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1.
J Am Chem Soc ; 145(29): 16069-16080, 2023 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-37450839

RESUMO

Electrophilic small molecules that can reversibly modify proteins are of growing interest in drug discovery. However, the ability to study reversible covalent probes in live cells can be limited by their reversible reactivity after cell lysis and in proteomic workflows, leading to scrambling and signal loss. We describe how thiomethyltetrazines function as reversible covalent warheads for cysteine modification, and this dynamic labeling behavior can be "switched off" via bioorthogonal chemistry inside live cells. Simultaneously, the tetrazine serves as a bioorthogonal reporter enabling the introduction of tags for fluorescent imaging or affinity purification. Thiomethyltetrazines can label isolated proteins, proteins in cellular lysates, and proteins in live cells with second-order rate constants spanning 2 orders of magnitude (k2, 1-100 M-1 s-1). Reversible modification by thiomethyltetrazines can be switched off upon the addition of trans-cyclooctene in live cells, converting the dynamic thiomethyltetrazine tag into a Diels-Alder adduct which is stable to lysis and proteomic workflows. Time-course quenching experiments were used to demonstrate temporal control over electrophilic modification. Moreover, it is shown that "locking in" the tag through Diels-Alder chemistry enables the identification of protein targets that are otherwise lost during sample processing. Three probes were further evaluated to identify unique pathways in a live-cell proteomic study. We anticipate that discovery efforts will be enabled by the trifold function of thiomethyltetrazines as electrophilic warheads, bioorthogonal reporters, and switches for "locking in" stability.


Assuntos
Cisteína , Compostos Heterocíclicos , Proteômica , Proteínas/química
2.
Nature ; 523(7562): 561-7, 2015 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-26200343

RESUMO

G-protein-coupled receptors (GPCRs) signal primarily through G proteins or arrestins. Arrestin binding to GPCRs blocks G protein interaction and redirects signalling to numerous G-protein-independent pathways. Here we report the crystal structure of a constitutively active form of human rhodopsin bound to a pre-activated form of the mouse visual arrestin, determined by serial femtosecond X-ray laser crystallography. Together with extensive biochemical and mutagenesis data, the structure reveals an overall architecture of the rhodopsin-arrestin assembly in which rhodopsin uses distinct structural elements, including transmembrane helix 7 and helix 8, to recruit arrestin. Correspondingly, arrestin adopts the pre-activated conformation, with a ∼20° rotation between the amino and carboxy domains, which opens up a cleft in arrestin to accommodate a short helix formed by the second intracellular loop of rhodopsin. This structure provides a basis for understanding GPCR-mediated arrestin-biased signalling and demonstrates the power of X-ray lasers for advancing the frontiers of structural biology.


Assuntos
Arrestina/química , Arrestina/metabolismo , Rodopsina/química , Rodopsina/metabolismo , Animais , Sítios de Ligação , Cristalografia por Raios X , Dissulfetos/química , Dissulfetos/metabolismo , Humanos , Lasers , Camundongos , Modelos Moleculares , Complexos Multiproteicos/biossíntese , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Ligação Proteica , Reprodutibilidade dos Testes , Transdução de Sinais , Raios X
3.
Proc Natl Acad Sci U S A ; 115(31): E7285-E7292, 2018 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-30012605

RESUMO

Proteolysis targeting chimeras (PROTACs) are heterobifunctional small molecules that simultaneously bind to a target protein and an E3 ligase, thereby leading to ubiquitination and subsequent degradation of the target. They present an exciting opportunity to modulate proteins in a manner independent of enzymatic or signaling activity. As such, they have recently emerged as an attractive mechanism to explore previously "undruggable" targets. Despite this interest, fundamental questions remain regarding the parameters most critical for achieving potency and selectivity. Here we employ a series of biochemical and cellular techniques to investigate requirements for efficient knockdown of Bruton's tyrosine kinase (BTK), a nonreceptor tyrosine kinase essential for B cell maturation. Members of an 11-compound PROTAC library were investigated for their ability to form binary and ternary complexes with BTK and cereblon (CRBN, an E3 ligase component). Results were extended to measure effects on BTK-CRBN cooperative interactions as well as in vitro and in vivo BTK degradation. Our data show that alleviation of steric clashes between BTK and CRBN by modulating PROTAC linker length within this chemical series allows potent BTK degradation in the absence of thermodynamic cooperativity.


