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1.
Biochemistry ; 62(3): 633-644, 2023 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-34985287

RESUMO

Autophagy-related proteins (Atgs) drive the lysosome-mediated degradation pathway, autophagy, to enable the clearance of dysfunctional cellular components and maintain homeostasis. In humans, this process is driven by the mammalian Atg8 (mAtg8) family of proteins comprising the LC3 and GABARAP subfamilies. The mAtg8 proteins play essential roles in the formation and maturation of autophagosomes and the capture of specific cargo through binding to the conserved LC3-interacting region (LIR) sequence within target proteins. Modulation of interactions of mAtg8 with its target proteins via small-molecule ligands would enable further interrogation of their function. Here we describe unbiased fragment and DNA-encoded library (DEL) screening approaches for discovering LC3 small-molecule ligands. Both strategies resulted in compounds that bind to LC3, with the fragment hits favoring a conserved hydrophobic pocket in mATG8 proteins, as detailed by LC3A-fragment complex crystal structures. Our findings demonstrate that the malleable LIR-binding surface can be readily targeted by fragments; however, rational design of additional interactions to drive increased affinity proved challenging. DEL libraries, which combine small, fragment-like building blocks into larger scaffolds, yielded higher-affinity binders and revealed an unexpected potential for reversible, covalent ligands. Moreover, DEL hits identified possible vectors for synthesizing fluorescent probes or bivalent molecules for engineering autophagic degradation of specific targets.


Assuntos
Autofagia , Proteínas Associadas aos Microtúbulos , Humanos , Animais , Proteínas Associadas aos Microtúbulos/metabolismo , Ligantes , Família da Proteína 8 Relacionada à Autofagia/química , Autofagossomos/metabolismo , Mamíferos/metabolismo
2.
J Biol Chem ; 293(43): 16803-16817, 2018 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-30206119

RESUMO

The caspase recruitment domain-containing protein 9 (CARD9)-B-cell lymphoma/leukemia 10 (Bcl10) signaling axis is activated in myeloid cells during the innate immune response to a variety of diverse pathogens. This signaling pathway requires a critical caspase recruitment domain (CARD)-CARD interaction between CARD9 and Bcl10 that promotes downstream activation of factors, including NF-κB and the mitogen-activated protein kinase (MAPK) p38. Despite these insights, CARD9 remains structurally uncharacterized, and little mechanistic understanding of its regulation exists. We unexpectedly found here that the CARD in CARD9 binds to Zn2+ with picomolar affinity-a concentration comparable with the levels of readily accessible Zn2+ in the cytosol. NMR solution structures of the CARD9-CARD in the apo and Zn2+-bound states revealed that Zn2+ has little effect on the ground-state structure of the CARD; yet the stability of the domain increased considerably upon Zn2+ binding, with a concomitant reduction in conformational flexibility. Moreover, Zn2+ binding inhibited polymerization of the CARD9-CARD into helical assemblies. Here, we also present a 20-Å resolution negative-stain EM (NS-EM) structure of these filamentous assemblies and show that they adopt a similar helical symmetry as reported previously for filaments of the Bcl10 CARD. Using both bulk assays and direct NS-EM visualization, we further show that the CARD9-CARD assemblies can directly template and thereby nucleate Bcl10 polymerization, a capacity considered critical to propagation of the CARD9-Bcl10 signaling cascade. Our findings indicate that CARD9 is a potential target of Zn2+-mediated signaling that affects Bcl10 polymerization in innate immune responses.


Assuntos
Proteína 10 de Linfoma CCL de Células B/metabolismo , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Zinco/metabolismo , Proteína 10 de Linfoma CCL de Células B/química , Proteína 10 de Linfoma CCL de Células B/genética , Proteínas Adaptadoras de Sinalização CARD/química , Proteínas Adaptadoras de Sinalização CARD/genética , Cristalografia por Raios X , Humanos , NF-kappa B/genética , NF-kappa B/metabolismo , Polimerização , Ligação Proteica , Domínios Proteicos , Transdução de Sinais , Zinco/química , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
3.
Proc Natl Acad Sci U S A ; 113(17): 4735-40, 2016 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-27071107

