ABSTRACT
Tumour oncogenes include transcription factors that co-opt the general transcriptional machinery to sustain the oncogenic state, but direct pharmacological inhibition of transcription factors has so far proven difficult. However, the transcriptional machinery contains various enzymatic cofactors that can be targeted for the development of new therapeutic candidates, including cyclin-dependent kinases (CDKs). Here we present the discovery and characterization of a covalent CDK7 inhibitor, THZ1, which has the unprecedented ability to target a remote cysteine residue located outside of the canonical kinase domain, providing an unanticipated means of achieving selectivity for CDK7. Cancer cell-line profiling indicates that a subset of cancer cell lines, including human T-cell acute lymphoblastic leukaemia (T-ALL), have exceptional sensitivity to THZ1. Genome-wide analysis in Jurkat T-ALL cells shows that THZ1 disproportionally affects transcription of RUNX1 and suggests that sensitivity to THZ1 may be due to vulnerability conferred by the RUNX1 super-enhancer and the key role of RUNX1 in the core transcriptional regulatory circuitry of these tumour cells. Pharmacological modulation of CDK7 kinase activity may thus provide an approach to identify and treat tumour types that are dependent on transcription for maintenance of the oncogenic state.
Subject(s)
Enzyme Inhibitors/pharmacology , Gene Expression Regulation, Neoplastic/drug effects , Phenylenediamines/pharmacology , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/enzymology , Pyrimidines/pharmacology , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Core Binding Factor Alpha 2 Subunit/metabolism , Cyclin-Dependent Kinases/antagonists & inhibitors , Cysteine/metabolism , Humans , Jurkat Cells , Phosphorylation/drug effectsABSTRACT
Clinical responses to anticancer therapies are often restricted to a subset of patients. In some cases, mutated cancer genes are potent biomarkers for responses to targeted agents. Here, to uncover new biomarkers of sensitivity and resistance to cancer therapeutics, we screened a panel of several hundred cancer cell lines--which represent much of the tissue-type and genetic diversity of human cancers--with 130 drugs under clinical and preclinical investigation. In aggregate, we found that mutated cancer genes were associated with cellular response to most currently available cancer drugs. Classic oncogene addiction paradigms were modified by additional tissue-specific or expression biomarkers, and some frequently mutated genes were associated with sensitivity to a broad range of therapeutic agents. Unexpected relationships were revealed, including the marked sensitivity of Ewing's sarcoma cells harbouring the EWS (also known as EWSR1)-FLI1 gene translocation to poly(ADP-ribose) polymerase (PARP) inhibitors. By linking drug activity to the functional complexity of cancer genomes, systematic pharmacogenomic profiling in cancer cell lines provides a powerful biomarker discovery platform to guide rational cancer therapeutic strategies.
Subject(s)
Drug Resistance, Neoplasm/genetics , Drug Screening Assays, Antitumor , Genes, Neoplasm/genetics , Genetic Markers/genetics , Genome, Human/genetics , Neoplasms/drug therapy , Neoplasms/genetics , Cell Line, Tumor , Cell Survival/drug effects , Drug Resistance, Neoplasm/drug effects , Gene Expression Regulation, Neoplastic/genetics , Genomics , Humans , Indoles/pharmacology , Neoplasms/pathology , Oncogene Proteins, Fusion/genetics , Pharmacogenetics , Phthalazines/pharmacology , Piperazines/pharmacology , Poly(ADP-ribose) Polymerase Inhibitors , Proto-Oncogene Protein c-fli-1/genetics , RNA-Binding Protein EWS/genetics , Sarcoma, Ewing/drug therapy , Sarcoma, Ewing/genetics , Sarcoma, Ewing/pathologyABSTRACT
BH3 mimetics such as ABT-263 induce apoptosis in a subset of cancer models. However, these drugs have shown limited clinical efficacy as single agents in small-cell lung cancer (SCLC) and other solid tumor malignancies, and rational combination strategies remain underexplored. To develop a novel therapeutic approach, we examined the efficacy of ABT-263 across >500 cancer cell lines, including 311 for which we had matched expression data for select genes. We found that high expression of the proapoptotic gene Bcl2-interacting mediator of cell death (BIM) predicts sensitivity to ABT-263. In particular, SCLC cell lines possessed greater BIM transcript levels than most other solid tumors and are among the most sensitive to ABT-263. However, a subset of relatively resistant SCLC cell lines has concomitant high expression of the antiapoptotic myeloid cell leukemia 1 (MCL-1). Whereas ABT-263 released BIM from complexes with BCL-2 and BCL-XL, high expression of MCL-1 sequestered BIM released from BCL-2 and BCL-XL, thereby abrogating apoptosis. We found that SCLCs were sensitized to ABT-263 via TORC1/2 inhibition, which led to reduced MCL-1 protein levels, thereby facilitating BIM-mediated apoptosis. AZD8055 and ABT-263 together induced marked apoptosis in vitro, as well as tumor regressions in multiple SCLC xenograft models. In a Tp53; Rb1 deletion genetically engineered mouse model of SCLC, the combination of ABT-263 and AZD8055 significantly repressed tumor growth and induced tumor regressions compared with either drug alone. Furthermore, in a SCLC patient-derived xenograft model that was resistant to ABT-263 alone, the addition of AZD8055 induced potent tumor regression. Therefore, addition of a TORC1/2 inhibitor offers a therapeutic strategy to markedly improve ABT-263 activity in SCLC.
