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1.
Cell Mol Gastroenterol Hepatol ; 11(1): 161-179, 2020 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-32810598

RESUMO

BACKGROUND & AIMS: Pancreatic ductal adenocarcinoma (PDAC) is resistant to most therapeutics owing to dense fibrotic stroma orchestrated by cancer-associated pancreatic stellate cells (CAPaSC). CAPaSC also support cancer cell growth, metastasis, and resistance to apoptosis. Currently, there is no effective therapy for PDAC that specifically targets CAPaSC. We previously reported a rationally designed protein, ProAgio, that targets integrin αvß3 at a novel site and induces apoptosis in integrin αvß3-expressing cells. Because both CAPaSC and angiogenic endothelial cells express high levels of integrin αvß3, we aimed to analyze the effects of ProAgio in PDAC tumor. METHODS: Expression of integrin αvß3 was examined in both patient tissue and cultured cells. The effects of ProAgio on CAPaSC were analyzed using an apoptosis assay kit. The effects of ProAgio in PDAC tumor were studied in 3 murine tumor models: subcutaneous xenograft, genetic engineered (KrasG12D; p53R172H; Pdx1-Cre, GEM-KPC) mice, and an orthotopic KrasG12D; p53R172H; Pdx1-Cre (KPC) model. RESULTS: ProAgio induces apoptosis in CAPaSC. ProAgio treatment significantly prolonged survival of a genetically engineered mouse-KPC and orthotopic KPC mice alone or in combination with gemcitabine (Gem). ProAgio specifically induced apoptosis in CAPaSC, resorbed collagen, and opened collapsed tumor vessels without an increase in angiogenesis in PDAC tumor, enabling drug delivery into the tumor. ProAgio decreased intratumoral insulin-like growth factor 1 levels as a result of depletion of CAPaSC and consequently decreased cytidine deaminase, a Gem metabolism enzyme in cancer cells, and thereby reduced resistance to Gem-induced apoptosis. CONCLUSIONS: Our study suggests that ProAgio is an effective PDAC treatment agent because it specifically depletes CAPaSC and eliminates tumor angiogenesis, thereby enhancing drug delivery and Gem efficacy in PDAC tumors.

2.
Molecules ; 25(9)2020 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-32375353

RESUMO

Calcium controls numerous biological processes by interacting with different classes of calcium binding proteins (CaBP's), with different affinities, metal selectivities, kinetics, and calcium dependent conformational changes. Due to the diverse coordination chemistry of calcium, and complexity associated with protein folding and binding cooperativity, the rational design of CaBP's was anticipated to present multiple challenges. In this paper we will first discuss applications of statistical analysis of calcium binding sites in proteins and subsequent development of algorithms to predict and identify calcium binding proteins. Next, we report efforts to identify key determinants for calcium binding affinity, cooperativity and calcium dependent conformational changes using grafting and protein design. Finally, we report recent advances in designing protein calcium sensors to capture calcium dynamics in various cellular environments.

3.
Artigo em Inglês | MEDLINE | ID: mdl-32126587

RESUMO

The extracellular matrix (ECM) consists of proteins and carbohydrates that supports different biological structures and processes such as tissue development, elasticity, and preservation of organ structure. Diseases involving inflammation, fibrosis, tumor invasion, and injury are all attributed to the transition of the ECM from homeostasis to remodeling, which can significantly change the biochemical and biomechanical features of ECM components. While contrast agents have played an indispensable role in facilitating clinical diagnosis of diseases using magnetic resonance imaging (MRI), there is a strong need to develop novel biomarker-targeted imaging probes for in vivo visualization of biological processes and pathological alterations at a cellular and molecular level, for both early diagnosis and monitoring drug treatment. Herein, we will first review the pathological accumulation and characterization of ECM proteins recognized as important molecular features of diseases. Developments in MRI probes targeting ECM proteins such as collagen, fibronectin, and elastin via conjugation of existing contrast agents to targeting moieties and their applications to various diseases, are also reviewed. We have also reviewed our progress in the development of collagen-targeted protein MRI contrast agent with significant improvement in relaxivity and metal binding specificity, and their applications in early detection of fibrosis and metastatic cancer. This article is categorized under: Diagnostic Tools > in vivo Nanodiagnostics and Imaging Biology-Inspired Nanomaterials > Peptide-Based Structures Biology-Inspired Nanomaterials > Protein and Virus-Based Structures.