Assuntos
Proteínas Tirosina Quinases/metabolismo , Proteólise , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Tirosina Quinase da Agamaglobulinemia , Animais , Células Cultivadas , Ligantes , Poliubiquitina/metabolismo , Ratos , Termodinâmica
4.
Org Biomol Chem ; 18(19): 3669-3673, 2020 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-32373883

RESUMO

A C-H functionalization strategy for the expedient access to photoreactive chemical probes of commonly found heterocyclic fragments or drug molecules of pharmaceutical relevance is described. A series of aryl glyoxylic acid reagents featuring pendant alkyne or azide clickable handles have been developed for application in the radical-mediated appendage of benzoyl fragments onto simple heteroaromatic fragments, as well as more complex drug-like compounds. This unprecedented strategy of chemical probe synthesis allows for direct access to photoreactive chemical probes without any requirement of fragment pre-functionalization or significant synthetic re-evaluation.

5.
J Proteome Res ; 14(5): 2287-97, 2015 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-25825992

RESUMO

Current methods for the large-scale characterization of disease states generally rely on the analysis of gene and/or protein expression levels. These existing methods fail to detect proteins with disease-related functions and unaltered expression levels. Here we describe the large-scale use of thermodynamic measurements of protein folding and stability for the characterization of disease states. Using the Stable Isotope Labeling with Amino Acids in Cell Culture and Stability of Proteins from Rates of Oxidation (SILAC-SPROX) technique, we assayed ∼800 proteins for protein folding and stability changes in three different cell culture models of breast cancer including the MCF-10A, MCF-7, and MDA-MB-231 cell lines. The thermodynamic stability profiles generated here created distinct molecular markers to differentiate the three cell lines, and a significant fraction (∼45%) of the differentially stabilized proteins did not have altered expression levels. Thus, the differential thermodynamic profiling strategy reported here created novel molecular signatures of breast cancer and provided additional insight into the molecular basis of the disease. Our results establish the utility of protein folding and stability measurements for the study of disease processes, and they suggest that such measurements may be useful for biomarker discovery in disease.


Assuntos
Biomarcadores Tumorais/isolamento & purificação , Neoplasias da Mama/genética , Proteínas de Neoplasias/isolamento & purificação , Proteoma/isolamento & purificação , Aminoácidos/metabolismo , Biomarcadores Tumorais/química , Biomarcadores Tumorais/genética , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Feminino , Expressão Gênica , Humanos , Marcação por Isótopo , Anotação de Sequência Molecular , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Oxirredução , Dobramento de Proteína , Estabilidade Proteica , Proteoma/química , Proteoma/genética , Proteômica/métodos , Termodinâmica
6.
J Proteome Res ; 14(11): 4571-80, 2015 Nov 06.
Artigo em Inglês | MEDLINE | ID: mdl-26398198

RESUMO

Liver receptor homologue-1 (LRH1) is an orphan nuclear receptor that has been shown to play a role in the transcriptional regulation of pathways involved in cancer. Elucidating the components of the LRH1 transcriptional complex to better understand endogenous regulation of the receptor as well as its role in cancer remains a high priority. A sub-cellular enrichment strategy coupled with proteomic approaches was employed to identify putative LRH1 co-regulators. Nuclear fractionation protocol was essential for detection of LRH1 peptides by mass spectrometry (MS), with most peptides being observed in the insoluble fraction (receptor bound to DNA). SERBP1 and ILF3 were identified as LRH1 interacting partners by both Western blot and MS/MS analysis. Receptor knockdown by siRNA showed an increase in SERBP1 expression, while ILF3 expression was unchanged. In contrast, receptor overexpression decreased only SERBP1 mRNA levels. Consistent with these data, in a promoter:reporter assay, binding of LRH1 to the promoter region of SERBP1 resulted in a decrease in the expression level of the reporter gene, subsequently inhibiting transcription. Given the receptor's role in cancer progression, the study here elucidates additional transcriptional machinery involved in LRH1 signaling and potentially provides new targets for therapeutics development.


