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1.
J Biol Chem ; 300(3): 105688, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38280431

RESUMO

Cytochrome b5 (b5) is known to stimulate some catalytic activities of cytochrome P450 (P450, CYP) enzymes, although mechanisms still need to be defined. The reactions most strongly enhanced by b5 are the 17,20-lyase reactions of P450 17A1 involved in steroid biosynthesis. We had previously used a fluorescently labeled human b5 variant (Alexa 488-T70C-b5) to characterize human P450 17A1-b5 interactions, but subsequent proteomic analyses indicated that lysines in b5 were also modified with Alexa 488 maleimide in addition to Cys-70, due to disulfide dimerization of the T70C mutant. A series of b5 variants were constructed with Cys replacements for the identified lysine residues and labeled with the dye. Fluorescence attenuation and the function of b5 in the steroid lyase reaction depended on the modified position. Apo-b5 (devoid of heme group) studies revealed the lack of involvement of the b5 heme in the fluorescence attenuation. A structural model of b5 with P450 17A1 was predicted using AlphaFold-Multimer algorithms/Rosetta docking, based upon the individual structures, which predicted several new contacts not previously reported, that is, interactions of b5 Glu-48:17A1 Arg-347, b5 Glu-49:17A1 Arg-449, b5 Asp-65:17A1 Arg-126, b5 Asp-65:17A1 Arg-125, and b5 Glu-61:17A1 Lys-91. Fluorescence polarization assays with two modified b5 variants yielded Kd values (for b5-P450 17A1) of 120 to 380 nM, the best estimate of binding affinity. We conclude that both monomeric and dimeric b5 can bind to P450 17A1 and stimulate activity. Results with the mutants indicate that several Lys residues in b5 are sensitive to the interaction with P450 17A1, including Lys-88 and Lys-91.


Assuntos
Citocromos b5 , Modelos Moleculares , Esteroide 17-alfa-Hidroxilase , Humanos , Citocromos b5/genética , Citocromos b5/metabolismo , Fluorescência , Heme , Proteômica , Esteroide 17-alfa-Hidroxilase/química , Esteroide 17-alfa-Hidroxilase/metabolismo , Ligação Proteica/genética , Ativação Enzimática/genética , Estrutura Quaternária de Proteína , Mutação
2.
Anal Chem ; 95(50): 18316-18325, 2023 12 19.
Artigo em Inglês | MEDLINE | ID: mdl-38049117

RESUMO

Correlating the structure and dynamics of proteins with biological function is critical to understanding normal and dysfunctional cellular mechanisms. We describe a quantitative method of hydroxyl radical generation via Fe(II)-ethylenediaminetetraacetic acid (EDTA)-catalyzed Fenton chemistry that provides ready access to protein oxidative footprinting using equipment commonly found in research and process control laboratories. Robust and reproducible dose-dependent oxidation of protein samples is observed and quantitated by mass spectrometry with as fine a single residue resolution. An oxidation analysis of lysozyme provides a readily accessible benchmark for our method. The efficacy of our oxidation method is demonstrated by mapping the interface of a RAS-monobody complex, the surface of the NIST mAb, and the interface between PRC2 complex components. These studies are executed using standard laboratory tools and a few pennies of reagents; the mass spectrometry analysis can be streamlined to map the protein structure with single amino acid residue resolution.


Assuntos
Radical Hidroxila , Proteínas , Ácido Edético/química , Radical Hidroxila/química , Proteínas/análise , Pegadas de Proteínas/métodos , Estresse Oxidativo , Oxirredução
3.
Bone ; 187: 117209, 2024 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-39047900

