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The complexity of the functional proteome extends considerably beyond the coding genome, resulting in millions of proteoforms. Investigation of proteoforms and their functional roles is important to understand cellular physiology and its deregulation in diseases but challenging to perform systematically. Here we applied thermal proteome profiling with deep peptide coverage to detect functional proteoform groups in acute lymphoblastic leukemia cell lines with different cytogenetic aberrations. We detected 15,846 proteoforms, capturing differently spliced, cleaved and post-translationally modified proteins expressed from 9,290 genes. We identified differential co-aggregation of proteoform pairs and established links to disease biology. Moreover, we systematically made use of measured biophysical proteoform states to find specific biomarkers of drug sensitivity. Our approach, thus, provides a powerful and unique tool for systematic detection and functional annotation of proteoform groups.
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Proteoma , Espectrometria de Massas em Tandem , Proteoma/metabolismo , Espectrometria de Massas em Tandem/métodos , Linhagem CelularRESUMO
Cancer heterogeneity at the proteome level may explain differences in therapy response and prognosis beyond the currently established genomic and transcriptomic-based diagnostics. The relevance of proteomics for disease classifications remains to be established in clinically heterogeneous cancer entities such as chronic lymphocytic leukemia (CLL). Here, we characterize the proteome and transcriptome alongside genetic and ex-vivo drug response profiling in a clinically annotated CLL discovery cohort (n = 68). Unsupervised clustering of the proteome data reveals six subgroups. Five of these proteomic groups are associated with genetic features, while one group is only detectable at the proteome level. This new group is characterized by accelerated disease progression, high spliceosomal protein abundances associated with aberrant splicing, and low B cell receptor signaling protein abundances (ASB-CLL). Classifiers developed to identify ASB-CLL based on its characteristic proteome or splicing signature in two independent cohorts (n = 165, n = 169) confirm that ASB-CLL comprises about 20% of CLL patients. The inferior overall survival in ASB-CLL is also independent of both TP53- and IGHV mutation status. Our multi-omics analysis refines the classification of CLL and highlights the potential of proteomics to improve cancer patient stratification beyond genetic and transcriptomic profiling.
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Leucemia Linfocítica Crônica de Células B , Proteogenômica , Humanos , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/metabolismo , Proteômica , Proteoma/genética , Mutação , Receptores de Antígenos de Linfócitos B/metabolismoRESUMO
Acute lymphoblastic leukemia (ALL) is the most common childhood cancer. Although standard-of-care chemotherapeutics are sufficient for most ALL cases, there are subsets of patients with poor response who relapse in disease. The biology underlying differences between subtypes and their response to therapy has only partially been explained by genetic and transcriptomic profiling. Here, we perform comprehensive multi-omic analyses of 49 readily available childhood ALL cell lines, using proteomics, transcriptomics, and pharmacoproteomic characterization. We connect the molecular phenotypes with drug responses to 528 oncology drugs, identifying drug correlations as well as lineage-dependent correlations. We also identify the diacylglycerol-analog bryostatin-1 as a therapeutic candidate in the MEF2D-HNRNPUL1 fusion high-risk subtype, for which this drug activates pro-apoptotic ERK signaling associated with molecular mediators of pre-B cell negative selection. Our data is the foundation for the interactive online Functional Omics Resource of ALL (FORALL) with navigable proteomics, transcriptomics, and drug sensitivity profiles at https://proteomics.se/forall .
Assuntos
Perfilação da Expressão Gênica , Leucemia-Linfoma Linfoblástico de Células Precursoras , Linhagem Celular , Humanos , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Proteômica , TranscriptomaRESUMO
The human mitochondrial ClpXP protease complex (HsClpXP) has recently attracted major attention as a target for novel anti-cancer therapies. Despite its important role in disease progression, the cellular role of HsClpXP is poorly characterized and only few small molecule inhibitors have been reported. Herein, we screened previously established S. aureus ClpXP inhibitors against the related human protease complex and identified potent small molecules against human ClpXP. The hit compounds showed anti-cancer activity in a panoply of leukemia, liver and breast cancer cell lines. We found that the bacterial ClpXP inhibitor 334 impairs the electron transport chain (ETC), enhances the production of mitochondrial reactive oxygen species (mtROS) and thereby promotes protein carbonylation, aberrant proteostasis and apoptosis. In addition, 334 induces cell death in re-isolated patient-derived xenograft (PDX) leukemia cells, potentiates the effect of DNA-damaging cytostatics and re-sensitizes resistant cancers to chemotherapy in non-apoptotic doses.