Assuntos
Regulação da Expressão Gênica , Peptídeos/análise , Proteínas de Ligação a RNA/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Transcrição Gênica , Sequência de Aminoácidos , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Fracionamento Químico , Células HEK293 , Hepatócitos/citologia , Hepatócitos/metabolismo , Humanos , Anotação de Sequência Molecular , Dados de Sequência Molecular , Proteínas do Fator Nuclear 90/genética , Proteínas do Fator Nuclear 90/metabolismo , Peptídeos/genética , Peptídeos/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Plasmídeos/química , Plasmídeos/metabolismo , Regiões Promotoras Genéticas , Proteína-Arginina N-Metiltransferases/genética , Proteína-Arginina N-Metiltransferases/metabolismo , Proteólise , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/genética , Receptores Citoplasmáticos e Nucleares/antagonistas & inibidores , Receptores Citoplasmáticos e Nucleares/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Transdução de Sinais , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transfecção
7.
Mol Pharmacol ; 87(2): 296-304, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25473120

RESUMO

The orphan nuclear receptor liver receptor homolog 1 (LRH-1; NR5A2) is a potent regulator of cholesterol metabolism and bile acid homeostasis. Recently, LRH-1 has been shown to play an important role in intestinal inflammation and in the progression of estrogen receptor positive and negative breast cancers and pancreatic cancer. Structural studies have revealed that LRH-1 can bind phospholipids and the dietary phospholipid dilauroylphosphatidylcholine activates LRH-1 activity in rodents. Here we characterize the activity of a novel synthetic nonphospholipid small molecule repressor of LRH-1, SR1848 (6-[4-(3-chlorophenyl)piperazin-1-yl]-3-cyclohexyl-1H-pyrimidine-2,4-dione). In cotransfection studies, SR1848 reduced LRH-1-dependent expression of a reporter gene and in cells that endogenously express LRH-1 dose dependently reduced the expression of cyclin-D1 and -E1, resulting in inhibition of cell proliferation. The cellular effects of SR1848 treatment are recapitulated after transfection of cells with small-interfering RNA targeting LRH-1. Immunocytochemistry analysis shows that SR1848 induces rapid translocation of nuclear LRH-1 to the cytoplasm. Combined, these results suggest that SR1848 is a functional repressor of LRH-1 that impacts expression of genes involved in proliferation in LRH-1-expressing cancers. Thus, SR1848 represents a novel chemical scaffold for the development of therapies targeting malignancies driven by LRH-1.


Assuntos
Proliferação de Células/fisiologia , Pirimidinas/metabolismo , Receptores Citoplasmáticos e Nucleares/antagonistas & inibidores , Receptores Citoplasmáticos e Nucleares/metabolismo , Animais , Proliferação de Células/efeitos dos fármacos , Células HEK293 , Células Hep G2 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Pirimidinas/química , Pirimidinas/farmacologia
8.
J Proteome Res ; 13(12): 5801-11, 2014 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-25285362

RESUMO

The genes encoding many viral proteins such as HIV-1 envelope glycoprotein gp120 have a tendency for codons that are poorly used by the human genome. Why these codons are frequently present in the HIV genome is not known. The presence of these codons limits expression of HIV-1 gp120 for biochemical studies. The poor codons are replaced by synonymous codons that are frequently present in the highly expressed human genes to overexpress this protein. Whether this codon optimization affects functional properties of gp120 such as its N-linked glycosylation is unknown. We applied a bottom-up mass-spectrometry-based workflow for the direct measurement of deglycosylated and unglycosylated peptides with putative N-linked glycosylation sites, that is, NxS/T motifs. Using this mass-spectrometry approach in combination with ELISA, it is found that codon optimization significantly reduces the frequency with which the dolichol pyrophosphate-linked oligosaccharide is added by the catalytic subunits of oligosaccharide transferase complex to the glycosylation sites. This reduction affects binding of glycan-dependent broadly neutralizing antibodies. These data are essential for biochemical studies of gp120 and successful development of a vaccine against HIV-1. Furthermore, they demonstrate a mass-spectrometry approach for studying the site-specific N-linked glycosylation efficiency of glycoproteins.