4.
Sci Adv ; 6(6): eaav7504, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32083172

RESUMO

Liver metastases often progress from primary cancers including uveal melanoma (UM), breast, and colon cancer. Molecular biomarker imaging is a new non-invasive approach for detecting early stage tumors. Here, we report the elevated expression of chemokine receptor 4 (CXCR4) in liver metastases in UM patients and metastatic UM mouse models, and development of a CXCR4-targeted MRI contrast agent, ProCA32.CXCR4, for sensitive MRI detection of UM liver metastases. ProCA32.CXCR4 exhibits high relaxivities (r 1 = 30.9 mM-1 s-1, r 2 = 43.2 mM-1 s-1, 1.5 T; r 1 = 23.5 mM-1 s-1, r 2 = 98.6 mM-1 s-1, 7.0 T), strong CXCR4 binding (K d = 1.10 ± 0.18 µM), CXCR4 molecular imaging capability in metastatic and intrahepatic xenotransplantation UM mouse models. ProCA32.CXCR4 enables detecting UM liver metastases as small as 0.1 mm3. Further development of the CXCR4-targeted imaging agent should have strong translation potential for early detection, surveillance, and treatment stratification of liver metastases patients.


Assuntos
Biomarcadores , Meios de Contraste , Neoplasias Hepáticas/diagnóstico por imagem , Neoplasias Hepáticas/metabolismo , Imagem por Ressonância Magnética , Imagem Molecular , Receptores CXCR4/metabolismo , Animais , Meios de Contraste/química , Modelos Animais de Doenças , Detecção Precoce de Câncer , Expressão Gênica , Humanos , Neoplasias Hepáticas/patologia , Imagem por Ressonância Magnética/métodos , Camundongos , Modelos Moleculares , Metástase Neoplásica , Ligação Proteica , Curva ROC , Receptores CXCR4/química , Receptores CXCR4/genética , Reprodutibilidade dos Testes , Relação Estrutura-Atividade
5.
Int J Mol Sci ; 22(1)2020 Dec 30.
Artigo em Inglês | MEDLINE | ID: mdl-33396740

RESUMO

Calmodulin (CaM) is an important intracellular protein that binds Ca2+ and functions as a critical second messenger involved in numerous biological activities through extensive interactions with proteins and peptides. CaM's ability to adapt to binding targets with different structures is related to the flexible central helix separating the N- and C-terminal lobes, which allows for conformational changes between extended and collapsed forms of the protein. CaM-binding targets are most often identified using prediction algorithms that utilize sequence and structural data to predict regions of peptides and proteins that can interact with CaM. In this review, we provide an overview of different CaM-binding proteins, the motifs through which they interact with CaM, and shared properties that make them good binding partners for CaM. Additionally, we discuss the historical and current methods for predicting CaM binding, and the similarities and differences between these methods and their relative success at prediction. As new CaM-binding proteins are identified and classified, we will gain a broader understanding of the biological processes regulated through changes in Ca2+ concentration through interactions with CaM.


Assuntos
Proteínas de Ligação a Calmodulina/química , Proteínas de Ligação a Calmodulina/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Sítios de Ligação , Cálcio/química , Calmodulina/química , Análise por Conglomerados , Análise Discriminante , Humanos , Aprendizado de Máquina , Cadeias de Markov , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Relação Estrutura-Atividade , Máquina de Vetores de Suporte
6.
Nat Commun ; 10(1): 4777, 2019 10 29.
Artigo em Inglês | MEDLINE | ID: mdl-31664017