RESUMO

Detailed understanding of how conformational dynamics orchestrates function in allosteric regulation of recognition and catalysis remains ambiguous. Here, we simulate CypA using multiple-microsecond-long atomistic molecular dynamics in explicit solvent and carry out NMR experiments. We analyze a large amount of time-dependent multidimensional data with a coarse-grained approach and map key dynamical features within individual macrostates by defining dynamics in terms of residue-residue contacts. The effects of substrate binding are observed to be largely sensed at a location over 15 Å from the active site, implying its importance in allostery. Using NMR experiments, we confirm that a dynamic cluster of residues in this distal region is directly coupled to the active site. Furthermore, the dynamical network of interresidue contacts is found to be coupled and temporally dispersed, ranging over 4 to 5 orders of magnitude. Finally, using network centrality measures we demonstrate the changes in the communication network, connectivity, and influence of CypA residues upon substrate binding, mutation, and during catalysis. We identify key residues that potentially act as a bottleneck in the communication flow through the distinct regions in CypA and, therefore, as targets for future mutational studies. Mapping these dynamical features and the coupling of dynamics to function has crucial ramifications in understanding allosteric regulation in enzymes and proteins, in general.


Assuntos
Aminoácidos/química , Ciclofilina A/química , Ciclofilina A/ultraestrutura , Espectroscopia de Ressonância Magnética/métodos , Modelos Químicos , Simulação de Dinâmica Molecular , Aminoácidos/genética , Sítios de Ligação , Catálise , Ciclofilina A/genética , Mutação , Ligação Proteica , Conformação Proteica , Relação Estrutura-Atividade
4.
Proc Natl Acad Sci U S A ; 111(28): 10203-8, 2014 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-24982184

RESUMO

Proline isomerization is a ubiquitous process that plays a key role in the folding of proteins and in the regulation of their functions. Different families of enzymes, known as "peptidyl-prolyl isomerases" (PPIases), catalyze this reaction, which involves the interconversion between the cis and trans isomers of the N-terminal amide bond of the amino acid proline. However, complete descriptions of the mechanisms by which these enzymes function have remained elusive. We show here that cyclophilin A, one of the most common PPIases, provides a catalytic environment that acts on the substrate through an electrostatic handle mechanism. In this mechanism, the electrostatic field in the catalytic site turns the electric dipole associated with the carbonyl group of the amino acid preceding the proline in the substrate, thus causing the rotation of the peptide bond between the two residues. We identified this mechanism using a combination of NMR measurements, molecular dynamics simulations, and density functional theory calculations to simultaneously determine the cis-bound and trans-bound conformations of cyclophilin A and its substrate as the enzymatic reaction takes place. We anticipate that this approach will be helpful in elucidating whether the electrostatic handle mechanism that we describe here is common to other PPIases and, more generally, in characterizing other enzymatic processes.


Assuntos
Ciclofilina A/química , Simulação de Dinâmica Molecular , Prolina/química , Catálise , Humanos , Ressonância Magnética Nuclear Biomolecular , Eletricidade Estática
5.
Biochemistry ; 54(38): 5815-27, 2015 Sep 29.
Artigo em Inglês | MEDLINE | ID: mdl-26335054

RESUMO

Cyclophilins catalyze cis ↔ trans isomerization of peptidyl-prolyl bonds, influencing protein folding along with a breadth of other biological functions such as signal transduction. Here, we have determined the microscopic rate constants defining the full enzymatic cycle for three human cyclophilins and a more distantly related thermophilic bacterial cyclophilin when catalyzing interconversion of a biologically representative peptide substrate. The cyclophilins studied here exhibit variability in on-enzyme interconversion as well as an up to 2-fold range in rates of substrate binding and release. However, among the human cyclophilins, the microscopic rate constants appear to have been tuned to maintain remarkably similar isomerization rates without a concurrent conservation of apparent binding affinities. While the structures and active site compositions of the human cyclophilins studied here are highly conserved, we find that the enzymes exhibit significant variability in microsecond to millisecond time scale mobility, suggesting a role for the inherent conformational fluctuations that exist within the cyclophilin family as being functionally relevant in regulating substrate interactions. We have additionally modeled the relaxation dispersion profile given by the commonly employed Carr-Purcell-Meiboom-Gill relaxation dispersion (CPMG-RD) experiment when applied to a reversible enzymatic system such as cyclophilin isomerization and identified a significant limitation in the applicability of this approach for monitoring on-enzyme turnover. Specifically, we show both computationally and experimentally that the CPMG-RD experiment is sensitive to noncatalyzed substrate binding and release in reversible systems even at saturating substrate concentrations unless the on-enzyme interconversion rate is much faster than the substrate release rate.