Assuntos
Ensaio de Imunoadsorção Enzimática/métodos , Proteína gp120 do Envelope de HIV/metabolismo , Oligossacarídeos/metabolismo , Espectrometria de Massas em Tandem/métodos , Sequência de Aminoácidos , Sítios de Ligação/genética , Códon/genética , Eletroforese em Gel de Poliacrilamida , Glicoproteínas/genética , Glicoproteínas/metabolismo , Glicosilação , Células HEK293 , Proteína gp120 do Envelope de HIV/genética , Humanos , Dados de Sequência Molecular , Mutação , Peptídeos/análise , Peptídeos/metabolismo , Proteômica/métodos
9.
Proc Natl Acad Sci U S A ; 108(52): 21259-64, 2011 Dec 27.
Artigo em Inglês | MEDLINE | ID: mdl-22160701

RESUMO

Abscisic acid (ABA) is an essential hormone that controls plant growth, development, and responses to abiotic stresses. Central for ABA signaling is the ABA-mediated autoactivation of three monomeric Snf1-related kinases (SnRK2.2, -2.3, and -2.6). In the absence of ABA, SnRK2s are kept in an inactive state by forming physical complexes with type 2C protein phosphatases (PP2Cs). Upon relief of this inhibition, SnRK2 kinases can autoactivate through unknown mechanisms. Here, we report the crystal structures of full-length Arabidopsis thaliana SnRK2.3 and SnRK2.6 at 1.9- and 2.3-Å resolution, respectively. The structures, in combination with biochemical studies, reveal a two-step mechanism of intramolecular kinase activation that resembles the intermolecular activation of cyclin-dependent kinases. First, release of inhibition by PP2C allows the SnRK2s to become partially active because of an intramolecular stabilization of the catalytic domain by a conserved helix in the kinase regulatory domain. This stabilization enables SnRK2s to gain full activity by activation loop autophosphorylation. Autophosphorylation is more efficient in SnRK2.6, which has higher stability than SnRK2.3 and has well-structured activation loop phosphate acceptor sites that are positioned next to the catalytic site. Together, these data provide a structural framework that links ABA-mediated release of PP2C inhibition to activation of SnRK2 kinases.


Assuntos
Ácido Abscísico/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Modelos Moleculares , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais/fisiologia , Cristalização , Ativação Enzimática , Fosfoproteínas Fosfatases/metabolismo , Fosforilação , Proteína Fosfatase 2C , Difração de Raios X
10.
Proc Natl Acad Sci U S A ; 107(20): 9078-82, 2010 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-20439767

RESUMO

Knowledge about the protein targets of therapeutic agents is critical for understanding drug mode of action. Described here is a mass spectrometry-based proteomics method for identifying the protein target(s) of drug molecules that is potentially applicable to any drug compound. The method, which involves making thermodynamic measurements of protein-folding reactions in complex biological mixtures to detect protein-drug interactions, is demonstrated in an experiment to identify yeast protein targets of the immunosuppressive drug, cyclosporin A (CsA). Two of the ten protein targets identified in this proof of principle work were cyclophilin A and UDP-glucose-4-epimerase, both of which are known to interact with CsA, the former through a direct binding event (K(d) approximately 70 nM) and the latter through an indirect binding event. These two previously known protein targets validate the methodology and its ability to detect both the on- and off-target effects of protein-drug interactions. The other eight protein targets discovered here, which include several proteins involved in glucose metabolism, create a new framework in which to investigate the molecular basis of CsA side effects in humans.


