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1.
Front Epidemiol ; 3: 1146006, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-38455914

RESUMO

Objectives: To develop a biological diary (CoronaCal) that allows anyone in the community to collect and store serial saliva samples and chart symptoms on ordinary printer paper. Methods: Diaries were analyzed for the presence of SARS-CoV-2 RNA using established polymerase chain reaction (PCR) procedures. CoronaCal diaries were distributed to volunteer subjects in the community during the peak of the COVID-19 outbreak in New York. Volunteers collected their own daily saliva samples and self-reported symptoms. Results: SARS-CoV-2 RNA extracted from CoronaCals was measured using qPCR and RNA levels were correlated with reported symptoms. SARS-CoV-2 RNA was detected in CoronaCals from nine of nine people with COVID-19 symptoms or exposure to someone with COVID-19, and not in one asymptomatic person. CoronaCals were stored for up to 70 days at room temperature during collection and then frozen for up to four months before analysis, suggesting that SARS-CoV-2 RNA is stable once dried onto paper. Conclusions: Sampling saliva on simple paper provides a useful method to study the natural history and epidemiology of COVID-19. The CoronaCal collection and testing method is easy to implement, inexpensive, non-invasive and scalable. The approach can inform the historical and epidemiological understanding of infections in individuals and populations.

2.
Adv Sci (Weinh) ; 9(34): e2202342, 2022 12.
Artigo em Inglês | MEDLINE | ID: mdl-36257905

RESUMO

Type 2 diabetes mellitus (T2D) is a major public health concern and is characterized by sustained hyperglycemia due to insulin resistance and destruction of insulin-producing ß cells. One pathological hallmark of T2D is the toxic accumulation of human islet amyloid polypeptide (hIAPP) aggregates. Monomeric hIAPP is a hormone normally co-secreted with insulin. However, increased levels of hIAPP in prediabetic and diabetic patients can lead to the formation of hIAPP protofibrils, which are toxic to ß cells. Current therapies fail to address hIAPP aggregation and current screening modalities do not detect it. Using a stabilizing capping protein, monoclonal antibodies (mAbs) can be developed against a previously nonisolatable form of hIAPP protofibrils, which are protofibril specific and do not engage monomeric hIAPP. Shown here are two candidate mAbs that can detect hIAPP protofibrils in serum and hIAPP deposits in pancreatic islets in a mouse model of rapidly progressing T2D. Treatment of diabetic mice with the mAbs delays disease progression and dramatically increases overall survival. These results demonstrate the potential for using novel hIAPP protofibril-specific mAbs as a diagnostic screening tool for early detection of T2D, as well as therapeutically to preserve ß cell function and target one of the underlying pathological mechanisms of T2D.


Assuntos
Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 2 , Animais , Humanos , Camundongos , Diabetes Mellitus Tipo 2/diagnóstico , Diabetes Mellitus Tipo 2/terapia , Insulina , Polipeptídeo Amiloide das Ilhotas Pancreáticas
3.
iScience ; 25(4): 104060, 2022 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-35355518

RESUMO

The photoreceptor rhodopsin (Rho) becomes active when a tethered inverse agonist ligand (11CR) is photoconverted to an agonist (ATR). The ligand-binding pocket of inactive rhodopsin is completely enclosed, whereas active rhodopsin displays pores accessible from the lipid bilayer. Stabilization of active rhodopsin impedes 11CR binding and photoreceptor dark adaptation. Here, we used genetic code expansion and bioorthogonal labeling to engineer Rho mutants that serve as FRET sensors for measuring 11CR binding kinetics and energetics. We found that mutations that alter a channel between transmembrane helices 5 and 6 (TM5/6) dramatically affect 11CR binding kinetics but not agonist release kinetics. Our data provide direct experimental evidence for 11CR entry between TM5/6 in Rho that involves dynamic allosteric control of the ligand entry channel. Our findings provide a conceptual framework for understanding the function of G protein-coupled receptors with hydrophobic ligands that are hypothesized to enter their binding pockets through transmembrane pores.

