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1.
Nucleic Acids Res ; 51(22): 12443-12458, 2023 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-37930833

RESUMO

The dNTPase activity of tetrameric SAM and HD domain containing deoxynucleoside triphosphate triphosphohydrolase 1 (SAMHD1) plays a critical role in cellular dNTP regulation. SAMHD1 also associates with stalled DNA replication forks, DNA repair foci, ssRNA and telomeres. The above functions require nucleic acid binding by SAMHD1, which may be modulated by its oligomeric state. Here we establish in cryo-EM and biochemical studies that the guanine-specific A1 activator site of each SAMHD1 monomer is used to target the enzyme to guanine nucleotides within single-stranded (ss) DNA and RNA. Remarkably, nucleic acid strands containing a single guanine base induce dimeric SAMHD1, while two or more guanines with ∼20 nucleotide spacing induce a tetrameric form. A cryo-EM structure of ssRNA-bound tetrameric SAMHD1 shows how ssRNA strands bridge two SAMHD1 dimers and stabilize the structure. This ssRNA-bound tetramer is inactive with respect to dNTPase and RNase activity.


Assuntos
Proteínas Monoméricas de Ligação ao GTP , RNA , Guanina , Proteínas Monoméricas de Ligação ao GTP/genética , Nucleotídeos/metabolismo , Polímeros/metabolismo , Proteína 1 com Domínio SAM e Domínio HD/metabolismo
2.
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
3.
Nat Chem Biol ; 17(2): 152-160, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33199914

RESUMO

Heterobifunctional chimeric degraders are a class of ligands that recruit target proteins to E3 ubiquitin ligases to drive compound-dependent protein degradation. Advancing from initial chemical tools, protein degraders represent a mechanism of growing interest in drug discovery. Critical to the mechanism of action is the formation of a ternary complex between the target, degrader and E3 ligase to promote ubiquitination and subsequent degradation. However, limited insights into ternary complex structures exist, including a near absence of studies on one of the most widely co-opted E3s, cellular inhibitor of apoptosis 1 (cIAP1). In this work, we use a combination of biochemical, biophysical and structural studies to characterize degrader-mediated ternary complexes of Bruton's tyrosine kinase and cIAP1. Our results reveal new insights from unique ternary complex structures and show that increased ternary complex stability or rigidity need not always correlate with increased degradation efficiency.


Assuntos
Tirosina Quinase da Agamaglobulinemia/genética , Proteínas Inibidoras de Apoptose/genética , Cromatografia em Gel , Reagentes de Ligações Cruzadas , Humanos , Cinética , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica , Proteólise , Espectrometria de Massas por Ionização por Electrospray , Ubiquitina-Proteína Ligases , Ubiquitinação , Difração de Raios X
4.
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
5.
Anal Chem ; 86(14): 7041-8, 2014 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-24896224

RESUMO

Described here is the development of a mass spectrometry-based covalent labeling protocol that utilizes the reaction of dimethyl(2-hydroxy-5-nitrobenzyl)sulfonium bromide (HNSB) with tryptophan (Trp) residues to measure protein folding free energies (ΔG(f) values). In the protocol, the chemical denaturant dependence of the rate at which globally protected Trp residues in a protein react with HNSB is evaluated using either a matrix assisted laser desorption ionization time-of-flight analysis of the intact protein or a quantitative, bottom-up proteomics analysis using isobaric mass tags. In the proof-of-principle studies performed here, the protocol yielded accurate ΔG(f) values for the two-state folding proteins, lysozyme and cytochrome c. The protocol also yielded an accurate measure of the dissociation constant (K(d) value) for the binding of N,N',N″-triacetylchitotriose to lysozyme, and it successfully detected the binding of brinzolamide to BCA II, a non-two-state folding protein. The HNSB protocol can be used in combination with SPROX (stability of proteins from rates of oxidation), a previously reported technique that exploits the hydrogen peroxide oxidation of methionine (Met) residues in proteins to make ΔG(f) value measurements. Incorporating the HNSB protocol into SPROX increased the peptide and protein coverage in proteome-wide SPROX experiments by 50% and 25%, respectively. As part of this work, the precision of proteome-wide ΔG(f) value measurements using the combined HNSB and SPROX protocol is also evaluated.