Assuntos
Misturas Complexas/química , Preparações Farmacêuticas/metabolismo , Dobramento de Proteína , Proteínas/metabolismo , Proteômica/métodos , Cromatografia Líquida , Ciclosporina/metabolismo , Espectrometria de Massas , Termodinâmica , UDPglucose 4-Epimerase/metabolismo , Leveduras
11.
Nat Struct Mol Biol ; 30(1): 22-30, 2023 01.
Artigo em Inglês | MEDLINE | ID: mdl-36522428

RESUMO

Glycerol-3-phosphate acyltransferase (GPAT)1 is a mitochondrial outer membrane protein that catalyzes the first step of de novo glycerolipid biosynthesis. Hepatic expression of GPAT1 is linked to liver fat accumulation and the severity of nonalcoholic fatty liver diseases. Here we present the cryo-EM structures of human GPAT1 in substrate analog-bound and product-bound states. The structures reveal an N-terminal acyltransferase domain that harbors important catalytic motifs and a tightly associated C-terminal domain that is critical for proper protein folding. Unexpectedly, GPAT1 has no transmembrane regions as previously proposed but instead associates with the membrane via an amphipathic surface patch and an N-terminal loop-helix region that contains a mitochondrial-targeting signal. Combined structural, computational and functional studies uncover a hydrophobic pathway within GPAT1 for lipid trafficking. The results presented herein lay a framework for rational inhibitor development for GPAT1.


Assuntos
Fígado , Membranas Mitocondriais , Humanos , Fígado/metabolismo , Membranas Mitocondriais/metabolismo , Glicerol-3-Fosfato O-Aciltransferase/química , Glicerol-3-Fosfato O-Aciltransferase/metabolismo , Sequência de Aminoácidos
12.
Expert Rev Proteomics ; 8(1): 43-59, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21329427

RESUMO

Functional regulation of ligand-activated receptors is driven by alterations in the conformational dynamics of the protein upon ligand binding. Differential hydrogen/deuterium exchange (HDX) coupled with mass spectrometry has emerged as a rapid and sensitive approach for characterization of perturbations in conformational dynamics of proteins following ligand binding. While this technique is sensitive to detecting ligand interactions and alterations in receptor dynamics, it also can provide important mechanistic insights into ligand regulation. For example, HDX has been used to determine a novel mechanism of ligand activation of the nuclear receptor peroxisome proliferator activated receptor-γ, perform detailed analyses of binding modes of ligands within the ligand-binding pocket of two estrogen receptor isoforms, providing insight into selectivity, and helped classify different types of estrogen receptor-α ligands by correlating their pharmacology with the way they interact with the receptor based solely on hierarchical clustering of receptor HDX signatures. Beyond small-molecule-receptor interactions, this technique has also been applied to study protein-protein complexes, such as mapping antibody-antigen interactions. In this article, we summarize the current state of the differential HDX approaches and the future outlook. We summarize how HDX analysis of protein-ligand interactions has had an impact on biology and drug discovery.


Assuntos
Medição da Troca de Deutério/métodos , Espectrometria de Massas/métodos , Proteínas/química , Animais , Hormônios/química , Humanos , Hidrogênio/química , Ligantes , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Proteínas Quinases/química , Estrutura Terciária de Proteína , Receptores de Superfície Celular/química , Receptores Citoplasmáticos e Nucleares/química
13.
ACS Chem Biol ; 16(8): 1445-1455, 2021 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-34374519

RESUMO

Stability proteomics techniques that do not require drug modifications have emerged as an attractive alternative to affinity purification methods in drug target engagement studies. Two representative techniques include the chemical-denaturation-based SPROX (Stability of Proteins from Rates of Oxidation), which utilizes peptide-level quantification and thermal-denaturation-based TPP (Thermal Proteome Profiling), which utilizes protein-level quantification. Recently, the "OnePot" strategy was adapted for both SPROX and TPP to increase the throughput. When combined with the 2D setup which measures both the denaturation and the drug dose dimensions, the OnePot 2D format offers improved analysis specificity with higher resource efficiency. However, a systematic evaluation of the OnePot 2D format and a comparison between SPROX and TPP are still lacking. Here, we performed SPROX and TPP to identify protein targets of a well-studied pan-kinase inhibitor staurosporine with K562 lysate, in curve-fitting and OnePot 2D formats. We found that the OnePot 2D format provided ∼10× throughput, achieved ∼1.6× protein coverage and involves more straightforward data analysis. We also compared SPROX with the current "gold-standard" stability proteomics technique TPP in the OnePot 2D format. The protein coverage of TPP is ∼1.5 fold of SPROX; however, SPROX offers protein domain-level information, identifies comparable numbers of kinase hits, has higher signal (R value), and requires ∼3× less MS time. Unique SPROX hits encompass higher-molecular-weight proteins, compared to the unique TPP hits, and include atypical kinases. We also discuss hit stratification and prioritization strategies to promote the efficiency of hit followup.