4.
PLoS Biol ; 19(6): e3001305, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-34191794

RESUMO

Oxytocin/vasopressin-related neuropeptides are highly conserved and play major roles in regulating social behavior across vertebrates. However, whether their insect orthologue, inotocin, regulates the behavior of social groups remains unknown. Here, we show that in the clonal raider ant Ooceraea biroi, individuals that perform tasks outside the nest have higher levels of inotocin in their brains than individuals of the same age that remain inside the nest. We also show that older ants, which spend more time outside the nest, have higher inotocin levels than younger ants. Inotocin thus correlates with the propensity to perform tasks outside the nest. Additionally, increasing inotocin pharmacologically increases the tendency of ants to leave the nest. However, this effect is contingent on age and social context. Pharmacologically treated older ants have a higher propensity to leave the nest only in the presence of larvae, whereas younger ants seem to do so only in the presence of pupae. Our results suggest that inotocin signaling plays an important role in modulating behaviors that correlate with age, such as social foraging, possibly by modulating behavioral response thresholds to specific social cues. Inotocin signaling thereby likely contributes to behavioral individuality and division of labor in ant societies.


Assuntos
Formigas/fisiologia , Comportamento Animal/fisiologia , Ocitocina/metabolismo , Comportamento Social , Vasopressinas/metabolismo , Envelhecimento/fisiologia , Animais , Encéfalo/fisiologia , Células HEK293 , Humanos , Ocitocina/química , Vasopressinas/química
5.
J Biol Chem ; 296: 100163, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33288675

RESUMO

Uveal melanoma is the most common eye cancer in adults and is clinically and genetically distinct from skin cutaneous melanoma. In a subset of cases, the oncogenic driver is an activating mutation in CYSLTR2, the gene encoding the G protein-coupled receptor cysteinyl-leukotriene receptor 2 (CysLTR2). The mutant CYSLTR2 encodes for the CysLTR2-L129Q receptor, with the substitution of Leu to Gln at position 129 (3.43). The ability of CysLTR2-L129Q to cause malignant transformation has been hypothesized to result from constitutive activity, but how the receptor could escape desensitization is unknown. Here, we characterize the functional properties of CysLTR2-L129Q. We show that CysLTR2-L129Q is a constitutively active mutant that strongly drives Gq/11 signaling pathways. However, CysLTR2-L129Q only poorly recruits ß-arrestin. Using a modified Slack-Hall operational model, we quantified the constitutive activity for both pathways and conclude that CysLTR2-L129Q displays profound signaling bias for Gq/11 signaling pathways while escaping ß-arrestin-mediated downregulation. CYSLTR2 is the first known example of a G protein-coupled receptor driver oncogene that encodes a highly biased constitutively active mutant receptor. These results provide new insights into the mechanism of CysLTR2-L129Q oncoprotein signaling and suggest CYSLTR2 as a promising potential therapeutic target in uveal melanoma.


Assuntos
Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/genética , Regulação Neoplásica da Expressão Gênica , Receptores de Leucotrienos/genética , Transdução de Sinais/genética , beta-Arrestina 2/genética , Substituição de Aminoácidos , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Glutamina/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Cinética , Lisina/metabolismo , Melanoma/genética , Melanoma/metabolismo , Melanoma/patologia , Modelos Biológicos , Mutação , Ligação Proteica , Receptores de Leucotrienos/metabolismo , Receptores de Vasopressinas/genética , Receptores de Vasopressinas/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Neoplasias Uveais/genética , Neoplasias Uveais/metabolismo , Neoplasias Uveais/patologia , beta-Arrestina 2/metabolismo
6.
J Alzheimers Dis ; 72(2): 537-548, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31609694

RESUMO

The amyloid-ß protein precursor (AßPP) is critical in the pathophysiology of Alzheimer's disease (AD), since two-step proteolytic processing of AßPP generates the neurotoxic amyloid-ß peptide (Aß). We developed a dual fluorescence labeling system to study the exact subcellular location of γ-secretase cleavage of AßPP. The C-terminal tail of AßPP was fluorescently labeled using a SNAP-tag, while the Aß region of AßPP was fluorescently tagged with a dye at a genetically-encoded noncanonical amino acid (ncAA). The ncAA was introduced at specific positions in AßPP using a genetic code expansion strategy and afterwards, the reactive side-chain of the ncAA was coupled to the dye using a bioorthogonal labeling chemistry. In proof-of-concept experiments, HEK293T cells were transfected with plasmids containing engineered AßPP harboring an amber mutation and an amber codon suppression system with an evolved tRNA synthetase/tRNA pair and grown in the presence of a lysine-derived ncAA. Processing of the AßPP variants was validated with ELISA and immunoblotting, and seven AßPP mutants that showed similar cleavage pattern as wild-type AßPP were identified. The AßPP mutant was fluorescently labeled with 6-methyl-tetrazine-BDP-FL and TMR-Star at the ncAA and SNAP-tag, respectively. Using this approach, AßPP was fluorescently labeled at two sites in living cells with minimal background to allow monitoring of Aß and C-terminal cleavage products simultaneously. The method described provides a powerful tool to label Aß with minimal perturbations of its processing, thus enabling studies of the trafficking of the cleavage products of AßPP.