Subject(s)
Aniline Compounds/therapeutic use , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Lung Neoplasms/drug therapy , Small Cell Lung Carcinoma/drug therapy , Sulfonamides/therapeutic use , Aniline Compounds/pharmacology , Animals , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Apoptosis/drug effects , Apoptosis Regulatory Proteins/metabolism , Bcl-2-Like Protein 11 , Cell Line, Tumor , Dose-Response Relationship, Drug , Genetic Engineering , Humans , Inhibitory Concentration 50 , Lung Neoplasms/pathology , Mechanistic Target of Rapamycin Complex 1 , Mechanistic Target of Rapamycin Complex 2 , Membrane Proteins/metabolism , Mice , Morpholines/pharmacology , Morpholines/therapeutic use , Multiprotein Complexes/antagonists & inhibitors , Multiprotein Complexes/metabolism , Myeloid Cell Leukemia Sequence 1 Protein/metabolism , Proto-Oncogene Proteins/metabolism , Remission Induction , Small Cell Lung Carcinoma/pathology , Sulfonamides/pharmacology , TOR Serine-Threonine Kinases/antagonists & inhibitors , TOR Serine-Threonine Kinases/metabolism , Xenograft Model Antitumor AssaysABSTRACT
BACKGROUND: The development of resistance to chemotherapies represents a significant barrier to successful cancer treatment. Resistance mechanisms are complex, can involve diverse and often unexpected cellular processes, and can vary with both the underlying genetic lesion and the origin or type of tumor. For these reasons developing experimental strategies that could be used to understand, identify and predict mechanisms of resistance in different malignant cells would be a major advance. METHODS: Here we describe a gain-of-function forward genetic approach for identifying mechanisms of resistance. This approach uses a modified piggyBac transposon to generate libraries of mutagenized cells, each containing transposon insertions that randomly activate nearby gene expression. Genes of interest are identified using next-gen high-throughput sequencing and barcode multiplexing is used to reduce experimental cost. RESULTS: Using this approach we successfully identify genes involved in paclitaxel resistance in a variety of cancer cell lines, including the multidrug transporter ABCB1, a previously identified major paclitaxel resistance gene. Analysis of co-occurring transposons integration sites in single cell clone allows for the identification of genes that might act cooperatively to produce drug resistance a level of information not accessible using RNAi or ORF expression screening approaches. CONCLUSION: We have developed a powerful pipeline to systematically discover drug resistance in mammalian cells in vitro. This cost-effective approach can be readily applied to different cell lines, to identify canonical or context specific resistance mechanisms. Its ability to probe complex genetic context and non-coding genomic elements as well as cooperative resistance events makes it a good complement to RNAi or ORF expression based screens.
Subject(s)
Antineoplastic Agents, Phytogenic/pharmacology , DNA Mutational Analysis/methods , DNA Transposable Elements/genetics , Drug Resistance, Neoplasm/genetics , Neoplasms/drug therapy , Paclitaxel/pharmacology , Blotting, Western , Cell Line, Tumor , DNA Barcoding, Taxonomic/methods , Humans , Mutagenesis, Insertional/methods , Neoplasms/genetics , RNA, Messenger/analysisABSTRACT
Combination of anti-cancer drugs is broadly seen as way to overcome the often-limited efficacy of single agents. The design and testing of combinations are however very challenging. Here we present a uniquely large dataset screening over 5000 targeted agent combinations across 81 non-small cell lung cancer cell lines. Our analysis reveals a profound heterogeneity of response across the tumor models. Notably, combinations very rarely result in a strong gain in efficacy over the range of response observable with single agents. Importantly, gain of activity over single agents is more often seen when co-targeting functionally proximal genes, offering a strategy for designing more efficient combinations. Because combinatorial effect is strongly context specific, tumor specificity should be achievable. The resource provided, together with an additional validation screen sheds light on major challenges and opportunities in building efficacious combinations against cancer and provides an opportunity for training computational models for synergy prediction.