Assuntos
Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases/análise , Proteoma/análise , Proteômica/métodos , Estaurosporina/farmacologia , Humanos , Células K562 , Proteínas Quinases/metabolismo , Proteoma/metabolismo
14.
Anal Chem ; 82(13): 5573-81, 2010 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-20527820

RESUMO

Described here is a mass spectrometry-based screening assay for the detection of protein-ligand binding interactions in multicomponent protein mixtures. The assay utilizes an oxidation labeling protocol that involves using hydrogen peroxide to selectively oxidize methionine residues in proteins in order to probe the solvent accessibility of these residues as a function of temperature. The extent to which methionine residues in a protein are oxidized after specified reaction times at a range of temperatures is determined in a MALDI analysis of the intact proteins and/or an LC-MS analysis of tryptic peptide fragments generated after the oxidation reaction is quenched. Ultimately, the mass spectral data is used to construct thermal denaturation curves for the detected proteins. In this proof-of-principle work, the protocol is applied to a four-protein model mixture comprised of ubiquitin, ribonuclease A (RNaseA), cyclophilin A (CypA), and bovine carbonic anhydrase II (BCAII). The new protocol's ability to detect protein-ligand binding interactions by comparing thermal denaturation data obtained in the absence and in the presence of ligand is demonstrated using cyclosporin A (CsA) as a test ligand. The known binding interaction between CsA and CypA was detected using both the MALDI- and LC-MS-based readouts described here.


Assuntos
Ligantes , Proteínas/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Sequência de Aminoácidos , Animais , Anidrase Carbônica II/química , Anidrase Carbônica II/metabolismo , Bovinos , Cromatografia Líquida/métodos , Ciclofilina A/química , Ciclofilina A/metabolismo , Peróxido de Hidrogênio/química , Metionina/química , Dados de Sequência Molecular , Oxirredução , Ligação Proteica , Ribonuclease Pancreático/química , Ribonuclease Pancreático/metabolismo , Temperatura , Tripsina/metabolismo , Ubiquitina/química , Ubiquitina/metabolismo
15.
Elife ; 82019 10 03.
Artigo em Inglês | MEDLINE | ID: mdl-31580259

RESUMO

Alanine-serine-cysteine transporter 2 (ASCT2, SLC1A5) is the primary transporter of glutamine in cancer cells and regulates the mTORC1 signaling pathway. The SLC1A5 function involves finely tuned orchestration of two domain movements that include the substrate-binding transport domain and the scaffold domain. Here, we present cryo-EM structures of human SLC1A5 and its complex with the substrate, L-glutamine in an outward-facing conformation. These structures reveal insights into the conformation of the critical ECL2a loop which connects the two domains, thus allowing rigid body movement of the transport domain throughout the transport cycle. Furthermore, the structures provide new insights into substrate recognition, which involves conformational changes in the HP2 loop. A putative cholesterol binding site was observed near the domain interface in the outward-facing state. Comparison with the previously determined inward-facing structure of SCL1A5 provides a basis for a more integrated understanding of substrate recognition and transport mechanism in the SLC1 family.


Assuntos
Sistema ASC de Transporte de Aminoácidos/química , Sistema ASC de Transporte de Aminoácidos/metabolismo , Glutamina/química , Glutamina/metabolismo , Antígenos de Histocompatibilidade Menor/química , Antígenos de Histocompatibilidade Menor/metabolismo , Microscopia Crioeletrônica , Humanos , Ligação Proteica , Conformação Proteica
16.
Anal Chem ; 80(11): 4175-85, 2008 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-18457414