RESUMO

Early diagnosis and noninvasive detection of liver fibrosis and its heterogeneity remain as major unmet medical needs for stopping further disease progression toward severe clinical consequences. Here we report a collagen type I targeting protein-based contrast agent (ProCA32.collagen1) with strong collagen I affinity. ProCA32.collagen1 possesses high relaxivities per particle (r1 and r2) at both 1.4 and 7.0 T, which enables the robust detection of early-stage (Ishak stage 3 of 6) liver fibrosis and nonalcoholic steatohepatitis (Ishak stage 1 of 6 or 1 A Mild) in animal models via dual contrast modes. ProCA32.collagen1 also demonstrates vasculature changes associated with intrahepatic angiogenesis and portal hypertension during late-stage fibrosis, and heterogeneity via serial molecular imaging. ProCA32.collagen1 mitigates metal toxicity due to lower dosage and strong resistance to transmetallation and unprecedented metal selectivity for Gd3+ over physiological metal ions with strong translational potential in facilitating effective treatment to halt further chronic liver disease progression.


Assuntos
Meios de Contraste/química , Gadolínio/química , Hipertensão Portal/diagnóstico por imagem , Fígado/diagnóstico por imagem , Imagem por Ressonância Magnética/métodos , Doença Crônica , Diagnóstico Precoce , Humanos
7.
Biomaterials ; 224: 119478, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31542517

RESUMO

The Liver is the most common organ for metastasis for various cancers, including uveal melanoma, the most common primary intraocular tumor. Uveal melanoma metastasizes to the liver in ~90% of patients, and results in death in almost all cases due to late detection and lack of effective treatment. There is a pressing unmet medical need to develop MRI contrast agents and imaging methodologies with desired sensitivity and specificity to overcome the high heterogeneous background and in vivo properties as well as reduced toxicity. Herein, we report the development of a collagen targeting protein contrast agent (ProCA32.collagen1), since collagen is a diagnostic biomarker and therapeutic target for many types of primary and metastatic cancers and the tumor microenvironment. In addition to a strong affinity to collagen I, ProCA32.collagen1 possesses high relaxivities (r1 and r2 are 68.0 ±â€¯0.25 and 100.0 ±â€¯0.32 mM-1 s-1 at 1.4 T, respectively, and 42.6 ±â€¯1.0 and 217 ±â€¯2.4 mM-1s-1 at 7.0 T per particle). ProCA32.collagen1 also has strong serum stability against degradation, resistance to transmetallation, and 102 and 1013-fold higher metal selectivity for Gd3+ over Ca2+ and Zn2+, respectively, compared to clinical contrast agents. ProCA32.collagen1 does not exhibit any cell toxicity for various cell lines. Sensitive detection of liver lesions in animal models can be achieved using multiple imaging methodologies, taking advantage of the dual relaxation property of ProCA32.collagen1. ProCA32.collagen1 enables sensitive and early stage detection of hepatic micrometastasis as small as 0.144 mm2 and two different tumor growth patterns. Further development of ProCA32.collagen1 has the potential to greatly facilitate non-invasive, early detection and staging of primary and metastatic liver cancers, and devising effective treatments.


Assuntos
Colágeno/química , Meios de Contraste/química , Neoplasias Hepáticas/diagnóstico , Neoplasias Hepáticas/secundário , Imagem por Ressonância Magnética , Animais , Linhagem Celular , Sobrevivência Celular , Endocitose , Feminino , Humanos , Fígado/patologia , Camundongos Endogâmicos C57BL , Distribuição Tecidual
8.
Methods Mol Biol ; 1929: 111-125, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30710270

RESUMO

Early diagnosis, noninvasive detection, and staging of various diseases, remain one of the major clinical barriers to effective medical treatment and prevention of disease progression toward major clinical consequences. Molecular imaging technologies play an indispensable role in the clinical field in overcoming these major barriers. The increasing application of imaging techniques and agents in early detection of different diseases such as cancer has resulted in improved treatment response and clinical patient management. In this chapter we will first introduce criteria for the design and engineering of calcium-binding protein (CaBP) parvalbumin as a protein Gd-MRI contrast agent (ProCA) with unprecedented metal selectivity for Gd3+ over physiological metal ions. We will then discuss the further development of targeted MRI contrast agent for molecular imaging of PSMA biomarker for early detection of prostate cancer.