Assuntos
Ciclofilinas/metabolismo , Domínio Catalítico , Ciclofilinas/química , Humanos , Isomerismo , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Conformação Proteica
6.
Biochemistry ; 54(20): 3207-17, 2015 May 26.
Artigo em Inglês | MEDLINE | ID: mdl-25923019

RESUMO

Thermophilic proteins have found extensive use in research and industrial applications because of their high stability and functionality at elevated temperatures while simultaneously providing valuable insight into our understanding of protein folding, stability, dynamics, and function. Cyclophilins, constituting a ubiquitously expressed family of peptidyl-prolyl isomerases with a range of biological functions and disease associations, have been utilized both for conferring stress tolerances and in exploring the link between conformational dynamics and enzymatic function. To date, however, no active thermophilic cyclophilin has been fully biophysically characterized. Here, we determine the structure of a thermophilic cyclophilin (GeoCyp) from Geobacillus kaustophilus, characterize its dynamic motions over several time scales using an array of methodologies that include chemical shift-based methods and relaxation experiments over a range of temperatures, and measure catalytic activity over a range of temperatures to compare its structure, dynamics, and function to those of a mesophilic counterpart, human cyclophilin A (CypA). Unlike those of most thermophile/mesophile pairs, GeoCyp catalysis is not substantially impaired at low temperatures as compared to that of CypA, retaining ~70% of the activity of its mesophilic counterpart. Examination of substrate-bound ensembles reveals a mechanism by which the two cyclophilins may have adapted to their environments through altering dynamic loop motions and a critical residue that acts as a clamp to regulate substrate binding differentially in CypA and GeoCyp. Fast time scale (pico- to nanosecond) dynamics are largely conserved between the two proteins, in accordance with the high degree of structural similarity, although differences do exist in their temperature dependencies. Slower (microsecond) time scale motions are likewise localized to similar regions in the two proteins with some variability in their magnitudes yet do not exhibit significant temperature dependencies in either enzyme.


Assuntos
Ciclofilinas/química , Domínio Catalítico , Temperatura Baixa , Estabilidade Enzimática , Geobacillus/enzimologia , Cinética , Simulação de Dinâmica Molecular , Ligação Proteica , Estrutura Secundária de Proteína , Homologia Estrutural de Proteína
7.
J Biol Chem ; 289(23): 16349-61, 2014 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-24755226

RESUMO

Eya proteins are essential co-activators of the Six family of transcription factors and contain a unique tyrosine phosphatase domain belonging to the haloacid dehalogenase family of phosphatases. The phosphatase activity of Eya is important for the transcription of a subset of Six1-target genes, and also directs cells to the repair rather than apoptosis pathway upon DNA damage. Furthermore, Eya phosphatase activity has been shown to mediate transformation, invasion, migration, and metastasis of breast cancer cells, making it a potential new drug target for breast cancer. We have previously identified a class of N-arylidenebenzohydrazide compounds that specifically inhibit the Eya2 phosphatase. Herein, we demonstrate that these compounds are reversible inhibitors that selectively inhibit the phosphatase activity of Eya2, but not Eya3. Our mutagenesis results suggest that this class of compounds does not bind to the active site and the binding does not require the coordination with Mg(2+). Moreover, these compounds likely bind within a site on the opposite face of the active site, and function as allosteric inhibitors. We also demonstrate that this class of compounds inhibits Eya2 phosphatase-mediated cell migration, setting the foundation for these molecules to be developed into chemical probes for understanding the specific function of the Eya2 phosphatase and to serve as a prototype for the development of Eya2 phosphatase specific anti-cancer drugs.