Assuntos
Dobramento de Proteína , Proteômica/métodos , Triptofano/química , Sequência de Aminoácidos , Citocromos c/química , Metionina/química , Dados de Sequência Molecular , Muramidase/química , Estabilidade Proteica , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Compostos de Sulfônio/química , Termodinâmica
6.
iScience ; 27(4): 109593, 2024 Apr 19.
Artigo em Inglês | MEDLINE | ID: mdl-38632987

RESUMO

Precise regulation of Type I interferon signaling is crucial for combating infection and cancer while avoiding autoimmunity. Type I interferon signaling is negatively regulated by USP18. USP18 cleaves ISG15, an interferon-induced ubiquitin-like modification, via its canonical catalytic function, and inhibits Type I interferon receptor activity through its scaffold role. USP18 loss-of-function dramatically impacts immune regulation, pathogen susceptibility, and tumor growth. However, prior studies have reached conflicting conclusions regarding the relative importance of catalytic versus scaffold function. Here, we develop biochemical and cellular methods to systematically define the physiological role of USP18. By comparing a patient-derived mutation impairing scaffold function (I60N) to a mutation disrupting catalytic activity (C64S), we demonstrate that scaffold function is critical for cancer cell vulnerability to Type I interferon. Surprisingly, we discovered that human USP18 exhibits minimal catalytic activity, in stark contrast to mouse USP18. These findings resolve human USP18's mechanism-of-action and enable USP18-targeted therapeutics.

8.
bioRxiv ; 2023 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-37398126

RESUMO

The dNTPase activity of tetrameric SAM and HD domain containing deoxynucleoside triphosphate triphosphohydrolase 1 (SAMHD1) plays a critical role in cellular dNTP regulation. SAMHD1 also associates with stalled DNA replication forks, DNA repair foci, ssRNA, and telomeres. The above functions require nucleic acid binding by SAMHD1, which may be modulated by its oligomeric state. Here we establish that the guanine-specific A1 activator site of each SAMHD1 monomer is used to target the enzyme to guanine nucleotides within single-stranded (ss) DNA and RNA. Remarkably, nucleic acid strands containing a single guanine base induce dimeric SAMHD1, while two or more guanines with ~20 nucleotide spacing induce a tetrameric form. A cryo-EM structure of ssRNA-bound tetrameric SAMHD1 shows how ssRNA strands bridge two SAMHD1 dimers and stabilize the structure. This ssRNA-bound tetramer is inactive with respect to dNTPase and RNase activity.

9.
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
10.
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
11.
J Am Soc Mass Spectrom ; 27(10): 1670-6, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27530778

RESUMO

Geldanamycin is a natural product with well-established and potent anti-cancer activities. Heat shock protein 90 (Hsp90) is the known target of geldanamycin, which directly binds to Hsp90's N-terminal ATP binding domain and inhibits Hsp90's ATPase activity. The affinity of geldanamycin for Hsp90 has been measured in multiple studies. However, there have been large discrepancies between the reported dissociation constants (i.e., Kd values), which have ranged from low nanomolar to micromolar. Here the stability of proteins from rates of oxidation (SPROX) technique was used in combination with an isobaric mass tagging strategy to measure the binding affinity of geldanamycin to unpurified Hsp90 in an MCF-7 cell lysate. The Kd values determined here were dependent on how long geldanamycin was equilibrated with the lysate prior to SPROX analysis. The Kd values determined using equilibration times of 0.5 and 24 h were 1 and 0.03 µM, respectively. These Kd values, which are similar to those previously reported in a geldanamycin-Hsp90 binding study that involved the use of a fluorescently labeled geldanamycin analogue, establish that the slow-tight binding behavior previously observed for the fluorescently labeled geldanamycin analogue is not an artifact of the fluorescent label, but rather an inherent property of the geldanamycin-Hsp90 binding interaction. The slow-tight binding property of this complex may be related to time-dependent conformational changes in Hsp90 and/or to time-dependent chemical changes in geldanamycin, both of which have been previously proposed to explain the slow-tight binding behavior of the geldanamycin-Hsp90 complex. Graphical Abstract ᅟ.


Assuntos
Benzoquinonas/química , Proteínas de Choque Térmico HSP90/química , Lactamas Macrocíclicas/química , Espectrometria de Massas , Proteômica , Ligação Proteica , Termodinâmica
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