RESUMO

Complications of diabetes is a major health problem affecting multiple organs including bone, where the chronic disease increases the risk of fragility fractures. One hypothesis suggests a pathogenic role for hyperglycemia-induced modification of proteins, a.k.a. advanced glycation end products (AGEs), resulting in structural and functional damage to bone extracellular matrix (ECM). Evidence supporting this hypothesis has been limited by the lack of comprehensive information about the location of AGEs that accumulate in vivo at specific sites within the proteins of bone ECM. Analyzing extracts from cortical bone of cadaveric femurs by liquid chromatography tandem mass spectrometry, we generated a quantitative AGE map of human collagen I for male and female adult donors with and without diabetes. The map describes the chemical nature, sequence position, and levels of four major physiological AGEs, e.g. carboxymethyllysine, and an AGE precursor fructosyllysine within the collagen I triple-helical region. The important features of the map are: 1) high map reproducibility in the individual bone extracts, i.e. 20 male and 20 female donors; 2) localization of modifications to distinct clusters: 10 clusters containing 34 AGE sites in male donors and 9 clusters containing 28 sites in female donors; 3) significant increases in modification levels in diabetes at multiple sites: 26 out of 34 sites in males and in 17 out of 28 sites in females; and 4) generally higher modification levels in male vs. female donors. Moreover, the AGE levels at multiple individual sites correlated with total bone pentosidine levels in male but not in female donors. Molecular dynamics simulations and molecular modeling predicted significant impact of modifications on solvent exposure, charge distribution, and hydrophobicity of the triple helix as well as disruptions to the structure of collagen I fibril. In summary, the AGE map of collagen I revealed diabetes-induced, sex-specific non-enzymatic modifications at distinct triple helical sites that can disrupt collagen structure, thus proposing a specific mechanism of AGE contribution to diabetic complications in human bone.

4.
BBA Adv ; 3: 100079, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37082268

RESUMO

Accumulation of advanced glycation end products (AGEs), particularly in long-lived extracellular matrix proteins, has been implicated in pathogenesis of diabetic complications and in aging. Knowledge about specific locations of AGEs and their precursors within protein primary structure is critical for understanding their physiological and pathophysiological impact. However, the information on specific AGE sites is lacking. Here, we identified sequence positions of four major AGEs, carboxymethyllysine, carboxyethyllysine, 5-hydro-5-methyl imidazolone, and 5-hydro-imidazolone, and an AGE precursor fructosyllysine within the triple helical region of collagen I from cortical bone of human femurs. The presented map provides a basis for site-specific quantitation of AGEs and other non-enzymatic post-translational modifications and identification of those sites affected by aging, diabetes, and other diseases such as osteoporosis; it can also help in guiding future studies of AGE impact on structure and function of collagen I in bone.

5.
Sci Adv ; 8(13): eabm0314, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35353580

RESUMO

Replication-coupled DNA repair and damage tolerance mechanisms overcome replication stress challenges and complete DNA synthesis. These pathways include fork reversal, translesion synthesis, and repriming by specialized polymerases such as PRIMPOL. Here, we investigated how these pathways are used and regulated in response to varying replication stresses. Blocking lagging-strand priming using a POLα inhibitor slows both leading- and lagging-strand synthesis due in part to RAD51-, HLTF-, and ZRANB3-mediated, but SMARCAL1-independent, fork reversal. ATR is activated, but CHK1 signaling is dampened compared to stalling both the leading and lagging strands with hydroxyurea. Increasing CHK1 activation by overexpressing CLASPIN in POLα-inhibited cells promotes replication elongation through PRIMPOL-dependent repriming. CHK1 phosphorylates PRIMPOL to promote repriming irrespective of the type of replication stress, and this phosphorylation is important for cellular resistance to DNA damage. However, PRIMPOL activation comes at the expense of single-strand gap formation, and constitutive PRIMPOL activity results in reduced cell fitness.


Assuntos
Replicação do DNA , DNA Polimerase Dirigida por DNA , Dano ao DNA , Reparo do DNA , DNA Polimerase Dirigida por DNA/genética , Fosforilação
6.
Protein Pept Lett ; 26(1): 61-69, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30543161

