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1.
Mol Cell Proteomics ; 21(4): 100217, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35217172

RESUMO

Immunosuppressive factors in the tumor microenvironment (TME) impair T cell function and limit the antitumor immune response. T cell surface receptors and surface proteins that influence interactions and function in the TME are proven targets for cancer immunotherapy. However, how the entire surface proteome remodels in primary human T cells in response to specific suppressive factors in the TME remains to be broadly and systematically characterized. Here, using a reductionist cell culture approach with primary human T cells and stable isotopic labeling with amino acids in cell culture-based quantitative cell surface capture glycoproteomics, we examined how two immunosuppressive TME factors, regulatory T cells (Tregs) and hypoxia, globally affect the activated CD8+ surface proteome (surfaceome). Surprisingly, coculturing primary CD8+ T cells with Tregs only modestly affected the CD8+ surfaceome but did partially reverse activation-induced surfaceomic changes. In contrast, hypoxia drastically altered the CD8+ surfaceome in a manner consistent with both metabolic reprogramming and induction of an immunosuppressed state. The CD4+ T cell surfaceome similarly responded to hypoxia, revealing a common hypoxia-induced surface receptor program. Our surfaceomics findings suggest that hypoxic environments create a challenge for T cell activation. These studies provide global insight into how Tregs and hypoxia remodel the T cell surfaceome and we believe represent a valuable resource to inform future therapeutic efforts to enhance T cell function.


Assuntos
Proteoma , Linfócitos T Reguladores , Linfócitos T CD8-Positivos , Humanos , Hipóxia , Microambiente Tumoral
2.
Proc Natl Acad Sci U S A ; 118(8)2021 02 23.
Artigo em Inglês | MEDLINE | ID: mdl-33536314

RESUMO

N terminomics is a powerful strategy for profiling proteolytic neo-N termini, but its application to cell surface proteolysis has been limited by the low relative abundance of plasma membrane proteins. Here we apply plasma membrane-targeted subtiligase variants (subtiligase-TM) to efficiently and specifically capture cell surface N termini in live cells. Using this approach, we sequenced 807 cell surface N termini and quantified changes in their abundance in response to stimuli that induce proteolytic remodeling of the cell surface proteome. To facilitate exploration of our datasets, we developed a web-accessible Atlas of Subtiligase-Captured Extracellular N Termini (ASCENT; http://wellslab.org/ascent). This technology will facilitate greater understanding of extracellular protease biology and reveal neo-N termini biomarkers and targets in disease.


Assuntos
Membrana Celular/metabolismo , Mapeamento de Peptídeos/métodos , Peptídeo Sintases/metabolismo , Subtilisinas/metabolismo , Células HEK293 , Humanos , Mutação , Peptídeo Sintases/genética , Processamento de Proteína Pós-Traducional , Proteólise , Subtilisinas/genética
3.
Chem Rev ; 120(6): 3127-3160, 2020 03 25.
Artigo em Inglês | MEDLINE | ID: mdl-31663725

RESUMO

Subtiligase-catalyzed peptide ligation is a powerful approach for site-specific protein bioconjugation, synthesis and semisynthesis of proteins and peptides, and chemoproteomic analysis of cellular N termini. Here, we provide a comprehensive review of the subtiligase technology, including its development, applications, and impacts on protein science. We highlight key advantages and limitations of the tool and compare it to other peptide ligase enzymes. Finally, we provide a perspective on future applications and challenges and how they may be addressed.


Assuntos
Biocatálise , Peptídeo Sintases/metabolismo , Peptídeos/química , Subtilisinas/metabolismo , Peptídeos/síntese química
4.
Biochem J ; 478(17): 3179-3184, 2021 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-34492095

RESUMO

Apoptosis is a cell death program that is executed by the caspases, a family of cysteine proteases that typically cleave after aspartate residues during a proteolytic cascade that systematically dismantles the dying cell. Extensive signaling crosstalk occurs between caspase-mediated proteolysis and kinase-mediated phosphorylation, enabling integration of signals from multiple pathways into the decision to commit to apoptosis. A new study from Maluch et al. examines how phosphorylation within caspase cleavage sites impacts the efficiency of substrate cleavage. The results demonstrate that while phosphorylation in close proximity to the scissile bond is generally inhibitory, it does not necessarily abrogate substrate cleavage, but instead attenuates the rate. In some cases, this inhibition can be overcome by additional favorable substrate features. These findings suggest potential nuanced physiological roles for phosphorylation of caspase substrates with exciting implications for targeting caspases with chemical probes and therapeutics.