RESUMO

Described here is a new technique, termed SPROX (stability of proteins from rates of oxidation), that can be used to measure the thermodynamic stability of proteins and protein-ligand complexes. SPROX utilizes hydrogen peroxide in the presence of increasing concentrations of a chemical denaturant to oxidize proteins. The extent of oxidation at a given oxidation time is determined as a function of the denaturant concentration using either electrospray or matrix-assisted laser desorption/ionization mass spectrometry. Ultimately, the denaturant concentration dependence of the oxidation reaction rate is used to evaluate a folding free energy (DeltaG(f)) and m value (deltaDeltaG(f)/delta[Den]) for the protein's folding/unfolding reaction. Measurements of such SPROX-derived DeltaG(f) and m values on proteins in the presence and absence of ligands can also be used to evaluate protein-ligand affinities (e.g., DeltaDeltaG(f) and Kd values). Presented here are SPROX results obtained on four model protein systems including ubiquitin, ribonuclease A (RNaseA), cyclophilin A (CypA), and bovine carbonic anhydrase II (BCAII). SPROX-derived DeltaG(f) and m values on these proteins are compared to values obtained using more established techniques (e.g., CD spectroscopy and SUPREX). The dissociation constants of several known protein-ligand complexes involving these proteins were also determined using SPROX and compared to previously reported values. The complexes included the CypA-cyclosporin A complex and the BCAII-4-carboxybenzenesulfonamide complex. The accuracy and precision of SPROX-derived thermodynamic parameters for the model proteins and protein-ligand complexes in this study are discussed as well as the caveats of the technique.


Assuntos
Proteínas/química , Proteínas/metabolismo , Animais , Bovinos , Estabilidade Enzimática , Cinética , Ligantes , Oxirredução , Ligação Proteica , Desnaturação Proteica , Dobramento de Proteína , Sensibilidade e Especificidade , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Termodinâmica
17.
Sci Rep ; 8(1): 4664, 2018 03 16.
Artigo em Inglês | MEDLINE | ID: mdl-29549268

RESUMO

Cystic Fibrosis (CF) is caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR). Mutations associated with CF cause loss-of-function in CFTR leading to salt imbalance in epithelial tissues. Kalydeco (also called VX-770 or ivacaftor) was approved for CF treatment in 2012 but little is known regarding the compound's interactions with CFTR including the site of binding or mechanisms of action. In this study we use hydrogen/deuterium exchange (HDX) coupled with mass spectrometry to assess the conformational dynamics of a thermostabilized form of CFTR in apo and ligand-bound states. We observe HDX protection at a known binding site for AMPPNP and significant protection for several regions of CFTR in the presence of Kalydeco. The ligand-induced changes of CFTR in the presence of Kalydeco suggest a potential binding site.


Assuntos
Difosfato de Adenosina/metabolismo , Adenilil Imidodifosfato/metabolismo , Aminofenóis/farmacologia , Regulador de Condutância Transmembrana em Fibrose Cística/química , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Quinolonas/farmacologia , Aminofenóis/química , Sítios de Ligação , Microscopia Crioeletrônica , Medição da Troca de Deutério , Humanos , Modelos Moleculares , Ligação Proteica , Conformação Proteica/efeitos dos fármacos , Estabilidade Proteica , Quinolonas/química , Termodinâmica
18.
Methods Mol Biol ; 1732: 29-55, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29480467

RESUMO

Protein-ligand interactions can be evaluated by a number of different biophysical methods. Here we describe some of the experimental methods that we have used to generate AMPK protein reagents and characterize its interactions with direct synthetic activators. Recombinant heterotrimeric AMPK complexes were generated using standard molecular biology methods by expression either in insect cells via infection with three different viruses or more routinely in Escherichia coli with a tricistronic expression vector. Hydrogen/deuterium exchange (HDX) coupled with mass spectrometry was used to probe protein conformational changes and potential binding sites of activators on AMPK. X-ray crystallographic studies were carried out on crystals of AMPK with bound ligands to reveal detailed molecular interactions formed by AMPK activators at near-atomic resolution. In order to gain insights into the mechanism of enzyme activation and to probe the effects of AMPK activators on kinetic parameters such as Michaelis-Menten constant (K m ) or maximal reaction velocity (V max), we performed classical enzyme kinetic studies using radioactive 33P-ATP-based filter assay. Equilibrium dissociation constants (K D ) and on and off rates of ligand binding were obtained by application of surface plasmon resonance (SPR) technique.