Assuntos
Meios de Contraste/síntese química , Gadolínio/química , Calicreínas/metabolismo , Parvalbuminas/química , Antígeno Prostático Específico/metabolismo , Neoplasias da Próstata/diagnóstico por imagem , Engenharia de Proteínas/métodos , Animais , Cálcio/metabolismo , Linhagem Celular Tumoral , Meios de Contraste/química , Meios de Contraste/farmacologia , Detecção Precoce de Câncer , Humanos , Imagem por Ressonância Magnética/métodos , Masculino , Camundongos , Modelos Moleculares , Conformação Molecular , Imagem Molecular/métodos , Transplante de Neoplasias , Neoplasias da Próstata/metabolismo
10.
Neurotherapeutics ; 15(3): 770-784, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29869055

RESUMO

Ischemic stroke remains a serious threat to human life. Generation of neuronal and vascular cells is an endogenous regenerative mechanism in the adult brain, which may contribute to tissue repair after stroke. However, the regenerative activity is typically insufficient for significant therapeutic effects after brain injuries. Pyruvate kinase isoform M2 (PKM2) is a key regulator for energy metabolism. PKM2 also has nonmetabolic roles involving regulations of gene expression, cell proliferation, and migration in cancer cells as well as noncancerous cells. In a focal ischemic stroke mouse model, recombinant PKM2 (rPKM2) administration (160 ng/kg, intranasal delivery) at 1 h after stroke showed the significant effect of a reduced infarct volume of more the 60%. Delayed treatment of rPKM2, however, lost the acute neuroprotective effect. We then tested a novel hypothesis that delayed treatment of PKM2 might show proregenerative effects for long-term functional recovery and this chronic action could be mediated by its downstream STAT3 signaling. rPKM2 (160 ng/kg) was delivered to the brain using noninvasive intranasal administration 24 h after the stroke and repeated every other day. Western blot analysis revealed that, 7 days after the stroke, the levels of PKM2 and phosphorylated STAT3 and the expression of angiogenic factors VEGF, Ang-1, and Tie-2 in the peri-infarct region were significantly increased in the rPKM2 treatment group compared with those of the stroke vehicle group. To label proliferating cells, 5-bromo-2'-deoxyuridine (BrdU, 50 mg/kg, i.p.) was injected every day starting 3 days after stroke. At 14 days after stroke, immunohistochemistry showed that rPKM2 increased cell homing of doublecortin (DCX)-positive neuroblasts to the ischemic cortex. In neural progenitor cell (NPC) cultures, rPKM2 (0.4-4 nM) increased the expression of integrin ß1 and the activation/phosphorylation of focal adhesion kinase (FAK). A mediator role of FAK in PKM2-promoted cell migration was verified in FAK-knockout fibroblast cultures. In the peri-infarct region of the brain, increased numbers of Glut-1/BrdU and NeuN/BrdU double-positive cells indicated enhanced angiogenesis and neurogenesis, respectively, compared to stroke vehicle mice. Using Laser Doppler imaging, we observed better recovery of the local blood flow in the peri-infarct region of rPKM2-treated mice 14 days after stroke. Meanwhile, rPKM2 improved the sensorimotor functional recovery measured by the adhesive removal test. Inhibiting the STAT3 phosphorylation/activation by the STAT3 inhibitor, BP-1-102 (3 mg/kg/day, o.g.), abolished all beneficial effects of rPKM2 in the stroke mice. Taken together, this investigation provides the first evidence demonstrating that early treatment of rPKM2 shows an acute neuroprotective effect against ischemic brain damage, whereas delayed rPKM2 treatment promotes regenerative activities in the poststroke brain leading to better functional recovery. The underlying mechanism involves activation of the STAT3 and FAK signals in the poststroke brain.