Assuntos
Caspases/metabolismo , Fosfotransferases/metabolismo , Proteólise , Transdução de Sinais/fisiologia , Apoptose/fisiologia , Ácido Aspártico/metabolismo , Humanos , Cinética , Fosforilação/fisiologia , Processamento de Proteína Pós-Traducional/fisiologia , Especificidade por Substrato/fisiologia
5.
J Membr Biol ; 254(2): 119-125, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33599828

RESUMO

Mass spectrometry-based proteomics has been used successfully to identify substrates for proteases. Identification of protease substrates at the cell surface, however, can be challenging since cleavages are less abundant compared to other cellular events. Precise methods are required to delineate cleavage events that take place in these compartmentalized areas. This article by up-and-coming scientist Dr. Amy Weeks, an Assistant Professor at the University of Wisconsin-Madison, provides an overview of methods developed to identify protease substrates and their cleavage sites at the membrane. An overview is presented with the pros and cons for each method and in particular the N-terminomics subtiligase-TM method, developed by Dr. Weeks as a postdoctoral fellow in the lab of Dr. Jim Wells at University of California, San Francisco, is described in detail. Subtiligase-TM is a genetically engineered subtilisin protease variant that acts to biotinylate newly generated N termini, hence revealing new cleavage events in the presence of a specific enzyme, and furthermore can precisely identify the cleavage P1 site. Importantly, this proteomics method is compatible with living cells. This method will open the door to understanding protein shedding events at the biological membrane controlled by proteases to regulate biological processes.


Assuntos
Peptídeo Hidrolases , Proteômica , Subtilisinas , Peptídeo Hidrolases/química , Peptídeo Hidrolases/genética , Peptídeo Sintases , Proteômica/métodos , Subtilisinas/química , Subtilisinas/genética
6.
Proc Natl Acad Sci U S A ; 115(10): E2193-E2201, 2018 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-29453276

RESUMO

Fluorinated small molecules play an important role in the design of bioactive compounds for a broad range of applications. As such, there is strong interest in developing a deeper understanding of how fluorine affects the interaction of these ligands with their targets. Given the small number of fluorinated metabolites identified to date, insights into fluorine recognition have been provided almost entirely by synthetic systems. The fluoroacetyl-CoA thioesterase (FlK) from Streptomyces cattleya thus provides a unique opportunity to study an enzyme-ligand pair that has been evolutionarily optimized for a surprisingly high 106 selectivity for a single fluorine substituent. In these studies, we synthesize a series of analogs of fluoroacetyl-CoA and acetyl-CoA to generate nonhydrolyzable ester, amide, and ketone congeners of the thioester substrate to isolate the role of fluorine molecular recognition in FlK selectivity. Using a combination of thermodynamic, kinetic, and protein NMR experiments, we show that fluorine recognition is entropically driven by the interaction of the fluorine substituent with a key residue, Phe-36, on the lid structure that covers the active site, resulting in an ∼5- to 20-fold difference in binding (KD). Although the magnitude of discrimination is similar to that found in designed synthetic ligand-protein complexes where dipolar interactions control fluorine recognition, these studies show that hydrophobic and solvation effects serve as the major determinant of naturally evolved fluorine selectivity.