Assuntos
Precursor de Proteína beta-Amiloide/química , Doença de Alzheimer/metabolismo , Aminoácidos/química , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Clonagem Molecular , Códon/genética , Corantes Fluorescentes , Células HEK293 , Humanos , Mutação/genética , Plasmídeos , Engenharia de Proteínas
7.
Biophys J ; 117(5): 903-919, 2019 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-31421836

RESUMO

The chemokine receptor CCR5 is a drug target to prevent transmission of HIV/AIDS. We studied four analogs of the native chemokine regulated, on activation, normal T-cell-expressed, and secreted (RANTES) (CCL5) that have anti-HIV potencies of around 25 pM, which is more than four orders of magnitude higher than that of RANTES itself. It has been hypothesized that the ultrahigh potency of the analogs is due to their ability to bind populations of receptors not accessible to native chemokines. To test this hypothesis, we developed a homogeneous dual-color fluorescence cross-correlation spectroscopy assay for saturation- and competition-binding experiments. The fluorescence cross-correlation spectroscopy assay has the advantage that it does not rely on competition with radioactively labeled native chemokines used in conventional assays. We prepared site-specifically labeled fluorescent analogs using native chemical ligation of synthetic peptides, followed by bioorthogonal fluorescent labeling. We engineered a mammalian cell expression construct to provide fluorescently labeled CCR5, which was purified using a tandem immunoaffinity and size-exclusion chromatography approach to obtain monomeric fluorescent CCR5 in detergent solution. We found subnanomolar binding affinities for the two analogs 5P12-RANTES and 5P14-RANTES and about 20-fold reduced affinities for PSC-RANTES and 6P4-RANTES. Using homologous and heterologous competition experiments with unlabeled chemokine analogs, we conclude that the analogs all bind at the same binding site, whereas the native chemokines (RANTES and MIP-1α) fail to displace bound fluorescent analogs even at tens of micromolar concentrations. Our results can be rationalized with de novo structural models of the N-terminal tails of the synthetic chemokines that adopt a different binding mode as compared to the parent compound.


Assuntos
Quimiocinas/metabolismo , HIV-1/metabolismo , Receptores CCR5/metabolismo , Ligação Competitiva , Quimiocina CCL5/metabolismo , Células HEK293 , Humanos , Ligantes , Modelos Biológicos , Ligação Proteica
8.
iScience ; 11: 366-374, 2019 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-30660104

RESUMO

A recent phylogenetic analysis showed global co-evolution of G protein-coupled receptors (GPCRs) and receptor-activity-modifying proteins (RAMPs) suggesting global interactions between these two protein families. Experimental validation of these findings is challenging because in humans whereas there are only three genes encoding RAMPs, there are about 800 genes encoding GPCRs. Here, we report an experimental approach to evaluate GPCR-RAMP interactions. As a proof-of-concept experiment, we over-expressed RAMP2 in HEK293T cells and evaluated the effect on the transcriptional levels of 14 representative GPCRs that were selected based on the earlier phylogenetic analysis. We utilized a multiplexed error-correcting fluorescence in situ hybridization (MERFISH) method to detect message levels for individual GPCRs in single cells. The MERFISH results showed changes in GPCR message levels with RAMP2 over-expression in a concordant pattern that was predicted by the earlier phylogenetic analysis. These results provide additional evidence that GPCR-RAMP interactions are more widespread than previously appreciated and that these interactions have functional consequences.