Assuntos
Proteína-Tirosina Quinases de Adesão Focal/genética , Infarto da Artéria Cerebral Média/tratamento farmacológico , Neovascularização Fisiológica/efeitos dos fármacos , Neurogênese/efeitos dos fármacos , Piruvato Quinase , Recuperação de Função Fisiológica/efeitos dos fármacos , Fator de Transcrição STAT3/genética , Animais , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Circulação Cerebrovascular/efeitos dos fármacos , Modelos Animais de Doenças , Fibroblastos/efeitos dos fármacos , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Infarto da Artéria Cerebral Média/fisiopatologia , Camundongos , Camundongos Endogâmicos C57BL , Fosfopiruvato Hidratase/metabolismo , Piruvato Quinase/farmacologia , Piruvato Quinase/uso terapêutico , Fator de Transcrição STAT3/metabolismo , Células-Tronco/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos
11.
J Proteome Res ; 17(4): 1700-1711, 2018 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-29518331

RESUMO

Regulator of G Protein Signaling 14 (RGS14) is a complex scaffolding protein that integrates G protein and MAPK signaling pathways. In the adult mouse brain, RGS14 is predominantly expressed in hippocampal CA2 neurons where it naturally inhibits synaptic plasticity and hippocampus-dependent learning and memory. However, the signaling proteins that RGS14 natively engages to regulate plasticity are unknown. Here, we show that RGS14 exists in a high-molecular-weight protein complex in brain. To identify RGS14 neuronal interacting partners, endogenous RGS14 immunoprecipitated from mouse brain was subjected to mass spectrometry and proteomic analysis. We find that RGS14 interacts with key postsynaptic proteins that regulate plasticity. Gene ontology analysis reveals the most enriched RGS14 interactors have functional roles in actin-binding, calmodulin(CaM)-binding, and CaM-dependent protein kinase (CaMK) activity. We validate these findings using biochemical assays that identify interactions with two previously unknown binding partners. We report that RGS14 directly interacts with Ca2+/CaM and is phosphorylated by CaMKII in vitro. Lastly, we detect that RGS14 associates with CaMKII and CaM in hippocampal CA2 neurons. Taken together, these findings demonstrate that RGS14 is a novel CaM effector and CaMKII phosphorylation substrate thereby providing new insight into mechanisms by which RGS14 controls plasticity in CA2 neurons.


Assuntos
Química Encefálica , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Calmodulina/metabolismo , Hipocampo/química , Proteínas RGS/metabolismo , Animais , Região CA2 Hipocampal/citologia , Cálcio/metabolismo , Hipocampo/metabolismo , Camundongos , Plasticidade Neuronal , Neurônios/metabolismo , Fosforilação , Ligação Proteica , Proteômica
12.
Ophthalmology ; 125(4): 597-605, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29122287

RESUMO

PURPOSE: The purpose of this study was to correlate magnetic resonance imaging (MRI) radiographic results with histopathologic growth patterns of metastatic uveal melanoma (UM) to the liver. DESIGN: Clinicopathologic correlation. PARTICIPANTS: Patients with metastatic UM to the liver. METHODS: A retrospective review of MRI images of patients with metastatic UM to the liver at a single institution between 2004 and 2016 was performed. The MRI growth patterns were classified as nodular or diffuse. The histopathologic findings of core liver biopsies of liver metastases identified by needle localization in a subset of these patients were reviewed. The core samples were evaluated by routine light microscopy, including immunohistochemical/immunofluorescent staining for CD31, CD105, and HMB45, and classified as exhibiting an infiltrative or nodular growth pattern. MAIN OUTCOME MEASURES: Magnetic resonance images and core biopsy findings. RESULTS: A total of 32 patients were identified with metastatic UM to the liver that was imaged by MRI, and 127 lesions were identified. A total of 46 lesions were classified by MRI as infiltrative and 81 as nodular. There were 9 needle-localized core biopsies that corresponded to MRI of metastatic lesions. Of these 9 lesions, 3 that were classified as infiltrative on MRI exhibited stage I infiltrative histologic growth patterns; of the remaining 6 that were classified as nodular by MRI, 5 histologically demonstrated stage II or stage III infiltrative growth patterns and 1 histologically demonstrated a nodular growth pattern. CONCLUSIONS: Magnetic resonance imaging of hepatic infiltrative growth patterns of metastatic UM corresponded to stage I histologic infiltrative growth in the sinusoidal spaces, whereas MRI nodular growth patterns corresponded to stage II/III histologic infiltrative growth that replaced the hepatic lobule or histologic nodular growth in the portal triad that effaced adjacent hepatic parenchyma.


