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1.
Nat Commun ; 12(1): 5885, 2021 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-34620873

RESUMO

Pathogenic fungi exhibit a heavy burden on medical care and new therapies are needed. Here, we develop the fungal specific enzyme sterylglucosidase 1 (Sgl1) as a therapeutic target. Sgl1 converts the immunomodulatory glycolipid ergosterol 3ß-D-glucoside to ergosterol and glucose. Previously, we found that genetic deletion of Sgl1 in the pathogenic fungus Cryptococcus neoformans (Cn) results in ergosterol 3ß-D-glucoside accumulation, renders Cn non-pathogenic, and immunizes mice against secondary infections by wild-type Cn, even in condition of CD4+ T cell deficiency. Here, we disclose two distinct chemical classes that inhibit Sgl1 function in vitro and in Cn cells. Pharmacological inhibition of Sgl1 phenocopies a growth defect of the Cn Δsgl1 mutant and prevents dissemination of wild-type Cn to the brain in a mouse model of infection. Crystal structures of Sgl1 alone and with inhibitors explain Sgl1's substrate specificity and enable the rational design of antifungal agents targeting Sgl1.

2.
Nat Commun ; 12(1): 4718, 2021 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-34354069

RESUMO

Phospholipid synthesis and fat storage as triglycerides are regulated by lipin phosphatidic acid phosphatases (PAPs), whose enzymatic PAP function requires association with cellular membranes. Using hydrogen deuterium exchange mass spectrometry, we find mouse lipin 1 binds membranes through an N-terminal amphipathic helix, the Ig-like domain and HAD phosphatase catalytic core, and a middle lipin (M-Lip) domain that is conserved in mammalian and mammalian-like lipins. Crystal structures of the M-Lip domain reveal a previously unrecognized protein fold that dimerizes. The isolated M-Lip domain binds membranes both in vitro and in cells through conserved basic and hydrophobic residues. Deletion of the M-Lip domain in lipin 1 reduces PAP activity, membrane association, and oligomerization, alters subcellular localization, diminishes acceleration of adipocyte differentiation, but does not affect transcriptional co-activation. This establishes the M-Lip domain as a dimeric protein fold that binds membranes and is critical for full functionality of mammalian lipins.


Assuntos
Fosfatidato Fosfatase/química , Células 3T3-L1 , Adipogenia , Sequência de Aminoácidos , Animais , Membrana Celular/metabolismo , Sequência Conservada , Cristalografia por Raios X , Células HEK293 , Humanos , Espectrometria de Massa com Troca Hidrogênio-Deutério , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Modelos Moleculares , Simulação de Dinâmica Molecular , Fosfatidato Fosfatase/genética , Fosfatidato Fosfatase/metabolismo , Ligação Proteica , Domínios Proteicos , Dobramento de Proteína , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Deleção de Sequência , Homologia de Sequência de Aminoácidos , Transcrição Genética
3.
Methods Mol Biol ; 2263: 465-485, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33877613

RESUMO

Cellular membranes are a central hub for initiation and execution of many signaling processes. Integral to these processes being accomplished appropriately is the highly controlled recruitment and assembly of proteins at membrane surfaces. The study of the molecular mechanisms that mediate protein-membrane interactions can be facilitated by utilizing hydrogen-deuterium exchange mass spectrometry (HDX-MS). HDX-MS is a robust analytical technique that allows for the measurement of the exchange rate of backbone amide hydrogens with solvent to make inferences about protein structure and conformation. This chapter discusses the use of HDX-MS as a tool to study the conformational changes that occur within peripheral membrane proteins upon association with membrane. Particular reference will be made to the analysis of the protein kinase Akt and its activation upon binding phosphatidylinositol (3,4,5) tris-phosphate (PIP3)-containing membranes to illustrate specific methodological principles.