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Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Proteínas Mitocondriais/antagonistas & inibidores , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Staphylococcus aureusRESUMO
Molecular classification of acute myeloid leukemia (AML) aids prognostic stratification and clinical management. Our aim in this study is to identify transcriptome-wide mRNAs that are specific to each of the molecular subtypes of AML. We analyzed RNA-sequencing data of 955 AML samples from three cohorts, including the BeatAML project, the Cancer Genome Atlas, and a cohort of Swedish patients to provide a comprehensive transcriptome-wide view of subtype-specific mRNA expression. We identified 729 subtype-specific mRNAs, discovered in the BeatAML project and validated in the other two cohorts. Using unique proteomics data, we also validated the presence of subtype-specific mRNAs at the protein level, yielding a rich collection of potential protein-based biomarkers for the AML community. To enable the exploration of subtype-specific mRNA expression by the broader scientific community, we provide an interactive resource to the public.
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Leucemia Mieloide Aguda/genética , RNA Mensageiro/biossíntese , RNA Neoplásico/biossíntese , Transcriptoma , Biomarcadores Tumorais , Genes Neoplásicos , Humanos , Leucemia Mieloide Aguda/classificação , Leucemia Mieloide Aguda/metabolismo , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Proteínas de Fusão Oncogênica/biossíntese , Proteínas de Fusão Oncogênica/genética , Proteoma , RNA Mensageiro/genética , RNA Neoplásico/genética , RNA-Seq , Estudos Retrospectivos , SuéciaRESUMO
Legionella pneumophila infects eukaryotic cells by forming a replicative organelle - the Legionella containing vacuole. During this process, the bacterial protein DrrA/SidM is secreted and manipulates the activity and post-translational modification (PTM) states of the vesicular trafficking regulator Rab1. As a result, Rab1 is modified with an adenosine monophosphate (AMP), and this process is referred to as AMPylation. Here, we use a chemical approach to stabilise low-affinity Rab:DrrA complexes in a site-specific manner to gain insight into the molecular basis of the interaction between the Rab protein and the AMPylation domain of DrrA. The crystal structure of the Rab:DrrA complex reveals a previously unknown non-conventional Rab-binding site (NC-RBS). Biochemical characterisation demonstrates allosteric stimulation of the AMPylation activity of DrrA via Rab binding to the NC-RBS. We speculate that allosteric control of DrrA could in principle prevent random and potentially cytotoxic AMPylation in the host, thereby perhaps ensuring efficient infection by Legionella.
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Monofosfato de Adenosina/metabolismo , Proteínas de Bactérias/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Legionella pneumophila/patogenicidade , Doença dos Legionários/patologia , Proteínas rab1 de Ligação ao GTP/metabolismo , Regulação Alostérica , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/ultraestrutura , Sítios de Ligação/genética , Cristalografia por Raios X , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/isolamento & purificação , Fatores de Troca do Nucleotídeo Guanina/ultraestrutura , Guanosina Trifosfato/metabolismo , Humanos , Legionella pneumophila/metabolismo , Doença dos Legionários/microbiologia , Macrófagos Alveolares/metabolismo , Macrófagos Alveolares/microbiologia , Fagocitose , Ligação Proteica , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Proteínas rab1 de Ligação ao GTP/genética , Proteínas rab1 de Ligação ao GTP/isolamento & purificação , Proteínas rab1 de Ligação ao GTP/ultraestruturaRESUMO
The original version of this Article contained errors in Fig. 1 and Supplementary Fig. 3. In Fig. 1, the labels indicating the Cx32wt constructs in panels d and e were incorrectly shifted with respect to the relevant western blot lanes. In Supplementary Fig. 3, numbers of unique peptides and % sequence coverage were incorrectly reported as being for wt and L90H separately, and should refer to wt and L90H combined. These errors have been corrected in the PDF and HTML versions of the Article.