9.
J Biol Chem ; 292(52): 21623-21630, 2017 12 29.
Artigo em Inglês | MEDLINE | ID: mdl-29101232

RESUMO

How an optimal level of human dopamine D4 receptor (hD4R) is maintained in synaptic membranes is not known. We show here that hD4R is ubiquitinated in primary neurons. We go on to show that ubiquitin is attached to hD4R through isopeptide and ester bonds. When lysine (Lys) residues of the hD4R are substituted with arginine (Arg) residues, cellular hD4R protein levels increase. A synergistic effect on hD4R levels is noted when cytoplasmic serine (Ser) and threonine (Thr) residues are mutated. Chloroquine, an inhibitor of lysosomal degradation, did not have an effect on hD4R protein levels. However, treatment with bortezomib, an inhibitor of the 20S proteasome, caused a dose-dependent increase in hD4R protein levels. The effect of bortezomib was attenuated in the receptor variants that lacked Lys or Ser/Thr residues, and the hD4R mutant that lacked 17 cytoplasmic Lys, Ser, and Thr residues was nearly insensitive to bortezomib treatment. We conclude that both isopeptide and ester bond ubiquitination regulate proteasomal degradation of hD4R.


Assuntos
Receptores de Dopamina D4/metabolismo , Receptores de Dopamina D4/fisiologia , Sequência de Aminoácidos , Arginina , Bortezomib , Células HEK293 , Humanos , Lisina , Neurônios/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Receptores Dopaminérgicos , Receptores de Dopamina D4/química , Ubiquitina/metabolismo , Ubiquitinação
10.
Nat Genet ; 48(6): 675-80, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-27089179

RESUMO

Uveal melanomas are molecularly distinct from cutaneous melanomas and lack mutations in BRAF, NRAS, KIT, and NF1. Instead, they are characterized by activating mutations in GNAQ and GNA11, two highly homologous α subunits of Gαq/11 heterotrimeric G proteins, and in PLCB4 (phospholipase C ß4), the downstream effector of Gαq signaling. We analyzed genomics data from 136 uveal melanoma samples and found a recurrent mutation in CYSLTR2 (cysteinyl leukotriene receptor 2) encoding a p.Leu129Gln substitution in 4 of 9 samples that lacked mutations in GNAQ, GNA11, and PLCB4 but in 0 of 127 samples that harbored mutations in these genes. The Leu129Gln CysLT2R mutant protein constitutively activates endogenous Gαq and is unresponsive to stimulation by leukotriene. Expression of Leu129Gln CysLT2R in melanocytes enforces expression of a melanocyte-lineage signature, drives phorbol ester-independent growth in vitro, and promotes tumorigenesis in vivo. Our findings implicate CYSLTR2 as a uveal melanoma oncogene and highlight the critical role of Gαq signaling in uveal melanoma pathogenesis.


Assuntos
Melanoma/genética , Mutação , Receptores de Leucotrienos/genética , Neoplasias Uveais/genética , Animais , Cálcio/metabolismo , Feminino , Predisposição Genética para Doença , Células HEK293 , Humanos , Camundongos , Camundongos SCID
11.
Chembiochem ; 15(12): 1820-9, 2014 Aug 18.
Artigo em Inglês | MEDLINE | ID: mdl-25045132

RESUMO

Novel methods are required for site-specific, quantitative fluorescent labeling of G-protein-coupled receptors (GPCRs) and other difficult-to-express membrane proteins. Ideally, fluorescent probes should perturb the native structure and function as little as possible. We evaluated bioorthogonal reactions to label genetically encoded p-acetyl-L-phenylalanine (AcF) or p-azido-L-phenylalanine (azF) residues in receptors heterologously expressed in mammalian cells. We found that keto-selective reagents were not truly bioorthogonal, possibly owing to post-translational protein oxidation reactions. In contrast, the strain-promoted [3+2] azide-alkyne cycloaddition (SpAAC) with dibenzocyclooctyne (DIBO) reagents yielded stoichiometric conjugates with azF-rhodopsin while undergoing negligible background reactions. As one application of this technique, we used Alexa488-rhodopsin to measure the kinetics of ligand uptake and release in membrane-mimetic bicelles using a novel fluorescence-quenching assay.