Subject(s)
Carcinoma, Non-Small-Cell Lung , Lung Neoplasms , Humans , Carcinoma, Non-Small-Cell Lung/drug therapy , Carcinoma, Non-Small-Cell Lung/genetics , Lung Neoplasms/drug therapy , Lung Neoplasms/genetics , Drug CombinationsABSTRACT
ADAM metallopeptidase domain 9 (ADAM9) is a member of the ADAM family of multifunctional, multidomain type 1 transmembrane proteins. ADAM9 is overexpressed in many cancers, including non-small cell lung, pancreatic, gastric, breast, ovarian, and colorectal cancer, but exhibits limited expression in normal tissues. A target-unbiased discovery platform based on intact tumor and progenitor cell immunizations, followed by an IHC screen, led to the identification of anti-ADAM9 antibodies with selective tumor-versus-normal tissue binding. Subsequent analysis revealed anti-ADAM9 antibodies were efficiently internalized and processed by tumor cells making ADAM9 an attractive target for antibody-drug conjugate (ADC) development. Here, we describe the preclinical evaluation of IMGC936, a novel ADC targeted against ADAM9. IMGC936 is comprised of a high-affinity humanized antibody site-specifically conjugated to DM21-C, a next-generation linker-payload that combines a maytansinoid microtubule-disrupting payload with a stable tripeptide linker, at a drug antibody ratio of approximately 2.0. In addition, the YTE mutation (M252Y/S254T/T256E) was introduced into the CH2 domain of the antibody Fc to maximize in vivo plasma half-life and exposure. IMGC936 exhibited cytotoxicity toward ADAM9-positive human tumor cell lines, as well as bystander killing, potent antitumor activity in human cell line-derived xenograft and patient-derived xenograft tumor models, and an acceptable safety profile in cynomolgus monkeys with favorable pharmacokinetic properties. Our preclinical data provide a strong scientific rationale for the further development of IMGC936 as a therapeutic candidate for the treatment of ADAM9-positive cancers. A first-in-human study of IMGC936 in patients with advanced solid tumors has been initiated (NCT04622774).
Subject(s)
Immunoconjugates , ADAM Proteins , Cell Line, Tumor , Heterografts , Humans , Immunoconjugates/chemistry , Membrane Proteins/genetics , Xenograft Model Antitumor AssaysABSTRACT
Multidrug resistance (MDR) in cancer remains a major challenge for the success of chemotherapy. Natural products have been a rich source for the discovery of drugs against MDR cancers. Here, we applied high-throughput cytotoxicity screening of an in-house natural product library against MDR SGC7901/VCR cells and identified that the cyclodepsipeptide verucopeptin demonstrated notable antitumor potency. Cytological profiling combined with click chemistry-based proteomics revealed that ATP6V1G directly interacted with verucopeptin. ATP6V1G, a subunit of the vacuolar H+-ATPase (v-ATPase) that has not been previously targeted, was essential for SGC7901/VCR cell growth. Verucopeptin exhibited strong inhibition of both v-ATPase activity and mTORC1 signaling, leading to substantial pharmacological efficacy against SGC7901/VCR cell proliferation and tumor growth in vivo. Our results demonstrate that targeting v-ATPase via its V1G subunit constitutes a unique approach for modulating v-ATPase and mTORC1 signaling with great potential for the development of therapeutics against MDR cancers.