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A fundamental step in membrane protein biogenesis is their integration into the lipid bilayer with a defined orientation of each transmembrane segment. Despite this, it remains unclear how cells detect and handle failures in this process. Here we show that single point mutations in the membrane protein connexin 32 (Cx32), which cause Charcot-Marie-Tooth disease, can cause failures in membrane integration. This leads to Cx32 transport defects and rapid degradation. Our data show that multiple chaperones detect and remedy this aberrant behavior: the ER-membrane complex (EMC) aids in membrane integration of low-hydrophobicity transmembrane segments. If they fail to integrate, these are recognized by the ER-lumenal chaperone BiP. Ultimately, the E3 ligase gp78 ubiquitinates Cx32 proteins, targeting them for degradation. Thus, cells use a coordinated system of chaperones for the complex task of membrane protein biogenesis, which can be compromised by single point mutations, causing human disease.
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Bicamadas Lipídicas/metabolismo , Chaperonas Moleculares/metabolismo , Animais , Células COS , Doença de Charcot-Marie-Tooth/genética , Doença de Charcot-Marie-Tooth/metabolismo , Chlorocebus aethiops , Conexinas/genética , Conexinas/metabolismo , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Chaperona BiP do Retículo Endoplasmático , Junções Comunicantes/metabolismo , Células HEK293 , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Chaperonas Moleculares/genética , Mutação , Proteína beta-1 de Junções ComunicantesRESUMO
Pyridoxal phosphate (PLP) is an enzyme cofactor required for the chemical transformation of biological amines in many central cellular processes. PLP-dependent enzymes (PLP-DEs) are ubiquitous and evolutionarily diverse, making their classification based on sequence homology challenging. Here we present a chemical proteomic method for reporting on PLP-DEs using functionalized cofactor probes. We synthesized pyridoxal analogues modified at the 2'-position, which are taken up by cells and metabolized in situ. These pyridoxal analogues are phosphorylated to functional cofactor surrogates by cellular pyridoxal kinases and bind to PLP-DEs via an aldimine bond which can be rendered irreversible by NaBH4 reduction. Conjugation to a reporter tag enables the subsequent identification of PLP-DEs using quantitative, label-free mass spectrometry. Using these probes we accessed a significant portion of the Staphylococcus aureus PLP-DE proteome (73%) and annotate uncharacterized proteins as novel PLP-DEs. We also show that this approach can be used to study structural tolerance within PLP-DE active sites and to screen for off-targets of the PLP-DE inhibitor D-cycloserine.
Assuntos
Alanina Racemase/metabolismo , Coenzimas/química , Coenzimas/metabolismo , Dopa Descarboxilase/metabolismo , Glicina Hidroximetiltransferase/metabolismo , Ornitina Descarboxilase/metabolismo , Fosfato de Piridoxal/metabolismo , Transaminases/metabolismo , Alanina Racemase/química , Dopa Descarboxilase/química , Glicina Hidroximetiltransferase/química , Cinética , Modelos Moleculares , Estrutura Molecular , Ornitina Descarboxilase/química , Fosforilação , Fosfato de Piridoxal/química , Transaminases/químicaRESUMO
Caseinolytic proteaseâ P (ClpP) is the proteolytic component of the ClpXP protein degradation complex. Eukaryotic ClpP was recently found to act within the mitochondria-specific unfolded protein response (UPRmt ). However, its detailed function and dedicated regulation remain largely unexplored. A small molecule (D9) acts as a potent and species-selective activator of human ClpP (hClpP) by mimicking the natural chaperone ClpX. Structure-activity relationship studies highlight the importance of a halogenated benzyl motif within D9 that interacts with a unique aromatic amino acid network in hClpP. Mutational and structural studies suggest that this YYW motif tightly controls hClpP activity and regulates substrate turnover by interaction with cognate ligands. This signature motif is unique to ClpP from higher organisms and does not exist in tested bacterial homologues, allowing a species-selective analysis. Thus, D9 is a versatile tool to analyze mechanistic features of hClpP.