Assuntos
Fluorescência , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , Coloração e Rotulagem , Código Genético , Cinética , Modelos Moleculares , Receptores Acoplados a Proteínas G/genética , Espectrometria de Fluorescência
12.
J Am Chem Soc ; 135(51): 19064-7, 2013 Dec 26.
Artigo em Inglês | MEDLINE | ID: mdl-24295328

RESUMO

Ultraviolet (UV) cone pigments can provide insights into the molecular evolution of vertebrate vision since they are nearer to ancestral pigments than the dim-light rod photoreceptor rhodopsin. While visible-absorbing pigments contain an 11-cis retinyl chromophore with a protonated Schiff-base (PSB11), UV pigments uniquely contain an unprotonated Schiff-base (USB11). Upon F86Y mutation in model UV pigments, both the USB11 and PSB11 forms of the chromophore are found to coexist at physiological pH. The origin of this intriguing equilibrium remains to be understood at the molecular level. Here, we address this phenomenon and the role of the USB11 environment in spectral tuning by combining mutagenesis studies with spectroscopic (UV-vis) and theoretical [DFT-QM/MM (SORCI+Q//B3LYP/6-31G(d): Amber96)] analysis. We compare structural models of the wild-type (WT), F86Y, S90A and S90C mutants of Siberian hamster ultraviolet (SHUV) cone pigment to explore structural rearrangements that stabilize USB11 over PSB11. We find that the PSB11 forms upon F86Y mutation and is stabilized by an "inter-helical lock" (IHL) established by hydrogen-bonding networks between transmembrane (TM) helices TM6, TM2, and TM3 (including water w2c and amino acid residues Y265, F86Y, G117, S118, A114, and E113). The findings implicate the involvement of the IHL in constraining the displacement of TM6, an essential component of the activation of rhodopsin, in the spectral tuning of UV pigments.


Assuntos
Modelos Moleculares , Pigmentos da Retina/química , Opsinas de Bastonetes/química , Raios Ultravioleta , Animais , Cricetinae , Cristalografia por Raios X , Evolução Molecular , Teoria Quântica , Bases de Schiff/química
13.
J Am Chem Soc ; 133(40): 15878-81, 2011 Oct 12.
Artigo em Inglês | MEDLINE | ID: mdl-21905700

RESUMO

Cell surface heptahelical G protein-coupled receptors (GPCRs) mediate critical cellular signaling pathways and are important pharmaceutical drug targets. (1) In addition to traditional small-molecule approaches, lipopeptide-based GPCR-derived pepducins have emerged as a new class of pharmaceutical agents. (2, 3) To better understand how pepducins interact with targeted receptors, we developed a cell-based photo-cross-linking approach to study the interaction between the pepducin agonist ATI-2341 and its target receptor, chemokine C-X-C-type receptor 4 (CXCR4). A pepducin analogue, ATI-2766, formed a specific UV-light-dependent cross-link to CXCR4 and to mutants with truncations of the N-terminus, the known chemokine docking site. These results demonstrate that CXCR4 is the direct binding target of ATI-2341 and suggest a new mechanism for allosteric modulation of GPCR activity. Adaptation and application of our findings should prove useful in further understanding pepducin modulation of GPCRs as well as enable new experimental approaches to better understand GPCR signal transduction.


Assuntos
Peptídeos/química , Peptídeos/farmacologia , Receptores CXCR4/agonistas , Receptores CXCR4/metabolismo , Regulação Alostérica/efeitos dos fármacos , Sequência de Aminoácidos , Linhagem Celular , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Processos Fotoquímicos , Raios Ultravioleta
14.
J Biol Chem ; 286(37): 32188-97, 2011 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-21730065

RESUMO

G protein-coupled receptor hetero-oligomerization is emerging as an important regulator of ligand-dependent transmembrane signaling, but precisely how receptor heteromers affect receptor pharmacology remains largely unknown. In this study, we have attempted to identify the functional significance of the heteromeric complex between CXCR4 and CXCR7 chemokine receptors. We demonstrate that co-expression of CXCR7 with CXCR4 results in constitutive recruitment of ß-arrestin to the CXCR4·CXCR7 complex and simultaneous impairment of G(i)-mediated signaling. CXCR7/CXCR4 co-expression also results in potentiation of CXCL12 (SDF-1)-mediated downstream ß-arrestin-dependent cell signaling pathways, including ERK1/2, p38 MAPK, and SAPK as judged from the results of experiments using siRNA knockdown to deplete ß-arrestin. Interestingly, CXCR7/CXCR4 co-expression enhances cell migration in response to CXCL12 stimulation. Again, inhibition of ß-arrestin using either siRNA knockdown or a dominant negative mutant abrogates the enhanced CXCL12-dependent migration of CXCR4/CXCR7-expressing cells. These results show how CXCR7, which cannot signal directly through G protein-linked pathways, can nevertheless affect cellular signaling networks by forming a heteromeric complex with CXCR4. The CXCR4·CXCR7 heterodimer complex recruits ß-arrestin, resulting in preferential activation of ß-arrestin-linked signaling pathways over canonical G protein pathways. CXCL12-dependent signaling of CXCR4 and its role in cellular physiology, including cancer metastasis, should be evaluated in the context of potential functional hetero-oligomerization with CXCR7.