Assuntos
Acetilcoenzima A/química , Acetilcoenzima A/metabolismo , Flúor/química , Flúor/metabolismo , Streptomyces/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Entropia , Ressonância Magnética Nuclear Biomolecular , Fenilalanina/química , Ligação Proteica , Especificidade por Substrato
7.
Biochem Soc Trans ; 48(3): 1153-1165, 2020 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-32539119

RESUMO

Enzymes that catalyze peptide ligation are powerful tools for site-specific protein bioconjugation and the study of cellular signaling. Peptide ligases can be divided into two classes: proteases that have been engineered to favor peptide ligation, and protease-related enzymes with naturally evolved peptide ligation activity. Here, we provide a review of key natural peptide ligases and proteases engineered to favor peptide ligation activity. We cover the protein engineering approaches used to generate and improve these tools, along with recent biological applications, advantages, and limitations associated with each enzyme. Finally, we address future challenges and opportunities for further development of peptide ligases as tools for biological research.


Assuntos
Ligases/química , Peptídeo Hidrolases/química , Peptídeos/química , Engenharia de Proteínas/métodos , Transdução de Sinais , Animais , Catálise , Catepsina A/genética , Cisteína Endopeptidases/genética , Variação Genética , Humanos , Subtilisina/genética , Tripsina/genética
8.
Nat Chem Biol ; 14(1): 50-57, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29155430

RESUMO

Enzyme-catalyzed peptide ligation is a powerful tool for site-specific protein bioconjugation, but stringent enzyme-substrate specificity limits its utility. We developed an approach for comprehensively characterizing peptide ligase specificity for N termini using proteome-derived peptide libraries. We used this strategy to characterize the ligation efficiency for >25,000 enzyme-substrate pairs in the context of the engineered peptide ligase subtiligase and identified a family of 72 mutant subtiligases with activity toward N-terminal sequences that were previously recalcitrant to modification. We applied these mutants individually for site-specific bioconjugation of purified proteins, including antibodies, and in algorithmically selected combinations for sequencing of the cellular N terminome with reduced sequence bias. We also developed a web application to enable algorithmic selection of the most efficient subtiligase variant(s) for bioconjugation to user-defined sequences. Our methods provide a new toolbox of enzymes for site-specific protein modification and a general approach for rapidly defining and engineering peptide ligase specificity.


Assuntos
Biblioteca de Peptídeos , Peptídeo Sintases/genética , Peptídeo Sintases/metabolismo , Engenharia de Proteínas/métodos , Proteômica/métodos , Subtilisinas/genética , Subtilisinas/metabolismo , Sítios de Ligação , Cromatografia Líquida , Humanos , Células Jurkat , Cinética , Ligantes , Modelos Moleculares , Plasmídeos , Especificidade por Substrato , Espectrometria de Massas em Tandem
9.
Proc Natl Acad Sci U S A ; 109(48): 19667-72, 2012 Nov 27.
Artigo em Inglês | MEDLINE | ID: mdl-23150553

RESUMO

The investigation of unique chemical phenotypes has led to the discovery of enzymes with interesting behaviors that allow us to explore unusual function. The organofluorine-producing microbe Streptomyces cattleya has evolved a fluoroacetyl-CoA thioesterase (FlK) that demonstrates a surprisingly high level of discrimination for a single fluorine substituent on its substrate compared with the cellularly abundant hydrogen analog, acetyl-CoA. In this report, we show that the high selectivity of FlK is achieved through catalysis rather than molecular recognition, where deprotonation at the C(α) position to form a putative ketene intermediate only occurs on the fluorinated substrate, thereby accelerating the rate of hydrolysis 10(4)-fold compared with the nonfluorinated congener. These studies provide insight into mechanisms of catalytic selectivity in a native system where the existence of two reaction pathways determines substrate rather than product selection.


Assuntos
Flúor/química , Catálise , Hidrólise , Cinética
10.
Biochemistry ; 53(12): 2053-63, 2014 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-24635371