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Endopeptidase Clp/metabolismo , Endopeptidase Clp/química , Ativação Enzimática , Humanos , Relação Estrutura-AtividadeRESUMO
OBJECTIVE: Few studies have examined associations between patient knowledge of direct oral anticoagulants (DOAC) and clinical outcomes, mostly because of the lack of validated questionnaires for assessing knowledge. The aim of this study was to develop and validate a questionnaire to self-assess knowledge of DOAC. METHODS: Twelve anticoagulation experts participated in the questionnaire development process to ensure content validity. The Knowledge Of Direct Oral Anticoagulants (KODOA)-test was submitted to patients on DOAC and to pharmacists to assess construct validity. Responsiveness was evaluated after educational counseling. Test-retest reliability was assessed to ensure stability over time, and Cronbach's α was calculated for internal reliability. Index of difficulty and item discrimination (D-value) were calculated to assess the performance of single items. RESULTS: The KODOA-test contains 15 items with multiple-choice answers. Each correct answer scores 1 point (max. score of 15). The KODOA-test was administered to 32 patients on DOAC and 28 pharmacists. Pharmacists scored significantly higher than patients at baseline (median score 13.3 vs 10.0; p<0.001), supporting construct validity. Patient scores increased significantly after educational counseling (median score 11 [interquartile range 2] vs 14 [interquartile range 3]; p<0.001). Test-retest and Cronbach's α were acceptable with a Pearson's correlation of 0.8 and an α of 0.67. The index of difficulty for most items was satisfactory (0.38-0.72) and the mean D-value was 42.5%. CONCLUSION: The KODOA-test is a brief, valid, and reliable knowledge self-assessment questionnaire that may be used in clinical trials to investigate associations between knowledge increase and patient-related outcomes.
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Staphylococcus aureus is a major bacterial pathogen that invades and damages host tissue by the expression of devastating toxins. We here performed a phenotypic screen of 35 molecules that were structurally inspired by previous hydroxyamide-based S. aureus virulence inhibitors compiled from commercial sources or designed and synthesized de novo. One of the most potent compounds, AV73, not only reduced hemolytic alpha-hemolysin production in S. aureus but also impeded in vitro biofilm formation. The effect of AV73 on bacterial proteomes and extracellular protein levels was analyzed by quantitative proteomics and revealed a significant down-regulation of major virulence and biofilm promoting proteins. To elucidate the mode of action of AV73, target identification was performed using affinity-based protein profiling (AfBPP), where among others YidC was identified as a target.
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Adesinas Bacterianas/biossíntese , Antibacterianos/farmacologia , Toxinas Bacterianas/biossíntese , Biofilmes/efeitos dos fármacos , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Staphylococcus aureus , Biofilmes/crescimento & desenvolvimento , Staphylococcus aureus/patogenicidade , Staphylococcus aureus/fisiologiaRESUMO
Maintaining the cellular protein homeostasis means managing life on the brink of death. This balance is largely based on precise fine-tuning of enzyme activities. For instance, the ClpP protease possesses several conformational switches which are fundamental to regulating its activity. Efforts have focused on revealing the structural basis of ClpP's conformational control. In the last decade, several amino acid clusters have been identified and functionally linked to specific activation states. Researchers have now begun to couple these hotspots to one another, uncovering a global network of residues that switch in response to internal and external stimuli. For these studies, they used small molecules to mimic intermolecular interactions and point-mutational studies to shortcut regulating amino acid circuits.
Assuntos
Endopeptidase Clp/química , Endopeptidase Clp/metabolismo , Trifosfato de Adenosina/metabolismo , Aminoácidos/química , Aminoácidos/metabolismo , Animais , Humanos , Modelos Moleculares , Chaperonas Moleculares/metabolismo , Conformação Proteica , Multimerização ProteicaRESUMO
Caseinolytic proteases (ClpP) are important for recognition and controlled degradation of damaged proteins. While the majority of bacterial organisms utilize only a single ClpP, Listeria monocytogenes expresses two isoforms (LmClpP1 and LmClpP2). LmClpPs assemble into either a LmClpP2 homocomplex or a LmClpP1/2 heterooligomeric complex. The heterocomplex in association with the chaperone ClpX, exhibits a boost in proteolytic activity for unknown reasons. Here, we use a combined chemical and biochemical strategy to unravel two activation principles of LmClpPs. First, determination of apparent affinity constants revealed a 7-fold elevated binding affinity between the LmClpP1/2 heterocomplex and ClpX, compared to homooligomeric LmClpP2. This tighter interaction favors the formation of the proteolytically active complex between LmClpX and LmClpP1/2 and thereby accelerating the overall turnover. Second, screening a diverse library of fluorescent labeled peptides and proteins with various ClpP mutants allowed the individual analysis of substrate preferences for both isoforms within the heterocomplex. In addition to Leu and Met, LmClpP2 preferred a long aliphatic chain (2-Aoc) in the P1 position for cleavage. Strikingly, design and synthesis of a corresponding 2-Aoc chloromethyl ketone inhibitor resulted in stimulation of proteolysis by 160% when LmClpP2 was partially alkylated on 20% of the active sites. Determination of apparent affinity constants also revealed an elevated complex stability between partially modified LmClpP2 and the cognate chaperone LmClpX. Thus, the stimulation of proteolysis through enhanced binding to the chaperone seems to be a characteristic feature of LmClpPs.