Assuntos
Movimento Celular/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Proteínas Nucleares/antagonistas & inibidores , Proteínas Tirosina Fosfatases/antagonistas & inibidores , Regulação Alostérica , Sequência de Aminoácidos , Calorimetria , Linhagem Celular , Cristalografia por Raios X , Inibidores Enzimáticos/farmacologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Magnésio/metabolismo , Simulação de Acoplamento Molecular , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Proteínas Nucleares/fisiologia , Ligação Proteica , Proteínas Tirosina Fosfatases/química , Proteínas Tirosina Fosfatases/metabolismo , Proteínas Tirosina Fosfatases/fisiologia , Homologia de Sequência de Aminoácidos , Espectrofotometria Ultravioleta
8.
Commun Biol ; 4(1): 916, 2021 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-34316015

RESUMO

Apolipoprotein L1 (ApoL1) is a circulating innate immunity protein protecting against trypanosome infection. However, two ApoL1 coding variants are associated with a highly increased risk of chronic kidney disease. Here we present X-ray and NMR structures of the N-terminal domain (NTD) of ApoL1 and of its closest relative ApoL2. In both proteins, four of the five NTD helices form a four-helix core structure which is different from the classical four-helix bundle and from the pore-forming domain of colicin A. The reactivity with a conformation-specific antibody and structural models predict that this four-helix motif is also present in the NTDs of ApoL3 and ApoL4, suggesting related functions within the small ApoL family. The long helix 5 of ApoL1 is conformationally flexible and contains the BH3-like region. This BH3-like α-helix resembles true BH3 domains only in sequence and structure but not in function, since it does not bind to the pro-survival members of the Bcl-2 family, suggesting a Bcl-2-independent role in cytotoxicity. These findings should expedite a more comprehensive structural and functional understanding of the ApoL immune protein family.


Assuntos
Apolipoproteína L1/química , Apolipoproteínas L/química , Domínios Proteicos , Apolipoproteína L1/genética , Apolipoproteína L1/metabolismo , Apolipoproteínas L/genética , Apolipoproteínas L/metabolismo , Humanos
9.
Nat Commun ; 10(1): 3070, 2019 07 11.
Artigo em Inglês | MEDLINE | ID: mdl-31296852

RESUMO

CARD9 and CARD11 drive immune cell activation by nucleating Bcl10 polymerization, but are held in an autoinhibited state prior to stimulation. Here, we elucidate the structural basis for this autoinhibition by determining the structure of a region of CARD9 that includes an extensive interface between its caspase recruitment domain (CARD) and coiled-coil domain. We demonstrate, for both CARD9 and CARD11, that disruption of this interface leads to hyperactivation in cells and to the formation of Bcl10-templating filaments in vitro, illuminating the mechanism of action of numerous oncogenic mutations of CARD11. These structural insights enable us to characterize two similar, yet distinct, mechanisms by which autoinhibition is relieved in the course of canonical CARD9 or CARD11 activation. We also dissect the molecular determinants of helical template assembly by solving the structure of the CARD9 filament. Taken together, these findings delineate the structural mechanisms of inhibition and activation within this protein family.


Assuntos
Proteínas Adaptadoras de Sinalização CARD/ultraestrutura , Guanilato Ciclase/ultraestrutura , Domínios Proteicos , Proteína 10 de Linfoma CCL de Células B/metabolismo , Proteínas Adaptadoras de Sinalização CARD/genética , Proteínas Adaptadoras de Sinalização CARD/imunologia , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Microscopia Crioeletrônica , Guanilato Ciclase/genética , Guanilato Ciclase/imunologia , Guanilato Ciclase/metabolismo , Células HEK293 , Humanos , Mutação , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica em alfa-Hélice , Multimerização Proteica/imunologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Transdução de Sinais/imunologia
10.
J Phys Chem B ; 122(8): 2368-2378, 2018 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-29376350

RESUMO

One-dimensional heteronuclear relaxation dispersion NMR spectroscopy at 13C natural abundance successfully characterized the dynamics of the hydrogen-deuterium exchange reaction occurring at the Nε position in l-arginine by monitoring Cδ in varying amounts of D2O. A small equilibrium isotope effect was observed and quantified, corresponding to ΔG = -0.14 kcal mol-1. A bimolecular rate constant of kD = 5.1 × 109 s-1 M-1 was determined from the pH*-dependence of kex (where pH* is the direct electrode reading of pH in 10% D2O and kex is the nuclear spin exchange rate constant), consistent with diffusion-controlled kinetics. The measurement of ΔG serves to bridge the millisecond time scale lifetimes of the detectable positively charged arginine species with the nanosecond time scale lifetime of the nonobservable low-populated neutral arginine intermediate species, thus allowing for characterization of the equilibrium lifetimes of the various arginine species in solution as a function of fractional solvent deuterium content. Despite the system being in fast exchange on the chemical shift time scale, the magnitude of the secondary isotope shift due to the exchange reaction at Nε was accurately measured to be 0.12 ppm directly from curve-fitting D2O-dependent dispersion data collected at a single static field strength. These results indicate that relaxation dispersion NMR spectroscopy is a robust and general method for studying base-catalyzed hydrogen-deuterium exchange chemistry at equilibrium.