RESUMO

The fluoroacetate-producing bacterium Streptomyces cattleya has evolved a fluoroacetyl-CoA thioesterase (FlK) that exhibits a remarkably high level of discrimination for its cognate substrate compared to the cellularly abundant analogue acetyl-CoA, which differs only by the absence of the fluorine substitution. A major determinant of FlK specificity derives from its ability to take advantage of the unique properties of fluorine to enhance the reaction rate, allowing fluorine discrimination under physiological conditions where both substrates are likely to be present at saturating concentrations. Using a combination of pH-rate profiles, pre-steady-state kinetic experiments, and Taft analysis of wild-type and mutant FlKs with a set of substrate analogues, we explore the role of fluorine in controlling the enzyme acylation and deacylation steps. Further analysis of chiral (R)- and (S)-[(2)H1]fluoroacetyl-CoA substrates demonstrates that a kinetic isotope effect (1.7 ± 0.2) is observed for only the (R)-(2)H1 isomer, indicating that deacylation requires recognition of the prochiral fluoromethyl group to position the α-carbon for proton abstraction. Taken together, the selectivity for the fluoroacetyl-CoA substrate appears to rely not only on the enhanced polarization provided by the electronegative fluorine substitution but also on molecular recognition of fluorine in both formation and breakdown of the acyl-enzyme intermediate to control active site reactivity. These studies provide insights into the basis of fluorine selectivity in a naturally occurring enzyme-substrate pair, with implications for drug design and the development of fluorine-selective biocatalysts.


Assuntos
Acetilcoenzima A/química , Acetilcoenzima A/metabolismo , Flúor/química , Flúor/metabolismo , Sítios de Ligação/fisiologia , Domínio Catalítico , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Streptomyces/enzimologia , Especificidade por Substrato
11.
bioRxiv ; 2024 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-38798401

RESUMO

In biological systems, ATP provides an energetic driving force for peptide bond formation, but protein chemists lack tools that emulate this strategy. Inspired by the eukaryotic ubiquitination cascade, we developed an ATP-driven platform for C-terminal activation and peptide ligation based on E. coli MccB, a bacterial ancestor of ubiquitin-activating (E1) enzymes that natively catalyzes C-terminal phosphoramidate bond formation. We show that MccB can act on non-native substrates to generate an O-AMPylated electrophile that can react with exogenous nucleophiles to form diverse C-terminal functional groups including thioesters, a versatile class of biological intermediates that have been exploited for protein semisynthesis. To direct this activity towards specific proteins of interest, we developed the Thioesterification C-terminal Handle (TeCH)-tag, a sequence that enables high-yield, ATP-driven protein bioconjugation via a thioester intermediate. By mining the natural diversity of the MccB family, we developed two additional MccB/TeCH-tag pairs that are mutually orthogonal to each other and to the E. coli system, facilitating the synthesis of more complex bioconjugates. Our method mimics the chemical logic of peptide bond synthesis that is widespread in biology for high-yield in vitro manipulation of protein structure with molecular precision.

12.
Cell Chem Biol ; 31(3): 534-549.e8, 2024 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-37816350

RESUMO

Proteomic profiling of protease-generated N termini provides key insights into protease function and specificity. However, current technologies have sequence limitations or require specialized synthetic reagents for N-terminal peptide isolation. Here, we introduce an N terminomics toolbox that combines selective N-terminal biotinylation using 2-pyridinecarboxaldehyde (2PCA) reagents with chemically cleavable linkers to enable efficient enrichment of protein N termini. By incorporating a commercially available alkyne-modified 2PCA in combination with Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC), our strategy eliminates the need for chemical synthesis of N-terminal probes. Using these reagents, we developed PICS2 (Proteomic Identification of Cleavage Sites with 2PCA) to profile the specificity of subtilisin/kexin-type proprotein convertases (PCSKs). We also implemented CHOPPER (chemical enrichment of protease substrates with purchasable, elutable reagents) for global sequencing of apoptotic proteolytic cleavage sites. Based on their broad applicability and ease of implementation, PICS2 and CHOPPER are useful tools that will advance our understanding of protease biology.


Assuntos
Peptídeo Hidrolases , Proteômica , Piridinas , Peptídeo Hidrolases/metabolismo , Química Click , Proteólise , Alcinos
13.
Curr Protoc ; 3(6): e798, 2023 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-37283519