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Comparator products should be the products that were shown to be safe and efficacious in pivotal clinical trials to ensure prescribability of generics. The use of a common comparator ensures switchability between generics. The selection of the comparator is a national responsibility and may be different between countries. This paper discusses the current recommendations on selection of comparators, the associated problems, and the possibility of harmonization. Most countries follow the World Health Organization (WHO) recommendations for selecting comparator products and require the comparator product to be obtained from their national markets to ensure switchability between the local comparator and their generics. These recommendations are only feasible in the few countries where the repetition of the bioequivalence study is economically feasible, but they are impracticable in all other countries. Furthermore, the exclusive use of the local comparator to ensure switchability is ethically and scientifically questionable. The innovator product from well-regulated markets should be the global comparator. This harmonization is feasible as the concept already applies in the WHO prequalification program. It is ineffectual to harmonize only the requirements for performing bioequivalence studies, if such a study has to be repeated for every single country simply because of the different comparator products.
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Medicamentos Genéricos/normas , Estudos de Equivalência como Asunto , Equivalência Terapêutica , Controle de Medicamentos e Entorpecentes , InternacionalidadeRESUMO
ß-Lactones have recently been introduced as the first selective ClpP inhibitors that attenuate virulence of both sensitive Staphylococcus aureus and multiresistant strains (MRSA). Although previous knockout studies showed that ClpP is essential for S. aureus alpha-toxin production, a link between ß-lactone inhibition and molecular virulence mechanisms has been lacking so far. We here perform a chemical-proteomic approach to elucidate antivirulence pathways. First, we demonstrate by gel-free activity-based protein profiling that ClpP is the predominant target of ß-lactones. Only a few off-targets were discovered, which, unlike ClpP, were not involved in the reduction of alpha-toxin expression. Second, in-depth mechanistic insight was provided by a full proteomic comparison between lactone treated and untreated S. aureus cells. Quantitative mass-spectrometric analysis revealed increased repressor of toxin (Rot) levels and a corresponding down-regulation of α-toxin, providing the first direct connection between the lactone-dependent phenotype and a corresponding cellular mechanism. By building up a quantitative virulence regulation network, we visualize the impact of ClpP inhibition in a systems biology context. Interestingly, a lack of in vitro Rot degradation by either ClpXP or ClpCP calls either for a proteolysis mechanism with yet unknown adaptor proteins or for an indirect mode of action that may involve ClpX-mediated RNA signaling and feedback circuits.
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Lactonas/farmacologia , Staphylococcus aureus/patogenicidade , Virulência/efeitos dos fármacos , Proteínas de Bactérias/análise , Proteínas de Bactérias/genética , Toxinas Bacterianas/genética , Endopeptidase Clp/efeitos dos fármacos , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Proteômica , Proteínas Repressoras/análise , Biologia de SistemasRESUMO
Affinity-based protein profiling (AfBPP) is a widely applied method for the target identification of bioactive molecules. Probes containing photocrosslinkers, such as benzophenones, diazirines, and aryl azides, irreversibly link the molecule of interest to its target protein upon irradiation with UV light. Despite their prevalent application, little is known about photocrosslinker-specific off-targets, affecting the reliability of results. Herein, we investigated background protein labeling by gel-free quantitative proteomics. Characteristic off-targets were identified for each photoreactive group and compiled in a comprehensive inventory. In a proof-of-principle study, H8, a protein kinaseâ A inhibitor, was equipped with a diazirine moiety. Application of this photoprobe revealed, by alignment with the diazirine background, unprecedented insight into its inâ situ proteome targets. Taken together, our findings guide the identification of biologically relevant binders in photoprobe experiments.