Assuntos
Arrestina/metabolismo , Movimento Celular/fisiologia , Sistema de Sinalização das MAP Quinases/fisiologia , Multimerização Proteica/fisiologia , Receptores CXCR4/metabolismo , Receptores CXCR/metabolismo , Arrestina/genética , Quimiocina CXCL12/genética , Quimiocina CXCL12/metabolismo , Células HEK293 , Humanos , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Estrutura Quaternária de Proteína , Receptores CXCR/genética , Receptores CXCR4/genética
15.
Proc Natl Acad Sci U S A ; 107(51): 22255-9, 2010 Dec 21.
Artigo em Inglês | MEDLINE | ID: mdl-21139054

RESUMO

The G protein-coupled receptor (GPCR), chemokine CXC-type receptor 4 (CXCR4), and its ligand, CXCL12, mediate the retention of polymorphonuclear neutrophils (PMNs) and hematopoietic stem and progenitor cells (HSPCs) in the bone marrow. Agents that disrupt CXCL12-mediated chemoattraction of CXCR4-expressing cells mobilize PMNs and HSPCs into the peripheral circulation and are therapeutically useful for HSPC collection before autologous bone marrow transplantation (ABMT). Our aim was to develop unique CXCR4-targeted therapeutics using lipopeptide GPCR modulators called pepducins. A pepducin is a synthetic molecule composed of a peptide derived from the amino acid sequence of one of the intracellular (IC) loops of a target GPCR coupled to a lipid tether. We prepared and screened a small CXCR4-targeted pepducin library and identified several pepducins with in vitro agonist activity, including ATI-2341, whose peptide sequence derives from the first IC loop. ATI-2341 induced CXCR4- and G protein-dependent signaling, receptor internalization, and chemotaxis in CXCR4-expressing cells. It also induced dose-dependent peritoneal recruitment of PMNs when administered i.p. to mice. However, when administered systemically by i.v. bolus, ATI-2341 acted as a functional antagonist and dose-dependently mediated release of PMNs from the bone marrow of both mice and cynomolgus monkeys. ATI-2341-mediated release of granulocyte/macrophage progenitor cells from the bone marrow was confirmed by colony-forming assays. We conclude that ATI-2341 is a potent and efficacious mobilizer of bone marrow PMNs and HSPCs and could represent a previously undescribed therapeutic approach for the recruitment of HSPCs before ABMT.


Assuntos
Mobilização de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/metabolismo , Peptídeos/farmacologia , Receptores CXCR4/agonistas , Transdução de Sinais/efeitos dos fármacos , Animais , Quimiotaxia/efeitos dos fármacos , Relação Dose-Resposta a Droga , Células HEK293 , Humanos , Leucócitos Mononucleares/metabolismo , Macaca fascicularis , Camundongos , Camundongos Endogâmicos BALB C , Receptores CXCR4/metabolismo
16.
Biochemistry ; 45(17): 5430-9, 2006 May 02.
Artigo em Inglês | MEDLINE | ID: mdl-16634624