RESUMO

Protein and peptide N termini are important targets for selective modification with chemoproteomics reagents and bioconjugation tools. The N-terminal ⍺-amine occurs only once in each polypeptide chain, making it an attractive target for protein bioconjugation. In cells, new N termini can be generated by proteolytic cleavage and captured by N-terminal modification reagents that enable proteome-wide identification of protease substrates through tandem mass spectrometry (LC-MS/MS). An understanding of the N-terminal sequence specificity of the modification reagents is critical for each of these applications. Proteome-derived peptide libraries in combination with LC-MS/MS are powerful tools for profiling the sequence specificity of N-terminal modification reagents. These libraries are highly diverse, and LC-MS/MS enables analysis of the modification efficiencies of tens of thousands of sequences in a single experiment. Proteome-derived peptide libraries are a powerful tool for profiling the sequence specificities of enzymatic and chemical peptide labeling reagents. Subtiligase, an enzymatic modification reagent, and 2-pyridinecarboxaldehyde (2PCA), a chemical modification reagent, are two reagents that have been developed for selective N-terminal peptide modification and can be studied using proteome-derived peptide libraries. This protocol outlines the steps for generating N-terminally diverse proteome-derived peptide libraries and for applying these libraries to profile the specificity of N-terminal modification reagents. Although we detail the steps for profiling the specificity of 2PCA and subtiligase in Escherichia coli and human cells, these protocols can easily be adapted to alternative proteome sources and other N-terminal peptide labeling reagents. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Generation of N-terminally diverse proteome-derived peptide libraries from E. coli Alternate Protocol: Generation of N-terminally diverse proteome-derived peptide libraries from human cells Basic Protocol 2: Characterizing the specificity of 2-pyridinecarboxaldehyde using proteome-derived peptide libraries Basic Protocol 3: Characterizing the specificity of subtiligase using proteome-derived peptide libraries.


Assuntos
Biblioteca de Peptídeos , Proteoma , Humanos , Proteoma/análise , Cromatografia Líquida/métodos , Escherichia coli/genética , Proteômica/métodos , Espectrometria de Massas em Tandem/métodos , Peptídeos/química , Peptídeos/metabolismo , Peptídeo Hidrolases/química , Peptídeo Hidrolases/metabolismo
14.
Biochemistry ; 51(34): 6827-37, 2012 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-22906002

RESUMO

The production of fatty acids is an important cellular pathway for both cellular function and the development of engineered pathways for the synthesis of advanced biofuels. Despite the conserved reaction chemistry of various fatty acid synthase systems, the individual isozymes that catalyze these steps are quite diverse in their structural and biochemical features and are important for controlling differences at the cellular level. One of the key steps in the fatty acid elongation cycle is the enoyl-ACP (CoA) reductase function that drives the equilibrium forward toward chain extension. In this work, we report the structural and biochemical characterization of the trans-enoyl-CoA reductase from Treponema denticola (tdTer), which has been utilized for the engineering of synthetic biofuel pathways with an order of magnitude increase in product titers compared to those of pathways constructed with other enoyl-CoA reductase components. The crystal structure of tdTer was determined to 2.00 Å resolution and shows that the Ter enzymes are distinct from members of the FabI, FabK, and FabL families but are highly similar to members of the FabV family. Further biochemical studies show that tdTer uses an ordered bi-bi mechanism initiated by binding of the NADH redox cofactor, which is consistent with the behavior of other enoyl-ACP (CoA) reductases. Mutagenesis of the substrate binding loop, characterization of enzyme activity with respect to crotonyl-CoA, hexenoyl-CoA, and dodecenoyl-CoA substrates, and product inhibition by lauroyl-CoA suggest that this region is important for controlling chain length specificity, with the major portal playing a more important role for longer chain length substrates.


Assuntos
Proteínas de Bactérias/química , Enoil-(Proteína de Transporte de Acila) Redutase (NADH)/química , Treponema denticola/enzimologia , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cristalografia por Raios X , Enoil-(Proteína de Transporte de Acila) Redutase (NADH)/genética , Enoil-(Proteína de Transporte de Acila) Redutase (NADH)/metabolismo , Cinética , Dados de Sequência Molecular , Filogenia , Especificidade por Substrato , Treponema denticola/química , Treponema denticola/classificação , Treponema denticola/genética
15.
Methods Mol Biol ; 2456: 71-83, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35612736

RESUMO

N terminomics methods combine selective isolation of protein N-terminal peptides with mass spectrometry (MS)-based proteomics for global profiling of proteolytic cleavage sites. However, traditional N terminomics workflows require cell lysis before N-terminal enrichment and provide poor coverage of N termini derived from cell surface proteins. Here, we describe application of subtiligase-TM, a plasma membrane-targeted peptide ligase, for selective biotinylation of cell surface N termini, enabling their enrichment and analysis by liquid chromatography-tandem MS (LC-MS/MS). This method provides increased coverage of and specificity for cell surface N termini and is compatible with existing quantitative LC-MS/MS workflows.