Subject(s)
Antineoplastic Agents/pharmacology , Biological Products/pharmacology , Depsipeptides/pharmacology , Drug Resistance, Multiple/drug effects , Drug Resistance, Neoplasm/drug effects , Vacuolar Proton-Translocating ATPases/antagonists & inhibitors , Animals , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Biological Products/chemical synthesis , Biological Products/chemistry , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Cells, Cultured , Depsipeptides/chemical synthesis , Depsipeptides/chemistry , Drug Screening Assays, Antitumor , Female , Humans , Male , Mechanistic Target of Rapamycin Complex 1/antagonists & inhibitors , Mechanistic Target of Rapamycin Complex 1/metabolism , Mice , Mice, Inbred BALB C , Mice, Nude , Neoplasms, Experimental/drug therapy , Neoplasms, Experimental/metabolism , Neoplasms, Experimental/pathology , Protein Subunits/drug effects , Proteomics , Vacuolar Proton-Translocating ATPases/metabolismABSTRACT
PURPOSE: Effective targeted therapies are lacking for refractory and relapsed T-cell acute lymphoblastic leukemia (T-ALL). Suppression of the NOTCH pathway using gamma-secretase inhibitors (GSI) is toxic and clinically not effective. The goal of this study was to identify alternative therapeutic strategies for T-ALL. EXPERIMENTAL DESIGN: We performed a comprehensive analysis of our high-throughput drug screen across hundreds of human cell lines including 15 T-ALL models. We validated and further studied the top hit, navitoclax (ABT-263). We used multiple human T-ALL cell lines as well as primary patient samples, and performed both in vitro experiments and in vivo studies on patient-derived xenograft models. RESULTS: We found that T-ALL are hypersensitive to navitoclax, an inhibitor of BCL2 family of antiapoptotic proteins. Importantly, GSI-resistant T-ALL are also susceptible to navitoclax. Sensitivity to navitoclax is due to low levels of MCL-1 in T-ALL. We identify an unsuspected regulation of mTORC1 by the NOTCH pathway, resulting in increased MCL-1 upon GSI treatment. Finally, we show that pharmacologic inhibition of mTORC1 lowers MCL-1 levels and further sensitizes cells to navitoclax in vitro and leads to tumor regressions in vivo. CONCLUSIONS: Our results support the development of navitoclax, as single agent and in combination with mTOR inhibitors, as a new therapeutic strategy for T-ALL, including in the setting of GSI resistance.
Subject(s)
Amyloid Precursor Protein Secretases/genetics , Myeloid Cell Leukemia Sequence 1 Protein/genetics , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/drug therapy , Receptor, Notch1/genetics , Amyloid Precursor Protein Secretases/antagonists & inhibitors , Aniline Compounds/pharmacology , Animals , Apoptosis/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Drug Resistance, Neoplasm/drug effects , Drug Resistance, Neoplasm/genetics , Heterografts , Humans , Mechanistic Target of Rapamycin Complex 1/genetics , Mice , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/genetics , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/pathology , Signal Transduction/drug effects , Sulfonamides/pharmacologyABSTRACT
Personalized cancer therapy is based on a patient's tumor lineage, histopathology, expression analyses, and/or tumor DNA or RNA analysis. Here, we aim to develop an in vitro functional assay of a patient's living cancer cells that could complement these approaches. We present methods for developing cell cultures from tumor biopsies and identify the types of samples and culture conditions associated with higher efficiency of model establishment. Toward the application of patient-derived cell cultures for personalized care, we established an immunofluorescence-based functional assay that quantifies cancer cell responses to targeted therapy in mixed cell cultures. Assaying patient-derived lung cancer cultures with this method showed promise in modeling patient response for diagnostic use. This platform should allow for the development of co-clinical trial studies to prospectively test the value of drug profiling on tumor-biopsy-derived cultures to direct patient care.
Subject(s)
Antineoplastic Agents/therapeutic use , Biomarkers, Tumor/genetics , Lung Neoplasms/drug therapy , Neoplasms/drug therapy , Precision Medicine/methods , Primary Cell Culture/methods , Acrylamides , Aminopyridines , Anaplastic Lymphoma Kinase , Aniline Compounds , Biomarkers, Tumor/metabolism , Biopsy , Crizotinib , ErbB Receptors/genetics , ErbB Receptors/metabolism , Erlotinib Hydrochloride/therapeutic use , Feeder Cells/cytology , Fluorescent Antibody Technique/methods , Gene Expression , High-Throughput Screening Assays , Humans , Keratin-18/genetics , Keratin-18/metabolism , Keratin-8/genetics , Keratin-8/metabolism , Lactams , Lactams, Macrocyclic/therapeutic use , Lung Neoplasms/genetics , Lung Neoplasms/metabolism , Lung Neoplasms/pathology , Mutation , Neoplasms/classification , Neoplasms/genetics , Neoplasms/pathology , Piperazines/therapeutic use , Pyrazoles/therapeutic use , Pyridines/therapeutic use , Receptor Protein-Tyrosine Kinases/genetics , Receptor Protein-Tyrosine Kinases/metabolism , Tumor Cells, CulturedABSTRACT
Fewer than half of children with high-risk neuroblastoma survive. Many of these tumors harbor high-level amplification of MYCN, which correlates with poor disease outcome. Using data from our large drug screen we predicted, and subsequently demonstrated, that MYCN-amplified neuroblastomas are sensitive to the BCL-2 inhibitor ABT-199. This sensitivity occurs in part through low anti-apoptotic BCL-xL expression, high pro-apoptotic NOXA expression, and paradoxical, MYCN-driven upregulation of NOXA. Screening for enhancers of ABT-199 sensitivity in MYCN-amplified neuroblastomas, we demonstrate that the Aurora Kinase A inhibitor MLN8237 combines with ABT-199 to induce widespread apoptosis. In diverse models of MYCN-amplified neuroblastoma, including a patient-derived xenograft model, this combination uniformly induced tumor shrinkage, and in multiple instances led to complete tumor regression.