Assuntos
Neoplasias Hepáticas/diagnóstico por imagem , Neoplasias Hepáticas/secundário , Melanoma/diagnóstico por imagem , Melanoma/secundário , Neoplasias Uveais/patologia , Biomarcadores Tumorais/metabolismo , Biópsia , Endoglina/metabolismo , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Imuno-Histoquímica , Neoplasias Hepáticas/metabolismo , Imagem por Ressonância Magnética , Masculino , Melanoma/metabolismo , Antígenos Específicos de Melanoma/metabolismo , Pessoa de Meia-Idade , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Estudos Retrospectivos , Neoplasias Uveais/diagnóstico por imagem , Neoplasias Uveais/metabolismo
13.
Biochem J ; 474(24): 4035-4051, 2017 11 27.
Artigo em Inglês | MEDLINE | ID: mdl-28963343

RESUMO

Calmodulin (CaM) is an intracellular Ca2+ transducer involved in numerous activities in a broad Ca2+ signaling network. Previous studies have suggested that the Ca2+/CaM complex may participate in gap junction regulation via interaction with putative CaM-binding motifs in connexins; however, evidence of direct interactions between CaM and connexins has remained elusive to date due to challenges related to the study of membrane proteins. Here, we report the first direct interaction of CaM with Cx45 (connexin45) of γ-family in living cells under physiological conditions by monitoring bioluminescence resonance energy transfer. The interaction between CaM and Cx45 in cells is strongly dependent on intracellular Ca2+ concentration and can be blocked by the CaM inhibitor, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W7). We further reveal a CaM-binding site at the cytosolic loop (residues 164-186) of Cx45 using a peptide model. The strong binding (Kd ∼ 5 nM) observed between CaM and Cx45 peptide, monitored by fluorescence-labeled CaM, is found to be Ca2+-dependent. Furthermore, high-resolution nuclear magnetic resonance spectroscopy reveals that CaM and Cx45 peptide binding leads to global chemical shift changes of 15N-labeled CaM, but does not alter the size of the structure. Observations involving both N- and C-domains of CaM to interact with the Cx45 peptide differ from the embraced interaction with Cx50 from another connexin family. Such interaction further increases Ca2+ sensitivity of CaM, especially at the N-terminal domain. Results of the present study suggest that both helicity and the interaction mode of the cytosolic loop are likely to contribute to CaM's modulation of connexins.


Assuntos
Técnicas de Transferência de Energia por Ressonância de Bioluminescência/métodos , Cálcio/metabolismo , Calmodulina/metabolismo , Conexinas/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Calmodulina/química , Conexinas/química , Transferência de Energia , Células HEK293 , Células HeLa , Humanos , Cinética , Ligação Proteica , Conformação Proteica , Homologia de Sequência , Transdução de Sinais
14.
J Vis Exp ; (123)2017 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-28570539