Assuntos
Membrana Celular/metabolismo , Proteínas Proto-Oncogênicas c-akt/química , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fenômenos Biofísicos , Membrana Celular/química , Espectrometria de Massa com Troca Hidrogênio-Deutério , Simulação de Dinâmica Molecular , Fosfatos de Fosfatidilinositol/química , Ligação Proteica , Conformação Proteica , Proteômica
4.
Structure ; 28(7): 830-846.e9, 2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32433991

RESUMO

Liver receptor homolog-1 (LRH-1; NR5A2) is a nuclear receptor that regulates a diverse array of biological processes. In contrast to dimeric nuclear receptors, LRH-1 is an obligate monomer and contains a subtype-specific helix at the C terminus of the DNA-binding domain (DBD), termed FTZ-F1. Although detailed structural information is available for individual domains of LRH-1, it is unknown how these domains exist in the intact nuclear receptor. Here, we developed an integrated structural model of human full-length LRH-1 using a combination of HDX-MS, XL-MS, Rosetta computational docking, and SAXS. The model predicts the DBD FTZ-F1 helix directly interacts with ligand binding domain helix 2. We confirmed several other predicted inter-domain interactions via structural and functional analyses. Comparison between the LRH-1/Dax-1 co-crystal structure and the integrated model predicted and confirmed Dax-1 co-repressor to modulate LRH-1 inter-domain dynamics. Together, these data support individual LRH-1 domains interacting to influence receptor structure and function.

5.
Nat Commun ; 11(1): 1734, 2020 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-32242008

RESUMO

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

6.
Nat Commun ; 11(1): 1309, 2020 03 11.
Artigo em Inglês | MEDLINE | ID: mdl-32161260

RESUMO

Lipin/Pah phosphatidic acid phosphatases (PAPs) generate diacylglycerol to regulate triglyceride synthesis and cellular signaling. Inactivating mutations cause rhabdomyolysis, autoinflammatory disease, and aberrant fat storage. Disease-mutations cluster within the conserved N-Lip and C-Lip regions that are separated by 500-residues in humans. To understand how the N-Lip and C-Lip combine for PAP function, we determined crystal structures of Tetrahymena thermophila Pah2 (Tt Pah2) that directly fuses the N-Lip and C-Lip. Tt Pah2 adopts a two-domain architecture where the N-Lip combines with part of the C-Lip to form an immunoglobulin-like domain and the remaining C-Lip forms a HAD-like catalytic domain. An N-Lip C-Lip fusion of mouse lipin-2 is catalytically active, which suggests mammalian lipins function with the same domain architecture as Tt Pah2. HDX-MS identifies an N-terminal amphipathic helix essential for membrane association. Disease-mutations disrupt catalysis or destabilize the protein fold. This illustrates mechanisms for lipin/Pah PAP function, membrane association, and lipin-related pathologies.


Assuntos
Fosfatidato Fosfatase/metabolismo , Fosfatidato Fosfatase/ultraestrutura , Proteínas de Protozoários/ultraestrutura , Domínio Catalítico/genética , Cristalografia por Raios X , Células HEK293 , Humanos , Fosfatidato Fosfatase/genética , Fosfatidato Fosfatase/isolamento & purificação , Conformação Proteica em alfa-Hélice , Proteínas de Protozoários/genética , Proteínas de Protozoários/isolamento & purificação , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/ultraestrutura , Tetrahymena thermophila/enzimologia , Transfecção
7.
EMBO Rep ; 21(2): e48441, 2020 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-31829496

RESUMO

The lipid kinase PI4KB, which generates phosphatidylinositol 4-phosphate (PI4P), is a key enzyme in regulating membrane transport and is also hijacked by multiple picornaviruses to mediate viral replication. PI4KB can interact with multiple protein binding partners, which are differentially manipulated by picornaviruses to facilitate replication. The protein c10orf76 is a PI4KB-associated protein that increases PI4P levels at the Golgi and is essential for the viral replication of specific enteroviruses. We used hydrogen-deuterium exchange mass spectrometry to characterize the c10orf76-PI4KB complex and reveal that binding is mediated by the kinase linker of PI4KB, with formation of the heterodimeric complex modulated by PKA-dependent phosphorylation. Complex-disrupting mutations demonstrate that PI4KB is required for membrane recruitment of c10orf76 to the Golgi, and that an intact c10orf76-PI4KB complex is required for the replication of c10orf76-dependent enteroviruses. Intriguingly, c10orf76 also contributed to proper Arf1 activation at the Golgi, providing a putative mechanism for the c10orf76-dependent increase in PI4P levels at the Golgi.


