TLR2/4 inhibition.
Assuntos
Adiponectina/metabolismo , Citocinas/análise , Inflamação/prevenção & controle , Lactação/metabolismo , Lipídeos/análise , Leite/química , Adiponectina/genética , Animais , Animais Recém-Nascidos , Feminino , Inflamação/metabolismo , Glândulas Mamárias Animais/metabolismo , Camundongos , Camundongos Knockout , Camundongos TransgênicosRESUMO
The existence of nonannotated protein-coding human short open reading frames (sORFs) has been revealed through the direct detection of their sORF-encoded polypeptide (SEP) products. The discovery of novel SEPs increases the size of the genome and the proteome and provides insights into the molecular biology of mammalian cells, such as the prevalent usage of non-AUG start codons. Through modifications of the existing SEP-discovery workflow, we discover an additional 195 SEPs in K562 cells and extend this methodology to identify novel human SEPs in additional cell lines and human tissue for a final tally of 237 new SEPs. These results continue to expand the human genome and proteome and demonstrate that SEPs are a ubiquitous class of nonannotated polypeptides that require further investigation.
Assuntos
Neoplasias da Mama/química , Genoma Humano , Fases de Leitura Aberta , Peptídeos/análise , Proteoma/análise , Neoplasias da Mama/genética , Linhagem Celular , Cromatografia Líquida , Códon de Iniciação/química , Códon de Iniciação/genética , Feminino , Humanos , Células K562 , Peptídeos/química , Biossíntese de Proteínas , Proteoma/química , Espectrometria de Massas em TandemRESUMO
The application of ribosome profiling and mass spectrometry technologies has recently revealed that the human proteome is larger than previously appreciated. Short open reading frames (sORFs), which are difficult to identify using traditional gene-finding algorithms, constitute a significant fraction of unknown protein-coding genes. Thus, experimental approaches to identify sORFs provide invaluable insight into the protein-coding potential of genomes. Here, we report an affinity-based approach to enrich and identify cysteine-containing human sORF-encoded polypeptides (ccSEPs) from cells. This approach revealed 16 novel ccSEPs, each derived from an uncharacterized sORF, demonstrating its potential for discovering new genes. We validated expression of a SEP from its endogenous RNA, and demonstrated the specificity of our labeling approach using synthetic SEP. The discovery of additional human SEPs and their conservation indicate the potential importance of these molecules in biology.
Assuntos
Cisteína/genética , Fases de Leitura Aberta , Peptídeos/genética , Proteoma/genética , Proteômica/métodos , Sequência de Aminoácidos , Cisteína/química , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Peptídeos/químicaRESUMO
The crystal structure of Rad4/Rad23, the yeast homolog of the human nucleotide excision repair (NER) lesion recognition factor XPC-RAD23B ( Min , J. H. and Pavletich , N. P. ( 2007 ) Nature 449 , 570 - 575 ) reveals that the lesion-partner base is flipped out of the helix and binds to amino acids of the protein. This suggests the hypothesis that the flipping of this partner base must overcome a free energy barrier, which constitutes one element contributing to changes in the thermodynamic properties induced by the DNA damage and sensed by the recognition protein. We explored this hypothesis by computing complete flipping free energy profiles for two lesions derived from the procarcinogenic polycyclic aromatic hydrocarbons (PAHs), dibenzo[a,l]pyrene (DB[a,l]P) and benzo[a]pyrene (B[a]P), R-trans-anti-DB[a,l]P-N(6)-dA (R-DB[a,l]P-dA) and R-trans-anti-B[a]P-N(6)-dA (R-B[a]P-dA), and the corresponding unmodified duplex. The DB[a,l]P and B[a]P adducts differ in number and organization of their aromatic rings. We integrate these results with prior profiles for the R-trans-anti-DB[a,l]P-dG adduct ( Zheng , H. et al. ( 2010 ) Chem. Res. Toxicol. 23 , 1868 - 1870 ). All adopt conformational themes involving intercalation of the PAH aromatic ring system into the DNA duplex; however, R-DB[a,l]P-dA and R-B[a]P-dA intercalate from the major groove, while R-DB[a,l]P-dG intercalates from the minor groove. These structural differences produce different computed van der Waals stacking interaction energies between the flipping partner base with the lesion aromatic ring system and adjacent bases; we find that the better the stacking, the higher the relative flipping free energy barrier and hence lower flipping probability. The better relative NER susceptibilities correlate with greater ease of flipping in these three differently intercalated lesions. In addition to partner base flipping, the Rad4/Rad23 crystal structure shows that a protein-ß-hairpin, BHD3, intrudes from the major groove side between the DNA strands at the lesion site. We present a molecular modeling study for the R-DB[a,l]P-dG lesion in Rad4/Rad23 showing BHD3 ß-hairpin intrusion with lesion eviction, and we hypothesize that lesion steric effects play a role in the recognition of intercalated adducts.