RESUMO

Intracellular calcium (Ca2+) transients evoked by extracellular stimuli initiate a multitude of biological processes in living organisms. At the center of intracellular calcium release are the major intracellular calcium storage organelles, the endoplasmic reticulum (ER) and the more specialized sarcoplasmic reticulum (SR) in muscle cells. The dynamic release of calcium from these organelles is mediated by the ryanodine receptor (RyR) and the inositol 1,4,5-triphosphate receptor (IP3R) with refilling occurring through the sarco/endoplasmic reticulum calcium ATPase (SERCA) pump. A genetically encoded calcium sensor (GECI) called CatchER was created to monitor the rapid calcium release from the ER/SR. Here, the detailed protocols for the transfection and expression of the improved, ER/SR-targeted GECI CatchER+ in HEK293 and C2C12 cells and its application in monitoring IP3R, RyR, and SERCA pump-mediated calcium transients in HEK293 cells using fluorescence microscopy is outlined. The receptor agonist or inhibitor of choice is dispersed in the chamber solution and the intensity changes are recorded in real time. With this method, a decrease in ER calcium is seen with RyR activation with 4-chloro-m-cresol (4-cmc), the indirect activation of IP3R with adenosine triphosphate (ATP), and inhibition of the SERCA pump with cyclopiazonic acid (CPA). We also discuss protocols for determining the in situ Kd and quantifying basal [Ca2+] in C2C12 cells. In summary, these protocols, used in conjunction with CatchER+, can elicit receptor mediated calcium release from the ER with future application in studying ER/SR calcium related pathologies.


Assuntos
Cálcio/metabolismo , Retículo Endoplasmático/metabolismo , Retículo Sarcoplasmático/metabolismo , Trifosfato de Adenosina/farmacologia , Animais , Linhagem Celular , Cresóis/farmacologia , Humanos , Indóis/farmacologia , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Camundongos , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo
15.
Int J Mol Sci ; 18(5)2017 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-28489021

RESUMO

The flow of intracellular calcium (Ca2+) is critical for the activation and regulation of important biological events that are required in living organisms. As the major Ca2+ repositories inside the cell, the endoplasmic reticulum (ER) and the sarcoplasmic reticulum (SR) of muscle cells are central in maintaining and amplifying the intracellular Ca2+ signal. The morphology of these organelles, along with the distribution of key calcium-binding proteins (CaBPs), regulatory proteins, pumps, and receptors fundamentally impact the local and global differences in Ca2+ release kinetics. In this review, we will discuss the structural and morphological differences between the ER and SR and how they influence localized Ca2+ release, related diseases, and the need for targeted genetically encoded calcium indicators (GECIs) to study these events.


Assuntos
Sinalização do Cálcio , Hipertermia Maligna/genética , Miotonia Congênita/genética , Retículo Sarcoplasmático/metabolismo , Taquicardia/genética , Animais , Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Humanos , Hipertermia Maligna/metabolismo , Miotonia Congênita/metabolismo , Retículo Sarcoplasmático/genética , Taquicardia/metabolismo
16.
Int J Mol Sci ; 18(3)2017 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-28335551

RESUMO

Metabotropic glutamate receptors (mGluRs) associated with the slow phase of the glutamatergic signaling pathway in neurons of the central nervous system have gained importance as drug targets for chronic neurodegenerative diseases. While extracellular Ca2+ was reported to exhibit direct activation and modulation via an allosteric site, the identification of those binding sites was challenged by weak binding. Herein, we review the discovery of extracellular Ca2+ in regulation of mGluRs, summarize the recent developments in probing Ca2+ binding and its co-regulation of the receptor based on structural and biochemical analysis, and discuss the molecular basis for Ca2+ to regulate various classes of drug action as well as its importance as an allosteric modulator in mGluRs.


Assuntos
Cálcio/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Regulação Alostérica , Animais , Sinalização do Cálcio , Humanos , Neurônios/metabolismo , Receptores de Glutamato Metabotrópico/agonistas , Receptores de Glutamato Metabotrópico/antagonistas & inibidores
17.
Front Physiol ; 7: 441, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27746744