Assuntos
Enterovirus , Animais , Enterovirus/genética , Enterovirus/metabolismo , Complexo de Golgi/metabolismo , Fosfatos de Fosfatidilinositol , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Ligação Proteica , Células Sf9 , Replicação Viral
8.
J Med Chem ; 62(13): 6241-6261, 2019 07 11.
Artigo em Inglês | MEDLINE | ID: mdl-31244112

RESUMO

The phosphoinositide 3-kinase (PI3K)/mechanistic target of rapamycin (mTOR) pathway is frequently overactivated in cancer, and drives cell growth, proliferation, survival, and metastasis. Here, we report a structure-activity relationship study, which led to the discovery of a drug-like adenosine 5'-triphosphate-site PI3K/mTOR kinase inhibitor: (S)-4-(difluoromethyl)-5-(4-(3-methylmorpholino)-6-morpholino-1,3,5-triazin-2-yl)pyridin-2-amine (PQR530, compound 6), which qualifies as a clinical candidate due to its potency and specificity for PI3K and mTOR kinases, and its pharmacokinetic properties, including brain penetration. Compound 6 showed excellent selectivity over a wide panel of kinases and an excellent selectivity against unrelated receptor enzymes and ion channels. Moreover, compound 6 prevented cell growth in a cancer cell line panel. The preclinical in vivo characterization of compound 6 in an OVCAR-3 xenograft model demonstrated good oral bioavailability, excellent brain penetration, and efficacy. Initial toxicity studies in rats and dogs qualify 6 for further development as a therapeutic agent in oncology.


Assuntos
Aminopiridinas/farmacologia , Antineoplásicos/farmacologia , Morfolinas/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase/farmacologia , Piridinas/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Triazinas/farmacologia , Aminopiridinas/síntese química , Aminopiridinas/metabolismo , Animais , Antineoplásicos/síntese química , Antineoplásicos/metabolismo , Encéfalo/metabolismo , Linhagem Celular Tumoral , Cães , Feminino , Humanos , Masculino , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Microssomos Hepáticos/metabolismo , Simulação de Acoplamento Molecular , Estrutura Molecular , Morfolinas/síntese química , Morfolinas/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase/síntese química , Inibidores de Fosfoinositídeo-3 Quinase/metabolismo , Ligação Proteica , Piridinas/síntese química , Piridinas/metabolismo , Ratos Wistar , Relação Estrutura-Atividade , Serina-Treonina Quinases TOR/metabolismo , Triazinas/síntese química , Triazinas/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
9.
J Biol Chem ; 294(18): 7488-7502, 2019 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-30890560

RESUMO

Neutral sphingomyelinase 2 (nSMase2) produces the bioactive lipid ceramide and has important roles in neurodegeneration, cancer, and exosome formation. Although nSMase2 has low basal activity, it is fully activated by phosphatidylserine (PS). Previous work showed that interdomain interactions within nSMase2 are needed for PS activation. Here, we use multiple approaches, including small angle X-ray scattering, hydrogen-deuterium exchange-MS, circular dichroism and thermal shift assays, and membrane yeast two-hybrid assays, to define the mechanism mediating this interdomain interactions within nSMase2. In contrast to what we previously assumed, we demonstrate that PS binding at the N-terminal and juxtamembrane regions of nSMase2 rather acts as a conformational switch leading to interdomain interactions that are critical to enzyme activation. Our work assigns a unique function for a class of linkers of lipid-activated, membrane-associated proteins. It indicates that the linker actively participates in the activation mechanism via intramolecular interactions, unlike the canonical linkers that typically aid protein dimerization or localization.