Subject(s)
Apoptosis/genetics , Neuroblastoma/drug therapy , Aniline Compounds/therapeutic use , Antineoplastic Agents/therapeutic use , Cell Line, Tumor , Humans , N-Myc Proto-Oncogene Protein , Neuroblastoma/genetics , Neuroblastoma/pathology , Nuclear Proteins , Oncogene Proteins , Sulfonamides/therapeutic useABSTRACT
HECT domain ubiquitin ligases (HECT E3s), typified by human E6AP and yeast Rsp5p, are unique among the several classes of known ubiquitin ligases in that they participate directly in the chemistry of substrate ubiquitination reactions. This chapter discusses strategies for the expression of active HECT E3s and the assays that are available for analyzing E2 interaction, ubiquitin-thioester formation, and substrate ubiquitination.
Subject(s)
Ubiquitin-Protein Ligases/biosynthesis , Ubiquitin-Protein Ligases/genetics , Endosomal Sorting Complexes Required for Transport , Humans , Protein Structure, Tertiary , Saccharomyces cerevisiae/enzymology , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/analysis , Saccharomyces cerevisiae Proteins/biosynthesis , Saccharomyces cerevisiae Proteins/genetics , Ubiquitin-Conjugating Enzymes/metabolism , Ubiquitin-Protein Ligase Complexes/analysis , Ubiquitin-Protein Ligase Complexes/biosynthesis , Ubiquitin-Protein Ligase Complexes/genetics , Ubiquitin-Protein Ligases/analysisABSTRACT
Colorectal cancers harboring KRAS or BRAF mutations are refractory to current targeted therapies. Using data from a high-throughput drug screen, we have developed a novel therapeutic strategy that targets the apoptotic machinery using the BCL-2 family inhibitor ABT-263 (navitoclax) in combination with a TORC1/2 inhibitor, AZD8055. This combination leads to efficient apoptosis specifically in KRAS- and BRAF-mutant but not wild-type (WT) colorectal cancer cells. This specific susceptibility results from TORC1/2 inhibition leading to suppression of MCL-1 expression in mutant, but not WT, colorectal cancers, leading to abrogation of BIM/MCL-1 complexes. This combination strategy leads to tumor regressions in both KRAS-mutant colorectal cancer xenograft and genetically engineered mouse models of colorectal cancer, but not in the corresponding KRAS-WT colorectal cancer models. These data suggest that the combination of BCL-2/BCL-XL inhibitors with TORC1/2 inhibitors constitutes a promising targeted therapy strategy to treat these recalcitrant cancers.