RESUMO

Ca2+-sensing receptors (CaSRs) play a central role in regulating extracellular calcium concentration ([Ca2+]o) homeostasis and many (patho)physiological processes in multiple organs. This regulation is orchestrated by a cooperative response to extracellular stimuli such as small changes in Ca2+, Mg2+, amino acids, and other ligands. In addition, CaSR is a pleiotropic receptor regulating several intracellular signaling pathways, including calcium mobilization and intracellular calcium oscillation. Nearly 200 mutations and polymorphisms have been found in CaSR in relation to a variety of human disorders associated with abnormal Ca2+ homeostasis. In this review, we summarize efforts directed at identifying binding sites for calcium and amino acids. Both homotropic cooperativity among multiple calcium binding sites and heterotropic cooperativity between calcium and amino acid were revealed using computational modeling, predictions, and site-directed mutagenesis coupled with functional assays. The hinge region of the bilobed Venus flytrap (VFT) domain of CaSR plays a pivotal role in coordinating multiple extracellular stimuli, leading to cooperative responses from the receptor. We further highlight the extensive number of disease-associated mutations that have also been shown to affect CaSR's cooperative action via several types of mechanisms. These results provide insights into the molecular bases of the structure and functional cooperativity of this receptor and other members of family C of the G protein-coupled receptors (cGPCRs) in health and disease states, and may assist in the prospective development of novel receptor-based therapeutics.

19.
Sci Adv ; 2(5): e1600241, 2016 05.
Artigo em Inglês | MEDLINE | ID: mdl-27386547

RESUMO

Ca(2+)-sensing receptors (CaSRs) modulate calcium and magnesium homeostasis and many (patho)physiological processes by responding to extracellular stimuli, including divalent cations and amino acids. We report the first crystal structure of the extracellular domain (ECD) of human CaSR bound with Mg(2+) and a tryptophan derivative ligand at 2.1 Å. The structure reveals key determinants for cooperative activation by metal ions and aromatic amino acids. The unexpected tryptophan derivative was bound in the hinge region between two globular ECD subdomains, and represents a novel high-affinity co-agonist of CaSR. The dissection of structure-function relations by mutagenesis, biochemical, and functional studies provides insights into the molecular basis of human diseases arising from CaSR mutations. The data also provide a novel paradigm for understanding the mechanism of CaSR-mediated signaling that is likely shared by the other family C GPCR [G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptor] members and can facilitate the development of novel CaSR-based therapeutics.


Assuntos
Íons/química , Magnésio/química , Receptores de Detecção de Cálcio/química , Triptofano/química , Linhagem Celular , Humanos , Íons/metabolismo , Ligantes , Magnésio/metabolismo , Modelos Moleculares , Conformação Molecular , Mutação , Ligação Proteica , Receptores de Detecção de Cálcio/agonistas , Receptores de Detecção de Cálcio/genética , Receptores de Detecção de Cálcio/metabolismo , Relação Estrutura-Atividade , Triptofano/análogos & derivados , Triptofano/metabolismo
20.
Nat Commun ; 7: 11675, 2016 05 31.
Artigo em Inglês | MEDLINE | ID: mdl-27241473

RESUMO

Integrin αvß3 expression is altered in various diseases and has been proposed as a drug target. Here we use a rational design approach to develop a therapeutic protein, which we call ProAgio, that binds to integrin αvß3 outside the classical ligand-binding site. We show ProAgio induces apoptosis of integrin αvß3-expressing cells by recruiting and activating caspase 8 to the cytoplasmic domain of integrin αvß3. ProAgio also has anti-angiogenic activity and strongly inhibits growth of tumour xenografts, but does not affect the established vasculature. Toxicity analyses demonstrate that ProAgio is not toxic to mice. Our study reports a new integrin-targeting agent with a unique mechanism of action, and provides a template for the development of integrin-targeting therapeutics.


Assuntos
Inibidores da Angiogênese/farmacologia , Apoptose/efeitos dos fármacos , Desenho de Fármacos , Integrina alfaVbeta3/metabolismo , Neovascularização Patológica/tratamento farmacológico , Sequência de Aminoácidos/genética , Inibidores da Angiogênese/química , Inibidores da Angiogênese/uso terapêutico , Animais , Sítios de Ligação/genética , Linhagem Celular , Feminino , Humanos , Ligantes , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Simulação de Acoplamento Molecular , Terapia de Alvo Molecular/métodos , Neoplasias/irrigação sanguínea , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Neovascularização Patológica/patologia , Ligação Proteica/genética , Engenharia de Proteínas/métodos , Estrutura Terciária de Proteína , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
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