Assuntos
Esfingomielina Fosfodiesterase/metabolismo , Regulação Alostérica , Aminoácidos/química , Domínio Catalítico , Ativação Enzimática , Humanos , Hidroxiureia/farmacologia , Mutação , Conformação Proteica , Saccharomyces cerevisiae/efeitos dos fármacos , Espalhamento a Baixo Ângulo , Esfingomielina Fosfodiesterase/química , Esfingomielina Fosfodiesterase/genética , Difração de Raios X
10.
J Biol Chem ; 294(12): 4621-4633, 2019 03 22.
Artigo em Inglês | MEDLINE | ID: mdl-30659094

RESUMO

Phosphoinositide 3-kinase ß (PI3Kß) is regulated by receptor tyrosine kinases (RTKs), G protein-coupled receptors (GPCRs), and small GTPases such as Rac1 and Rab5. Our lab previously identified two residues (Gln596 and Ile597) in the helical domain of the catalytic subunit (p110ß) of PI3Kß whose mutation disrupts binding to Rab5. To better define the Rab5-p110ß interface, we performed alanine-scanning mutagenesis and analyzed Rab5 binding with an in vitro pulldown assay with GST-Rab5GTP Of the 35 p110ß helical domain mutants assayed, 11 disrupted binding to Rab5 without affecting Rac1 binding, basal lipid kinase activity, or Gßγ-stimulated kinase activity. These mutants defined the Rab5-binding interface within p110ß as consisting of two perpendicular α-helices in the helical domain that are adjacent to the initially identified Gln596 and Ile597 residues. Analysis of the Rab5-PI3Kß interaction by hydrogen-deuterium exchange MS identified p110ß peptides that overlap with these helices; no interactions were detected between Rab5 and other regions of p110ß or p85α. Similarly, the binding of Rab5 to isolated p85α could not be detected, and mutations in the Ras-binding domain (RBD) of p110ß had no effect on Rab5 binding. Whereas soluble Rab5 did not affect PI3Kß activity in vitro, the interaction of these two proteins was critical for chemotaxis, invasion, and gelatin degradation by breast cancer cells. Our results define a single, discrete Rab5-binding site in the p110ß helical domain, which may be useful for generating inhibitors to better define the physiological role of Rab5-PI3Kß coupling in vivo.


Assuntos
Neoplasias da Mama/patologia , Invasividade Neoplásica , Fosfatidilinositol 3-Quinase/metabolismo , Proteínas rab5 de Ligação ao GTP/metabolismo , Sítios de Ligação , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Quimiotaxia , Gelatina/metabolismo , Células HEK293 , Humanos , Espectrometria de Massas/métodos , Mutação , Fosfatidilinositol 3-Quinase/genética , Ligação Proteica
11.
J Med Chem ; 61(23): 10463-10472, 2018 12 13.
Artigo em Inglês | MEDLINE | ID: mdl-30380865

RESUMO

Using a novel chemistry-based assay for identifying electrophilic natural products in unprocessed extracts, we identified the PI3-kinase/mTOR dual inhibitor neolymphostin A from Salinispora arenicola CNY-486. The method further showed that the vinylogous ester substituent on the neolymphostin core was the exact site for enzyme conjugation. Tandem MS/MS experiments on PI3Kα treated with the inhibitor revealed that neolymphostin covalently modified Lys802 with a shift in mass of +306 amu, corresponding to addition of the inhibitor and elimination of methanol. The binding pose of the inhibitor bound to PI3Kα was modeled, and hydrogen-deuterium exchange mass spectrometry experiments supported this model. Against a panel of kinases, neolymphostin showed good selectivity for PI3-kinase and mTOR. In addition, the natural product blocked AKT phosphorylation in live cells with an IC50 of ∼3 nM. Taken together, neolymphostin is the first reported example of a covalent kinase inhibitor from the bacterial domain of life.