RESUMO

The role of ionizable amino acid side chains in the bovine rhodopsin activation mechanism was studied in mutants E134Q, E134R/R135E, H211F, and E122Q. All mutants exhibited bathorhodopsin stability on the 30 ns to 1 micros time scale similar to that of the wild type. Lumirhodopsin decay was also similar to that of the wild type except for the H211F mutant where early decay (20 micros) to a second form of lumirhodopsin was seen, followed by formation of an extremely long-lived Meta I(480) product (34 ms), an intermediate which forms to a much reduced extent, if at all, in dodecyl maltoside suspensions of wild-type rhodopsin. A smaller amount of a similar long-lived Meta I(480) product was seen after photolysis of E122Q, but E134Q and E134R/R135Q displayed kinetics much more similar to those of the wild type under these conditions (i.e., no Meta I(480) product). These results support the idea that specific interaction of His211 and Glu122 plays a significant role in deprotonation of the retinylidene Schiff base and receptor activation. Proton uptake measurements using bromcresol purple showed that E122Q was qualitatively similar to wild-type rhodopsin, with at least one proton being released during lumirhodopsin decay per Meta I(380) intermediate formed, followed by uptake of at least two protons per rhodopsin bleached on a time scale of tens of milliseconds. Different results were obtained for H211F, E134Q, and E134R/R135E, which all released approximately two protons per rhodopsin bleached. These results show that several ionizable groups besides the Schiff base imine are affected by the structural changes involved in rhodopsin activation. At least two proton uptake groups and probably at least one proton release group in addition to the Schiff base are present in rhodopsin.


Assuntos
Prótons , Rodopsina/química , Rodopsina/genética , Animais , Bovinos , Ligação de Hidrogênio , Cinética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Fotoquímica , Bases de Schiff/química , Análise Espectral
17.
Science ; 311(5767): 1617-21, 2006 Mar 17.
Artigo em Inglês | MEDLINE | ID: mdl-16543463

RESUMO

The parietal-eye photoreceptor is unique because it has two antagonistic light signaling pathways in the same cell-a hyperpolarizing pathway maximally sensitive to blue light and a depolarizing pathway maximally sensitive to green light. Here, we report the molecular components of these two pathways. We found two opsins in the same cell: the blue-sensitive pinopsin and a previously unidentified green-sensitive opsin, which we name parietopsin. Signaling components included gustducin-alpha and Galphao, but not rod or cone transducin-alpha. Single-cell recordings demonstrated that Go mediates the depolarizing response. Gustducin-alpha resembles transducin-alpha functionally and likely mediates the hyperpolarizing response. The parietopsin-Go signaling pair provides clues about how rod and cone phototransduction might have evolved.


Assuntos
Evolução Biológica , Lagartos/fisiologia , Fenômenos Fisiológicos Oculares , Células Fotorreceptoras de Vertebrados/fisiologia , Opsinas de Bastonetes/fisiologia , Visão Ocular , 3',5'-GMP Cíclico Fosfodiesterases/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , GMP Cíclico/metabolismo , Subunidades alfa de Proteínas de Ligação ao GTP/genética , Subunidades alfa de Proteínas de Ligação ao GTP/fisiologia , Humanos , Lagartos/genética , Dados de Sequência Molecular , Técnicas de Patch-Clamp , Células Fotorreceptoras de Vertebrados/química , Opsinas de Bastonetes/análise , Opsinas de Bastonetes/genética , Transducina/genética , Transducina/fisiologia
18.
Biochemistry ; 43(39): 12614-21, 2004 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-15449951

RESUMO

The role of glutamic acid 181 in the bovine rhodopsin retinylidene chromophore pocket was studied by expressing E181 mutants in COS cells and measuring, as a function of time, the absorbance changes produced after excitation of lauryl maltoside pigment suspensions with 7 ns laser pulses. All mutants studied except E181D showed accelerated decay of bathorhodopsin compared to wild type. Even for E181D, an anomalously large blue shift was observed in the absorption spectrum of the bathorhodopsin decay product, BSI. These observations support the idea that E181 plays a significant role in the earliest stages of receptor activation. E181 mutations have a pronounced effect on the decay of the lumirhodopsin photointermediate, primarily affecting the size of the red shift that occurs in the lumirhodopsin I to lumirhodopsin II transition that takes place on the 10 micros time scale after wild-type photoexcitation. While the spectral change that occurs in the lumirhodopsin I to lumirhodopsin II transition in wild-type rhodopsin is very small ( approximately 2 nm), making it difficult to detect, it is larger in E181D ( approximately 6 nm), making it evident even in the lower signal-to-noise ratio measurements possible with rhodopsin mutants. The change seen is even larger for the E181F mutant where significant amounts of a deprotonated Schiff base intermediate are produced with the 10 micros time constant of lumirhodopsin II formation. The E181Q mutant shows lumirhodopsin decay more similar to wild-type behavior, and no lumirhodopsin I to lumirhodopsin II transition can be resolved. The addition of chloride ion to E181Q increases the lumirhodopsin I-lumirhodopsin II spectral shift and slows the deprotonation of the Schiff base. The latter result is consistent with the idea that a negative charge at position 181 contributes to protonated Schiff base stability in the later intermediates.