Subject(s)
Aniline Compounds/therapeutic use , Antineoplastic Agents/therapeutic use , Colorectal Neoplasms/drug therapy , Morpholines/therapeutic use , Multiprotein Complexes/antagonists & inhibitors , Protein Kinase Inhibitors/therapeutic use , Sulfonamides/therapeutic use , TOR Serine-Threonine Kinases/antagonists & inhibitors , Aniline Compounds/pharmacology , Animals , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Colorectal Neoplasms/genetics , Humans , Mechanistic Target of Rapamycin Complex 1 , Mechanistic Target of Rapamycin Complex 2 , Mice , Mice, Mutant Strains , Mice, Nude , Morpholines/pharmacology , Mutation , Myeloid Cell Leukemia Sequence 1 Protein/antagonists & inhibitors , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins B-raf/genetics , Proto-Oncogene Proteins c-bcl-2/antagonists & inhibitors , Proto-Oncogene Proteins p21(ras) , Sulfonamides/pharmacology , bcl-X Protein/antagonists & inhibitors , ras Proteins/geneticsABSTRACT
ISG15 is an interferon (IFN)-alpha/beta-induced ubiquitin-like protein that is conjugated to cellular proteins during innate immune responses to viral and bacterial infections. A recent proteomics study identified 158 human proteins targeted for ISG15 conjugation, including the ISG15 E1 and E2 enzymes (Ube1L and UbcH8, respectively) and a HECT E3 enzyme, Herc5. Like the genes encoding Ube1L and UbcH8, expression of Herc5 was also induced by IFN-beta, suggesting that Herc5 might be a component of the ISG15 conjugation system. Consistent with this, small interfering RNAs targeting Herc5 had a dramatic effect on overall ISG15 conjugation in human cells, abrogating conjugation to the vast majority of ISG15 target proteins in vivo. In addition, co-transfection of plasmids expressing ISG15, Ube1L, UbcH8, and Herc5 resulted in robust ISG15 conjugation in non-IFN-treated cells, while the active-site cysteine mutant of Herc5 or a mutant lacking the RCC1 repeat region did not support ISG15 conjugation. These results demonstrate that Herc5 is required for conjugation of ISG15 to a broad spectrum of target proteins in human cells.
Subject(s)
Cytokines/metabolism , Interferon-beta/pharmacology , Intracellular Signaling Peptides and Proteins/physiology , Humans , Intracellular Signaling Peptides and Proteins/chemistry , RNA, Small Interfering/pharmacology , Ubiquitins/metabolismABSTRACT
The reaction cycle of HECT domain ubiquitin ligases consists of three steps: 1) binding of an E2 protein, 2) transfer of ubiquitin from E2 to the HECT domain, and 3) transfer of ubiquitin to the substrate. We report the identification of a determinant that is specifically required for the last step of this cycle, a phenylalanine residue located four amino acids from the C terminus of most HECT domains, referred to here as the -4F. Alteration of this residue in human E6AP and Saccharomyces cerevisae Rsp5p did not affect ubiquitin-thioester formation, but effectively blocked substrate ubiquitination. Alteration of the -4F to alanine with concomitant substitution of a nearby residue to phenylalanine only partially restored Rsp5p activity, indicating that precise spatial placement of this residue is important. C-terminally extended E6AP and Rsp5p proteins were also defective for substrate ubiquitination, providing a likely biochemical understanding of a previously isolated Angelman syndrome-associated mutation of E6AP that alters the stop codon of an otherwise wild-type gene. We propose that the -4F may play a role in orienting ubiquitin when it is tethered to the HECT active site cysteine. This may be necessary to allow for approach of the incoming lysine epsilon-amino group of the substrate.
Subject(s)
Phenylalanine , Ubiquitin-Protein Ligases/metabolism , Ubiquitin/metabolism , Adaptor Proteins, Signal Transducing , Amino Acid Sequence , Amino Acid Substitution , Binding Sites , Carrier Proteins/metabolism , Catalysis , Conserved Sequence , Humans , Saccharomyces cerevisiae Proteins , Sequence Alignment , Trans-Activators , Ubiquitin-Protein Ligases/chemistry , Ubiquitin-Protein Ligases/geneticsABSTRACT
Initiation of protein synthesis is a major post-transcriptional regulatory step in gene expression. The initiator tRNA gene from Mycobacterium smegmatis, a fast-growing mycobacterium, was characterized and compared with its counterpart from Mycobacterium tuberculosis, a slow-growing mycobacterium. In both mycobacteria, the functional initiator tRNA genes were found in a single copy. Unlike the M. tuberculosis initiator tRNA, the CCA end of the M. smegmatis initiator is not encoded in the gene, and it is most likely added post-transcriptionally. Transcription start site mapping allowed accurate assignment of the hexameric -10 and -35 promoter elements for both genes. These elements of the M. smegmatis initiator tRNA gene contain single nucleotide changes compared to their respective counterparts in the M. tuberculosis gene. Chloramphenicol acetyl transferase reporter assays suggested that the promoter of the initiator tRNA gene from M. smegmatis is twice as strong as that of M. tuberculosis, irrespective of whether the assays were performed in the fast-growing homologous host (M. smegmatis) or the slow-growing heterologous host (M. tuberculosis). Characterization of the M. smegmatis metU promoter, in this study, provides a valuable tool for the expression of genes in mycobacteria.