Assuntos
Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Ésteres/química , Inibidores de Fosfoinositídeo-3 Quinase , Quinolinas/química , Quinolinas/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Inibidores Enzimáticos/metabolismo , Simulação de Acoplamento Molecular , Fosfatidilinositol 3-Quinases/química , Fosfatidilinositol 3-Quinases/metabolismo , Conformação Proteica , Quinolinas/metabolismo
12.
Nat Commun ; 9(1): 3772, 2018 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-30217979

RESUMO

The GTPase Rab11 plays key roles in receptor recycling, oogenesis, autophagosome formation, and ciliogenesis. However, investigating Rab11 regulation has been hindered by limited molecular detail describing activation by cognate guanine nucleotide exchange factors (GEFs). Here, we present the structure of Rab11 bound to the GEF SH3BP5, along with detailed characterization of Rab-GEF specificity. The structure of SH3BP5 shows a coiled-coil architecture that mediates exchange through a unique Rab-GEF interaction. Furthermore, it reveals a rearrangement of the switch I region of Rab11 compared with solved Rab-GEF structures, with a constrained conformation when bound to SH3BP5. Mutation of switch I provides insights into the molecular determinants that allow for Rab11 selectivity over evolutionarily similar Rab GTPases present on Rab11-positive organelles. Moreover, we show that GEF-deficient mutants of SH3BP5 show greatly decreased Rab11 activation in cellular assays of active Rab11. Overall, our results give molecular insight into Rab11 regulation, and how Rab-GEF specificity is achieved.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Fatores de Troca do Nucleotídeo Guanina/ultraestrutura , Proteínas rab de Ligação ao GTP/ultraestrutura , Cristalografia , Escherichia coli , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Estrutura Molecular , Mutação , Ligação Proteica , Proteínas rab de Ligação ao GTP/metabolismo
13.
J Mol Biol ; 430(18 Pt B): 3129-3142, 2018 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-30031006

RESUMO

Phosphatidylinositol 4-kinase IIIα (PI4KIIIα) is the lipid kinase primarily responsible for generating the lipid phosphatidylinositol 4-phosphate (PI4P) at the plasma membrane, which acts as the substrate for generation of the signaling lipids PIP2 and PIP3. PI4KIIIα forms a large heterotrimeric complex with two regulatory partners, TTC7 and FAM126. We describe using an integrated electron microscopy and hydrogen-deuterium exchange mass spectrometry (HDX-MS) approach to probe the architecture and dynamics of the complex of PI4KIIIα/TTC7/FAM126. HDX-MS reveals that the majority of the PI4KIIIα sequence was protected from exchange in short deuterium pulse experiments, suggesting presence of secondary structure, even in putative unstructured regions. Negative stain electron microscopy reveals the shape and architecture of the full-length complex, revealing an overall dimer of PI4KIIIα/TTC7/FAM126 trimers. HDX-MS reveals conformational changes in the TTC7/FAM126 complex upon binding PI4KIIIα, including both at the direct TTC7-PI4KIIIα interface and at the putative membrane binding surface. Finally, HDX-MS experiments of PI4KIIIα bound to the highly potent and selective inhibitor GSK-A1 compared to that bound to the non-specific inhibitor PIK93 revealed substantial conformational changes throughout an extended region of the kinase domain. Many of these changes were distant from the putative inhibitor binding site, showing a large degree of allosteric conformational changes that occur upon inhibitor binding. Overall, our results reveal novel insight into the regulation of PI4KIIIα by its regulatory proteins TTC7/FAM126, as well as additional dynamic information on how selective inhibition of PI4KIIIα is achieved.


Assuntos
1-Fosfatidilinositol 4-Quinase/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas/metabolismo , 1-Fosfatidilinositol 4-Quinase/química , Regulação Alostérica , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Modelos Moleculares , Complexos Multiproteicos/química , Complexos Multiproteicos/ultraestrutura , Ligação Proteica , Conformação Proteica , Multimerização Proteica , Proteínas/química , Proteínas Recombinantes
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