Assuntos
Ácido Glutâmico/genética , Rodopsina/análogos & derivados , Rodopsina/química , Rodopsina/genética , Substituição de Aminoácidos/genética , Animais , Ácido Aspártico/genética , Células COS , Bovinos , Ácido Glutâmico/química , Glutamina/genética , Mutagênese Sítio-Dirigida , Fenilalanina/genética , Fotólise , Prótons , Bases de Schiff , Espectrofotometria
19.
Biochemistry ; 43(34): 10867-76, 2004 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-15323547

RESUMO

The vibrational structure of the chromophore in the primary photoproduct of vision, bathorhodopsin, is examined to determine the cause of the anomalously decoupled and intense C(11)=C(12) hydrogen-out-of-plane (HOOP) wagging modes and their relation to energy storage in the primary photoproduct. Low-temperature (77 K) resonance Raman spectra of Glu181 and Ser186 mutants of bovine rhodopsin reveal only mild mutagenic perturbations of the photoproduct spectrum suggesting that dipolar, electrostatic, or steric interactions with these residues do not cause the HOOP mode frequencies and intensities. Density functional theory calculations are performed to investigate the effect of geometric distortion on the HOOP coupling. The decoupled HOOP modes can be simulated by imposing approximately 40 degrees twists in the same direction about the C(11)=C(12) and C(12)-C(13) bonds. Sequence comparison and examination of the binding site suggests that these distortions are caused by three constraints consisting of an electrostatic anchor between the protonated Schiff base and the Glu113 counterion, as well as steric interactions of the 9- and 13-methyl groups with surrounding residues. This distortion stores light energy that is used to drive the subsequent protein conformational changes that activate rhodopsin.


Assuntos
Rodopsina/análogos & derivados , Rodopsina/química , Rodopsina/metabolismo , Animais , Bovinos , Congelamento , Ácido Glutâmico/genética , Luz , Modelos Moleculares , Mutagênese Sítio-Dirigida , Fotoquímica , Conformação Proteica , Estrutura Secundária de Proteína , Prótons , Rodopsina/genética , Bases de Schiff/química , Serina/genética , Especificidade da Espécie , Análise Espectral Raman/métodos , Eletricidade Estática , Termodinâmica
20.
Proc Natl Acad Sci U S A ; 100(16): 9262-7, 2003 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-12835420

RESUMO

The biological function of Glu-181 in the photoactivation process of rhodopsin is explored through spectroscopic studies of site-specific mutants. Preresonance Raman vibrational spectra of the unphotolyzed E181Q mutant are nearly identical to spectra of the native pigment, supporting the view that Glu-181 is uncharged (protonated) in the dark state. The pH dependence of the absorption of the metarhodopsin I (Meta I)-like photoproduct of E181Q is investigated, revealing a dramatic shift of its Schiff base pKa compared with the native pigment. This result is most consistent with the assignment of Glu-181 as the primary counterion of the retinylidene protonated Schiff base in the Meta I state, implying that there is a counterion switch from Glu-113 in the dark state to Glu-181 in Meta I. We propose a model where the counterion switch occurs by transferring a proton from Glu-181 to Glu-113 through an H-bond network formed primarily with residues on extracellular loop II (EII). The resulting reorganization of EII is then coupled to movements of helix III through a conserved disulfide bond (Cys110-Cys187); this process may be a general element of G protein-coupled receptor activation.


Assuntos
Proteínas de Ligação ao GTP/química , Íons , Retina/metabolismo , Rodopsina/análogos & derivados , Rodopsina/química , Animais , Bovinos , Proteínas de Ligação ao GTP/fisiologia , Ácido Glutâmico/química , Concentração de Íons de Hidrogênio , Luz , Modelos Químicos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Ligação Proteica , Rodopsina/fisiologia , Análise Espectral Raman , Temperatura , Raios Ultravioleta
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