Assuntos
Espectrometria de Massas em Tandem , Membrana Celular , Cromatografia Líquida , Peptídeo Sintases , Proteólise , Subtilisinas
16.
ACS Cent Sci ; 8(10): 1447-1456, 2022 Oct 26.
Artigo em Inglês | MEDLINE | ID: mdl-36313159

RESUMO

Proteolytic cleavage of cell surface proteins triggers critical processes including cell-cell interactions, receptor activation, and shedding of signaling proteins. Consequently, dysregulated extracellular proteases contribute to malignant cell phenotypes including most cancers. To understand these effects, methods are needed that identify proteolyzed membrane proteins within diverse cellular contexts. Herein we report a proteomic approach, called cell surface N-terminomics, to broadly identify precise cleavage sites (neo-N-termini) on the surface of living cells. First, we functionalized the engineered peptide ligase, called stabiligase, with an N-terminal nucleophile that enables covalent attachment to naturally occurring glycans. Upon the addition of a biotinylated peptide ester, glycan-tethered stabiligase efficiently tags extracellular neo-N-termini for proteomic analysis. To demonstrate the versatility of this approach, we identified and characterized 1532 extracellular neo-N-termini across a panel of different cell types including primary immune cells. The vast majority of cleavages were not identified by previous proteomic studies. Lastly, we demonstrated that single oncogenes, KRAS(G12V) and HER2, induce extracellular proteolytic remodeling of proteins involved in cancerous cell growth, invasion, and migration. Cell surface N-terminomics is a generalizable platform that can reveal proteolyzed, neoepitopes to target using immunotherapies.

17.
Biochemistry ; 50(24): 5404-18, 2011 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-21591680

RESUMO

Living organisms have evolved a vast array of catalytic functions that make them ideally suited for the production of medicinally and industrially relevant small-molecule targets. Indeed, native metabolic pathways in microbial hosts have long been exploited and optimized for the scalable production of both fine and commodity chemicals. Our increasing capacity for DNA sequencing and synthesis has revealed the molecular basis for the biosynthesis of a variety of complex and useful metabolites and allows the de novo construction of novel metabolic pathways for the production of new and exotic molecular targets in genetically tractable microbes. However, the development of commercially viable processes for these engineered pathways is currently limited by our ability to quickly identify or engineer enzymes with the correct reaction and substrate selectivity as well as the speed by which metabolic bottlenecks can be determined and corrected. Efforts to understand the relationship among sequence, structure, and function in the basic biochemical sciences can advance these goals for synthetic biology applications while also serving as an experimental platform for elucidating the in vivo specificity and function of enzymes and reconstituting complex biochemical traits for study in a living model organism. Furthermore, the continuing discovery of natural mechanisms for the regulation of metabolic pathways has revealed new principles for the design of high-flux pathways with minimized metabolic burden and has inspired the development of new tools and approaches to engineering synthetic pathways in microbial hosts for chemical production.


Assuntos
Redes e Vias Metabólicas , Modelos Biológicos , Bioengenharia , Evolução Molecular Direcionada , Enzimas/genética , Enzimas/metabolismo , Ensaios de Triagem em Larga Escala , Metagenômica
18.
Curr Opin Chem Biol ; 60: 10-19, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-32768891

RESUMO

Post-translational modifications, complex formation, subcellular localization, and cell-type-specific expression create functionally distinct protein subpopulations that enable living systems to execute rapid and precise responses to changing conditions. Systems-level analysis of these subproteomes remains challenging, requiring preservation of spatial information or enrichment of species that are transient and present at low abundance. Engineered proteins have emerged as important tools for selective proteomics based on their capacity for highly specific molecular recognition and their genetic targetability. Here, we focus on new developments in protein engineering for selective proteomics of post-translational modifications, protein complexes, subcellular compartments, and cell types. We also address remaining challenges and future opportunities to integrate engineered protein tools across different subproteome scales to map the proteome with unprecedented depth and detail.