Assuntos
Deutério/química , Hidrogênio/química , Medição da Troca de Deutério , Concentração de Íons de Hidrogênio , Espectroscopia de Ressonância Magnética , Termodinâmica
11.
J Neurosurg ; 107(2): 347-51, 2007 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-17695389

RESUMO

OBJECT: Cerebrospinal fluid (CSF) rhinorrhea remains a significant cause of morbidity after resection of vestibular schwannomas (VSs), with rates of rhinorrhea after this procedure reported to range between 0 and 27%. The authors investigated whether reconstruction of the drilled posterior wall of the porus acusticus with hydroxyapatite cement (HAC) would decrease the incidence of postoperative CSF rhinorrhea. METHODS: A prospective observational study of 130 consecutive patients who underwent surgery for reconstruction of the posterior wall of the drilled porus acusticus with HAC was conducted between October 2002 and September 2005. All patients underwent a retrosigmoid transmeatal approach for VS resection and were followed up to document cases of CSF rhinorrhea, incisional CSF leak, meningitis, or rhinorrhea-associated meningitis. A cohort of 150 patients with VSs who were treated with the same surgical approach but without HAC reconstruction served as a control group. RESULTS: The authors found that HAC reconstruction of the porus acusticus wall significantly reduced the rate of postoperative CSF rhinorrhea in their patients. In the patients treated with HAC, rhinorrhea developed in only three patients (2.3%) compared with 18 patients (12%) in the control group. This was a statistically significant finding (p = 0.002, odds ratio = 5.8). CONCLUSIONS: The use of HAC in the reconstruction of the drilled posterior wall of the porus acusticus, occluding exposed air cells, greatly reduces the risk of CSF rhinorrhea.


Assuntos
Rinorreia de Líquido Cefalorraquidiano/etiologia , Rinorreia de Líquido Cefalorraquidiano/prevenção & controle , Hidroxiapatitas/uso terapêutico , Neuroma Acústico/cirurgia , Osso Petroso/cirurgia , Complicações Pós-Operatórias , Adulto , Seguimentos , Humanos , Pessoa de Meia-Idade , Neuroma Acústico/patologia , Estudos Prospectivos , Resultado do Tratamento
12.
Protein Sci ; 26(3): 600-610, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-28028839

RESUMO

IgA1 proteases (IgA1P) from diverse pathogenic bacteria specifically cleave human immunoglobulin A1 (IgA1) at the hinge region, thereby thwarting protective host immune responses. Streptococcus pneumoniae (S. pneumoniae) IgA1P shares no sequence conservation with serine or cysteine types of IgA1Ps or other known proteins, other than a conserved HExxH Zn-binding motif (1604-1608) found in metalloproteases. We have developed a novel expression system to produce the mature S. pneumoniae IgA1P and we have discovered that this form is both attached to the bacterial cell surface and released in its full form. Our data demonstrate that the S. pneumoniae IgA1P comprises two distinct regions that associate to form an active metalloprotease, the first such example of a metalloprotease that can be split in vitro and recombined to form an active enzyme. By capitalizing on this novel domain architecture, we show that the N-terminal region of S. pneumoniae IgA1P comprises the primary binding region for IgA1, although the C-terminal region of S. pneumoniae IgA1P is necessary for cleavage of IgA1. Our findings lend insight into the protein domain architecture of the S. pneumoniae IgA1P and function of this important virulence factor for S. pneumoniae infection.