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BACKGROUND: The scaling-up of access to antiretroviral therapy, particularly in low- to middle-income countries, was facilitated by the introduction and widespread use of generic antiretroviral medicines and fixed-dose combinations. Generic medicines are approved by regulatory authorities based on the demonstration of bioequivalence with the innovator or reference product, as well as meeting quality standards. In clinical practice, however, it is not unusual for generics to be interchanged between each other. This study investigated the differences in bioavailability between WHO-prequalified first-line antiretroviral generics by means of adjusted indirect comparisons to ensure interchangeability between these generics. METHODS: Data on 34 products containing emtricitabine, tenofovir disoproxil fumarate, lamivudine and efavirenz in single formulations or fixed-dose combinations were included in the analysis. The 90% CI for the adjusted indirect comparisons was calculated using the homoscedastic method that uses the conventional t-test, and assumes homogeneity of variances between the studies and small sample sizes. The combined standard deviation of both bioequivalence studies was calculated from the variability of each individual study. RESULTS: The adjusted indirect comparisons between generics showed that the differences, expressed as 90% CIs, are less than 30%. Confidence in the interchangeability of two generic products was reduced if the mean difference between the test and reference in the original studies is more than 10%. CONCLUSIONS: From a bioequivalence perspective, the generic antiretroviral medicines prequalified by WHO are interchangeable with the reference, as well as between each other without safety or efficacy concerns.
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Fármacos Anti-HIV/farmacocinética , Benzoxazinas/farmacocinética , Medicamentos Genéricos/farmacocinética , Emtricitabina/farmacocinética , Lamivudina/farmacocinética , Modelos Estatísticos , Tenofovir/farmacocinética , Alcinos , Área Sob a Curva , Disponibilidade Biológica , Ciclopropanos , Combinação de Medicamentos , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , Humanos , Equivalência Terapêutica , Organização Mundial da SaúdeRESUMO
ClpP is a self-compartmentalizing protease with crucial roles in bacterial and mitochondrial protein quality control. Although the ClpP homocomplex is composed of 14 equivalent active sites, it degrades a multitude of substrates to small peptides, demonstrating its capability to carry out diverse cleavage reactions. Here, we show that ClpP proteases from E. coli, S. aureus, and human mitochondria exhibit preferences for certain amino acids in the P1, P2, and P3 positions using a tailored fluorogenic substrate library. However, this high specificity is not retained during proteolysis of endogenous substrates as shown by mass spectrometric analysis of peptides produced in ClpXP-mediated degradation reactions. Our data suggest a mechanism that implicates the barrel-shaped architecture of ClpP not only in shielding the active sites to prevent uncontrolled proteolysis but also in providing high local substrate concentrations to enable efficient proteolytic processing. Furthermore, we introduce customized fluorogenic substrates with unnatural amino acids that greatly surpass the sensitivity of previously used tools. We used these to profile the activity of cancer-patient- and Perrault-syndrome-derived ClpP mutant proteins.
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Endopeptidase Clp/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Staphylococcus aureus/enzimologia , Sequência de Aminoácidos , Aminoácidos/química , Aminoácidos/metabolismo , Domínio Catalítico , Endopeptidase Clp/química , Escherichia coli/química , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Humanos , Mitocôndrias/química , Mitocôndrias/enzimologia , Mitocôndrias/metabolismo , Modelos Moleculares , Peptídeos/química , Peptídeos/metabolismo , Conformação Proteica , Staphylococcus aureus/química , Staphylococcus aureus/metabolismo , Especificidade por SubstratoRESUMO
PURPOSE: Adjusted indirect comparisons can be used to investigate bioequivalence between generic products that are bioequivalent with a common reference product. In previous work with generic tuberculosis medicines prequalified by the WHO, it was observed that although indirect comparisons are an effective approach for confirming the interchangeability of generics, the approach is subject to less precision than direct comparisons. The objective of this investigation was to explore this by examining the influence of point estimates and power of bioequivalence studies versus the reference on the ability to show equivalence in indirect comparisons. METHODS: Power was considered as a determining factor instead of variability and sample size, because sample size is calculated based on variability and desired power. Scenarios were computed combining a range of point estimate differences (0-14 %) and statistical power of the studies (50-99.99 %). RESULTS: The indirect comparisons could conclude equivalence between generics only when (a) point estimate differences between generics were low (≤ 5.5 %) for any sufficiently powered study (> 80 %), or (b) the differences were large (but less than 14 %) and both bioequivalence studies were overpowered (e.g., 10 % difference and power ≥ 95 %). CONCLUSIONS: In summary, the ability to demonstrate interchangeability between generics is dependent not only on the real differences between the products but also on the design of the original generic vs. reference bioequivalence studies being combined, as earmarked by their respective power.