Assuntos
Engenharia de Proteínas/métodos , Proteômica/métodos , Animais , Humanos , Espaço Intracelular/metabolismo , Processamento de Proteína Pós-Traducional
19.
Biochemistry ; 49(43): 9269-79, 2010 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-20836570

RESUMO

We have initiated a broad-based program aimed at understanding the molecular basis of fluorine specificity in enzymatic systems, and in this context, we report crystallographic and biochemical studies on a fluoroacetyl-coenzyme A (CoA) specific thioesterase (FlK) from Streptomyces cattleya. Our data establish that FlK is competent to protect its host from fluoroacetate toxicity in vivo and demonstrate a 10(6)-fold discrimination between fluoroacetyl-CoA (k(cat)/K(M) = 5 × 107 M⁻¹ s⁻¹) and acetyl-CoA (k(cat)/K(M) = 30 M⁻¹ s⁻¹) based on a single fluorine substitution that originates from differences in both substrate reactivity and binding. We show that Thr 42, Glu 50, and His 76 are key catalytic residues and identify several factors that influence substrate selectivity. We propose that FlK minimizes interaction with the thioester carbonyl, leading to selection against acetyl-CoA binding that can be recovered in part by new C═O interactions in the T42S and T42C mutants. We hypothesize that the loss of these interactions is compensated by the entropic driving force for fluorinated substrate binding in a hydrophobic binding pocket created by a lid structure, containing Val 23, Leu 26, Phe 33, and Phe 36, that is not found in other structurally characterized members of this superfamily. We further suggest that water plays a critical role in fluorine specificity based on biochemical and structural studies focused on the unique Phe 36 "gate" residue, which functions to exclude water from the active site. Taken together, the findings from these studies offer molecular insights into organofluorine recognition and design of fluorine-specific enzymes.


Assuntos
Acetilcoenzima A/química , Flúor/química , Tioléster Hidrolases/química , Acetilcoenzima A/metabolismo , Domínio Catalítico , Cristalografia por Raios X , Escherichia coli , Flúor/metabolismo , Cinética , Ligação Proteica , Conformação Proteica , Streptomyces/enzimologia , Especificidade por Substrato , Termodinâmica , Tioléster Hidrolases/metabolismo , Água/química
20.
Curr Protoc Chem Biol ; 12(1): e79, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32074409

RESUMO

Subtiligase is a powerful enzymatic tool for N-terminal modification of proteins and peptides. In a typical subtiligase-catalyzed N-terminal modification reaction, a peptide ester donor substrate is ligated onto the unblocked N terminus of a protein, resulting in the exchange of the ester bond in the donor substrate for an amide bond between the donor substrate and protein N terminus. Using this strategy, new chemical probes and payloads, such as fluorophores, affinity handles, cytotoxic drugs, and reactive functional groups, can be introduced site-specifically into proteins. While the efficiency of this reaction depends on the sequences to be ligated, a panel of mutants was recently developed that expands the scope of substrate sequences that are suitable for subtiligase modification. This article outlines the steps for applying subtiligase or specificity variants for both site-specific bioconjugation of purified proteins and for global modification of cellular N termini to enable their sequencing by tandem mass spectrometry. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Subtiligase-catalyzed site-specific protein bioconjugation Support Protocol 1: Expression and purification of subtiligase-His6 Support Protocol 2: Subtiligase substrate synthesis Basic Protocol 2: Subtiligase N terminomics using a cocktail of subtiligase specificity mutants.


Assuntos
Mutação , Peptídeo Sintases/genética , Peptídeo Sintases/metabolismo , Proteínas/química , Proteínas/metabolismo , Subtilisinas/genética , Subtilisinas/metabolismo , Modelos Moleculares , Especificidade por Substrato/genética , Espectrometria de Massas em Tandem
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