Assuntos
Dano ao DNA , Reparo do DNA , DNA/química , DNA/efeitos dos fármacos , Hidrocarbonetos Policíclicos Aromáticos/farmacologia , Reparo do DNA/efeitos dos fármacos , Humanos , Simulação de Dinâmica Molecular , Conformação de Ácido Nucleico , Relação Estrutura-Atividade , TermodinâmicaRESUMO
The structural origins of differences in susceptibilities of various DNA lesions to nucleotide excision repair (NER) are poorly understood. Here we compared, in the same sequence context, the relative NER dual incision efficiencies elicited by two stereochemically distinct pairs of guanine (N(2)-dG) and adenine (N(6)-dA) DNA lesions, derived from enantiomeric genotoxic diol epoxides of the highly tumorigenic fjord region polycyclic aromatic hydrocarbon dibenzo[a,l]pyrene (DB[a,l]P). Remarkably, in cell-free HeLa cell extracts, the guanine adduct with R absolute chemistry at the N(2)-dG linkage site is â¼35 times more susceptible to NER dual incisions than the stereochemically identical N(6)-dA adduct. For the guanine and adenine adducts with S stereochemistry, a similar but somewhat smaller effect (factor of â¼15) is observed. The striking resistance of the bulky N(6)-dA in contrast to the modest to good susceptibilities of the N(2)-dG adducts to NER is interpreted in terms of the balance between lesion-induced DNA distorting and DNA stabilizing van der Waals interactions in their structures, that are partly reflected in the overall thermal stabilities of the modified duplexes. Our results are consistent with the hypothesis that the high genotoxic activity of DB[a,l]P is related to the formation of NER-resistant and persistent DB[a,l]P-derived adenine adducts in cellular DNA.
Assuntos
Adenina/química , Benzopirenos/farmacologia , Adutos de DNA/química , Adutos de DNA/efeitos dos fármacos , Reparo do DNA , Guanina/química , Benzopirenos/toxicidade , Células HeLa , Humanos , Modelos Moleculares , Simulação de Dinâmica Molecular , Estrutura Molecular , Relação Estrutura-Atividade , TemperaturaRESUMO
The complete extent to which the human genome is translated into polypeptides is of fundamental importance. We report a peptidomic strategy to detect short open reading frame (sORF)-encoded polypeptides (SEPs) in human cells. We identify 90 SEPs, 86 of which are previously uncharacterized, which is the largest number of human SEPs ever reported. SEP abundances range from 10-1,000 molecules per cell, identical to abundances of known proteins. SEPs arise from sORFs in noncoding RNAs as well as multicistronic mRNAs, and many SEPs initiate with non-AUG start codons, indicating that noncanonical translation may be more widespread in mammals than previously thought. In addition, coding sORFs are present in a small fraction (8 out of 1,866) of long intergenic noncoding RNAs. Together, these results provide strong evidence that the human proteome is more complex than previously appreciated.