Assuntos
Proteínas de Bactérias/química , Serina Endopeptidases/química , Streptococcus pneumoniae/enzimologia , Fatores de Virulência/química , Motivos de Aminoácidos , Catálise , Domínios Proteicos
13.
Protein Sci ; 24(5): 651-60, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25564798

RESUMO

Crimean-Congo Hemorrhagic fever virus (CCHFV) is one of several lethal viruses that encodes for a viral ovarian tumor domain (vOTU), which serves to cleave and remove ubiquitin (Ub) and interferon stimulated gene product 15 (ISG15) from numerous proteins involved in cellular signaling. Such manipulation of the host cell machinery serves to downregulate the host response and, therefore, complete characterization of these proteases is important. While several structures of the CCHFV vOTU protease have been solved, both free and bound to Ub and ISG15, few structural differences have been found and little insight has been gained as to the structural plasticity of this protease. Therefore, we have used NMR relaxation experiments to probe the dynamics of CCHFV vOTU, both alone and in complex with Ub, discovering a highly dynamic protease that exhibits conformational exchange within the same regions found to engage its Ub substrate. These experiments reveal a structural plasticity around the N-terminal regions of CCHFV vOTU, which are unique to vOTUs, and provide a rationale for engaging multiple substrates with the same binding site.


Assuntos
Vírus da Febre Hemorrágica da Crimeia-Congo/química , Peptídeo Hidrolases/química , Estrutura Terciária de Proteína , Sítios de Ligação , Cristalografia por Raios X , Vírus da Febre Hemorrágica da Crimeia-Congo/patogenicidade , Febre Hemorrágica da Crimeia/virologia , Humanos , Ressonância Magnética Nuclear Biomolecular , Especificidade por Substrato , Ubiquitina/química
14.
Biomol NMR Assign ; 8(1): 23-7, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23138858

RESUMO

Cyclophilins catalyze the reversible peptidyl-prolyl isomerization of their substrates and are present across all kingdoms of life from humans to bacteria. Although numerous biological roles have now been discovered for cyclophilins, their function was initially ascribed to their chaperone-like activity in protein folding where they catalyze the often rate-limiting step of proline isomerization. This chaperone-like activity may be especially important under extreme conditions where cyclophilins are often over expressed, such as in tumors for human cyclophilins (Lee Archiv Pharm Res 33(2): 181-187, 2010), but also in organisms that thrive under extreme conditions, such as theromophilic bacteria. Moreover, the reversible nature of the peptidyl-prolyl isomerization reaction catalyzed by cyclophilins has allowed these enzymes to serve as model systems for probing the role of conformational changes during catalytic turnover (Eisenmesser et al. Science 295(5559): 1520-1523, 2002; Eisenmesser et al. Nature 438(7064): 117-121, 2005). Thus, we present here the resonance assignments of a thermophilic cyclophilin from Geobacillus kaustophilus derived from deep-sea sediment (Takami et al. Extremophiles 8(5): 351-356, 2004). This thermophilic cyclophilin may now be studied at a variety of temperatures to provide insight into the comparative structure, dynamics, and catalytic mechanism of cyclophilins.


Assuntos
Ciclofilina A/química , Geobacillus/enzimologia , Ressonância Magnética Nuclear Biomolecular , Temperatura , Sequência de Aminoácidos , Isótopos de Carbono , Estabilidade Enzimática , Humanos , Hidrogênio , Dados de Sequência Molecular , Isótopos de Nitrogênio , Estrutura Secundária de Proteína
15.
Protein Sci ; 23(4): 464-80, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24442768

RESUMO

Interleukin-8 (CXCL8, IL-8) is a proinflammatory chemokine important for the regulation of inflammatory and immune responses via its interaction with G-protein coupled receptors, including CXC receptor 1 (CXCR1). CXCL8 exists as both a monomer and as a dimer at physiological concentrations, yet the molecular basis of CXCL8 interaction with its receptor as well as the importance of CXCL8 dimer formation remain poorly characterized. Although several biological studies have indicated that both the CXCL8 monomer and dimer are active, biophysical studies have reported conflicting results regarding the binding of CXCL8 to CXCR1. To clarify this problem, we expressed and purified a peptide (hCXCR1pep) corresponding to the N-terminal region of human CXCR1 (hCXCR1) and utilized nuclear magnetic resonance (NMR) spectroscopy to interrogate the binding of wild-type CXCL8 and a previously reported mutant (CXCL8M) that stabilizes the monomeric form. Our data reveal that the CXCL8 monomer engages hCXCR1pep with a slightly higher affinity than the CXCL8 dimer, but that the CXCL8 dimer does not dissociate upon binding hCXCR1pep. These investigations also showed that CXCL8 is dynamic on multiple timescales, which may help explain the versatility in this interleukin for engaging its target receptors.