RESUMO

BACKGROUND: For almost four decades, hydroxyl radical chemically generated by Fenton chemistry has been a mainstay for the oxidative 'footprinting' of macromolecules. OBJECTIVE: In this article, we start by reviewing the application of chemical generation of hydroxyl radical to the development of oxidative footprinting of DNA and RNA and the subsequent application of the method to oxidative footprinting of proteins. We next discuss a novel strategy for generating hydroxyl radicals by Fenton chemistry that immobilizes catalytic iron on a solid surface (Pyrite Shrink Wrap laminate) for the application of nucleic acid and protein footprinting. METHOD: Pyrite Shrink-Wrap Laminate is fabricated by depositing pyrite (Fe-S2, aka 'fool's gold') nanocrystals onto thermolabile plastic (Shrinky Dink). The laminate can be thermoformed into a microtiter plate format into which samples are deposited for oxidation. RESULTS: We demonstrate the utility of the Pyrite Shrink-Wrap Laminate for the chemical generation of hydroxyl radicals by mapping the surface of the T-cell co-stimulatory protein Programmed Death - 1 (PD-1) and the interface of the complex with its ligand PD-L1. CONCLUSION: We have developed and validated an affordable and reliable benchtop method of hydroxyl radical generation that will broaden the application of protein oxidative footprinting. Due to the minimal equipment required to implement this method, it should be easily adaptable by many laboratories with access to mass spectrometry.


Assuntos
Radical Hidroxila , Espectrometria de Massas/métodos , Pegadas de Proteínas/métodos , DNA/análise , DNA/química , Radical Hidroxila/análise , Radical Hidroxila/química , Ferro/química , Oxirredução , Proteínas/análise , Proteínas/química , RNA/análise , RNA/química , Sulfetos/química
7.
Lab Chip ; 15(7): 1646-50, 2015 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-25666234

RESUMO

The structure of macromolecules and their complexes dictate their biological function. In "footprinting", the solvent accessibility of the residues that constitute proteins, DNA and RNA can be determined from their reactivity to an exogenous reagent such as the hydroxyl radical (·OH). While ·OH generation for protein footprinting is achieved by radiolysis, photolysis and electrochemistry, we present a simpler solution. A thin film of pyrite (cubic FeS2) nanocrystals deposited onto a shape memory polymer (commodity shrink-wrap film) generates sufficient ·OH via Fenton chemistry for oxidative footprinting analysis of proteins. We demonstrate that varying either time or H2O2 concentration yields the required ·OH dose-oxidation response relationship. A simple and scalable sample handling protocol is enabled by thermoforming the "pyrite shrink-wrap laminate" into a standard microtiter plate format. The low cost and malleability of the laminate facilitates its integration into high throughput screening and microfluidic devices.


Assuntos
Ferro/química , Pegadas de Proteínas/instrumentação , Pegadas de Proteínas/métodos , Sulfetos/química , Desenho de Equipamento , Peróxido de Hidrogênio , Proteínas/análise , Proteínas/química
8.
Cancer Discov ; 3(9): 1002-19, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23955273

RESUMO

UNLABELLED: Although aberrant DNA methylation patterning is a hallmark of cancer, the relevance of targeting DNA methyltransferases (DNMT) remains unclear for most tumors. In diffuse large B-cell lymphoma (DLBCL) we observed that chemoresistance is associated with aberrant DNA methylation programming. Prolonged exposure to low-dose DNMT inhibitors (DNMTI) reprogrammed chemoresistant cells to become doxorubicin sensitive without major toxicity in vivo. Nine genes were recurrently hypermethylated in chemoresistant DLBCL. Of these, SMAD1 was a critical contributor, and reactivation was required for chemosensitization. A phase I clinical study was conducted evaluating azacitidine priming followed by standard chemoimmunotherapy in high-risk patients newly diagnosed with DLBCL. The combination was well tolerated and yielded a high rate of complete remission. Pre- and post-azacitidine treatment biopsies confirmed SMAD1 demethylation and chemosensitization, delineating a personalized strategy for the clinical use of DNMTIs. SIGNIFICANCE: The problem of chemoresistant DLBCL remains the most urgent challenge in the clinical management of patients with this disease. We describe a mechanism-based approach toward the rational translation of DNMTIs for the treatment of high-risk DLBCL.


Assuntos
Azacitidina/uso terapêutico , Metilação de DNA/genética , Metilases de Modificação do DNA/antagonistas & inibidores , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Linfoma Difuso de Grandes Células B/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Antimetabólitos Antineoplásicos/uso terapêutico , Azacitidina/efeitos adversos , Linhagem Celular Tumoral , Dano ao DNA/efeitos dos fármacos , Metilases de Modificação do DNA/metabolismo , Doxorrubicina/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Epigênese Genética , Humanos , Pessoa de Meia-Idade , Interferência de RNA , RNA Interferente Pequeno , Proteína Smad1/genética , Adulto Jovem
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