Assuntos
Proteínas Quinases Ativadas por AMP/química , Medição da Troca de Deutério/métodos , Ativadores de Enzimas/química , Ressonância de Plasmônio de Superfície/métodos , Proteínas Quinases Ativadas por AMP/isolamento & purificação , Animais , Sítios de Ligação , Cristalografia por Raios X , Medição da Troca de Deutério/instrumentação , Ativação Enzimática , Ensaios Enzimáticos/instrumentação , Ensaios Enzimáticos/métodos , Cinética , Ligantes , Espectrometria de Massas/instrumentação , Espectrometria de Massas/métodos , Simulação de Acoplamento Molecular , Ligação Proteica , Estrutura Quaternária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Células Sf9 , Ressonância de Plasmônio de Superfície/instrumentação
19.
J Mol Biol ; 430(21): 4102-4118, 2018 10 19.
Artigo em Inglês | MEDLINE | ID: mdl-30120952

RESUMO

Photoreceptors of the squid Loligo pealei contain a G-protein-coupled receptor (GPCR) signaling system that activates phospholipase C in response to light. Analogous to the mammalian visual system, signaling of the photoactivated GPCR rhodopsin is terminated by binding of squid arrestin (sArr). sArr forms a light-dependent, high-affinity complex with squid rhodopsin, which does not require prior receptor phosphorylation for interaction. This is at odds with classical mammalian GPCR desensitization where an agonist-bound phosphorylated receptor is needed to break stabilizing constraints within arrestins, the so-called "three-element interaction" and "polar core" network, before a stable receptor-arrestin complex can be established. Biophysical and mass spectrometric analysis of the squid rhodopsin-arrestin complex indicates that in contrast to mammalian arrestins, the sArr C-tail is not involved in a stable three-element interaction. We determined the crystal structure of C-terminally truncated sArr that adopts a basal conformation common to arrestins and is stabilized by a series of weak but novel polar core interactions. Unlike mammalian arrestin-1, deletion of the sArr C-tail does not influence kinetic properties of complex formation of sArr with the receptor. Hydrogen-deuterium exchange studies revealed the footprint of the light-activated rhodopsin on sArr. Furthermore, double electron-electron resonance spectroscopy experiments provide evidence that receptor-bound sArr adopts a conformation different from the one known for arrestin-1 and molecular dynamics simulations reveal the residues that account for the weak three-element interaction. Insights gleaned from studying this system add to our general understanding of GPCR-arrestin interaction.


Assuntos
Arrestina/química , Arrestina/metabolismo , Decapodiformes/metabolismo , Domínios e Motivos de Interação entre Proteínas , Rodopsina/química , Rodopsina/metabolismo , Animais , Cinética , Modelos Moleculares , Simulação de Dinâmica Molecular , Fosforilação , Ligação Proteica , Conformação Proteica , Análise Espectral , Relação Estrutura-Atividade
20.
Methods Mol Biol ; 1513: 11-22, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-27807827

RESUMO

A powerful interplay exists between the recognition of gene families, sensitive techniques in proteomics, and the interrogation of protein function using chemical probes. The most prominent methods, such as affinity capture, activity-based protein profiling and photoaffinity labeling, are extensively reviewed in the literature. Here we briefly review additional methods developed in the past 15 years. These include "stability proteomics" methods such as proteomically analyzed cellular thermal shift assays and the use of chemical oxidation as a probe of structure, the use of multiple bead-linked kinase inhibitors to analyze inhibitor specificities, and advances in the use of proteolysis-targeting chimeras for selective protein elimination.


Assuntos
Antineoplásicos/farmacologia , Descoberta de Drogas/métodos , Proteínas de Neoplasias/genética , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases/genética , Proteômica/métodos , Bioensaio , Expressão Gênica , Humanos , Células K562 , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/metabolismo , Oxirredução , Desnaturação Proteica/efeitos dos fármacos , Proteínas Quinases/metabolismo , Estabilidade Proteica/efeitos dos fármacos , Termodinâmica
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