Assuntos
Interleucina-8/metabolismo , Peptídeos/metabolismo , Receptores de Interleucina-8A/química , Receptores de Interleucina-8A/metabolismo , Termodinâmica , Humanos , Interleucina-8/química , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Peptídeos/química
16.
Diabetes ; 61(10): 2495-505, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22698915

RESUMO

Obese white adipose tissue (AT) is characterized by large-scale infiltration of proinflammatory macrophages, in parallel with systemic insulin resistance; however, the cellular stimulus that initiates this signaling cascade and chemokine release is still unknown. The objective of this study was to determine the role of the phosphoinositide 3-kinase (PI3K) regulatory subunits on AT macrophage (ATM) infiltration in obesity. Here, we find that the Pik3r1 regulatory subunits (i.e., p85α/p55α/p50α) are highly induced in AT from high-fat diet-fed obese mice, concurrent with insulin resistance. Global heterozygous deletion of the Pik3r1 regulatory subunits (αHZ), but not knockout of Pik3r2 (p85ß), preserves whole-body, AT, and skeletal muscle insulin sensitivity, despite severe obesity. Moreover, ATM accumulation, proinflammatory gene expression, and ex vivo chemokine secretion in obese αHZ mice are markedly reduced despite endoplasmic reticulum (ER) stress, hypoxia, adipocyte hypertrophy, and Jun NH(2)-terminal kinase activation. Furthermore, bone marrow transplant studies reveal that these improvements in obese αHZ mice are independent of reduced Pik3r1 expression in the hematopoietic compartment. Taken together, these studies demonstrate that Pik3r1 expression plays a critical role in mediating AT insulin sensitivity and, more so, suggest that reduced PI3K activity is a key step in the initiation and propagation of the inflammatory response in obese AT.


Assuntos
Tecido Adiposo/metabolismo , Classe Ia de Fosfatidilinositol 3-Quinase/genética , Dieta Hiperlipídica , Resistência à Insulina/genética , Macrófagos/metabolismo , Obesidade/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Animais , Classe Ia de Fosfatidilinositol 3-Quinase/metabolismo , Citocinas/sangue , Insulina/sangue , Masculino , Camundongos , Camundongos Knockout , Obesidade/etiologia , Obesidade/genética , Fosfatidilinositol 3-Quinases/genética
17.
J Clin Invest ; 121(11): 4281-8, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21985785

RESUMO

Skeletal muscle insulin resistance is a key component of the etiology of type 2 diabetes. Caloric restriction (CR) enhances the sensitivity of skeletal muscle to insulin. However, the molecular signals within skeletal muscle linking CR to improved insulin action remain largely unknown. Recently, the mammalian ortholog of Sir2, sirtuin 1 (Sirt1), has been identified as a potential transducer of perturbations in cellular energy flux into subsequent metabolic adaptations, including modulation of skeletal muscle insulin action. Here, we have demonstrated that CR increases Sirt1 deacetylase activity in skeletal muscle in mice, in parallel with enhanced insulin-stimulated phosphoinositide 3-kinase (PI3K) signaling and glucose uptake. These adaptations in skeletal muscle insulin action were completely abrogated in mice lacking Sirt1 deacetylase activity. Mechanistically, Sirt1 was found to be required for the deacetylation and inactivation of the transcription factor Stat3 during CR, which resulted in decreased gene and protein expression of the p55α/p50α subunits of PI3K, thereby promoting more efficient PI3K signaling during insulin stimulation. Thus, these data demonstrate that Sirt1 is an integral signaling node in skeletal muscle linking CR to improved insulin action, primarily via modulation of PI3K signaling.


Assuntos
Restrição Calórica , Resistência à Insulina/fisiologia , Músculo Esquelético/metabolismo , Sirtuína 1/metabolismo , Animais , Sequência de Bases , Primers do DNA/genética , Diabetes Mellitus Tipo 2/etiologia , Diabetes Mellitus Tipo 2/metabolismo , Técnica Clamp de Glucose , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Sirtuína 1/deficiência , Sirtuína 1/genética
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