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1.
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
2.
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
3.
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
4.
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
6.
Nanoscale ; 8(25): 12668-82, 2016 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-26961235

RESUMO

Prostate-specific membrane antigen (PSMA) is one of the most specific cell surface markers for prostate cancer diagnosis and targeted treatment. However, achieving molecular imaging using non-invasive MRI with high resolution has yet to be achieved due to the lack of contrast agents with significantly improved relaxivity for sensitivity, targeting capabilities and metal selectivity. We have previously reported our creation of a novel class of protein Gd(3+) contrast agents, ProCA32, which displayed significantly improved relaxivity while exhibiting strong Gd(3+) binding selectivity over physiological metal ions. In this study, we report our effort in further developing biomarker-targeted protein MRI contrast agents for molecular imaging of PSMA. Among three PSMA targeted contrast agents engineered with addition of different molecular recognition sequences, ProCA32.PSMA exhibits a binding affinity of 1.1 ± 0.1 µM for PSMA while the metal binding affinity is maintained at 0.9 ± 0.1 × 10(-22) M. In addition, ProCA32.PSMA exhibits r1 of 27.6 mM(-1) s(-1) and r2 of 37.9 mM(-1) s(-1) per Gd (55.2 and 75.8 mM(-1) s(-1) per molecule r1 and r2, respectively) at 1.4 T. At 7 T, ProCA32.PSMA also has r2 of 94.0 mM(-1) s(-1) per Gd (188.0 mM(-1) s(-1) per molecule) and r1 of 18.6 mM(-1) s(-1) per Gd (37.2 mM(-1) s(-1) per molecule). This contrast capability enables the first MRI enhancement dependent on PSMA expression levels in tumor bearing mice using both T1 and T2-weighted MRI at 7 T. Further development of these PSMA-targeted contrast agents are expected to be used for the precision imaging of prostate cancer at an early stage and to monitor disease progression and staging, as well as determine the effect of therapeutic treatment by non-invasive evaluation of the PSMA level using MRI.


Assuntos
Antígenos de Superfície/análise , Meios de Contraste , Glutamato Carboxipeptidase II/análise , Imagem por Ressonância Magnética , Imagem Molecular , Neoplasias Experimentais/diagnóstico por imagem , Neoplasias da Próstata/diagnóstico por imagem , Animais , Linhagem Celular Tumoral , Gadolínio , Humanos , Masculino , Camundongos , Camundongos Nus
7.
Curr Protein Pept Sci ; 17(6): 519-33, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26721404

RESUMO

Prostate cancer is the most common cancer for man with a high mortality rate due to a lack of non-invasive accurate and sensitive molecular diagnostic methods. The molecular imaging of cancer biomarkers using MRI with its spatial and temporal resolution, however, is largely limited by the lack of contrast agents with high sensitivity, targeting specificity and deep tumor penetration. In this review, we will first overview the current stage of prostate cancer diagnosis and then review prostate cancer biomarkers and related imaging techniques. Since biomarker targeting moieties are essential for molecular imaging, we will use prostate-specific membrane antigen (PSMA) as an example to discuss different methods to characterize the interaction between biomarker and targeting moieties. At the end, we will review current progress of the development of targeted protein-based MRI contrast agents (ProCAs) for prostate cancer biomarkers with improved relaxivity and targeting capability.


Assuntos
Biomarcadores Tumorais , Meios de Contraste , Imagem por Ressonância Magnética , Imagem Molecular , Neoplasias da Próstata/diagnóstico por imagem , Neoplasias da Próstata/metabolismo , Proteínas/metabolismo , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/metabolismo , Antígenos de Neoplasias/química , Antígenos de Neoplasias/metabolismo , Diagnóstico por Imagem/métodos , Humanos , Ligantes , Imagem por Ressonância Magnética/métodos , Masculino , Imagem Molecular/métodos , Sondas Moleculares/química , Sondas Moleculares/metabolismo , Ligação Proteica , Proteínas/química , Compostos Radiofarmacêuticos/química , Compostos Radiofarmacêuticos/metabolismo , Receptores da Bombesina/química , Receptores da Bombesina/metabolismo
8.
Sci Rep ; 5: 16214, 2015 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-26577829

RESUMO

Gastrin-releasing peptide receptor (GRPR) is differentially expressed on the surfaces of various diseased cells, including prostate and lung cancer. However, monitoring temporal and spatial expression of GRPR in vivo by clinical MRI is severely hampered by the lack of contrast agents with high relaxivity, targeting capability and tumor penetration. Here, we report the development of a GRPR-targeted MRI contrast agent by grafting the GRPR targeting moiety into a scaffold protein with a designed Gd(3+) binding site (ProCA1.GRPR). In addition to its strong binding affinity for GRPR (Kd = 2.7 nM), ProCA1.GRPR has high relaxivity (r1 = 42.0 mM(-1)s(-1) at 1.5 T and 25 °C) and strong Gd(3+) selectivity over physiological metal ions. ProCA1.GRPR enables in vivo detection of GRPR expression and spatial distribution in both PC3 and H441 tumors in mice using MRI. ProCA1.GRPR is expected to have important preclinical and clinical implications for the early detection of cancer and for monitoring treatment effects.


Assuntos
Meios de Contraste , Imagem por Ressonância Magnética , Imagem Molecular , Neoplasias/diagnóstico , Neoplasias/metabolismo , Receptores da Bombesina/metabolismo , Animais , Sítios de Ligação , Biomarcadores , Linhagem Celular Tumoral , Meios de Contraste/química , Meios de Contraste/metabolismo , Meios de Contraste/farmacocinética , Modelos Animais de Doenças , Expressão Gênica , Xenoenxertos , Humanos , Ligantes , Imagem por Ressonância Magnética/métodos , Camundongos , Modelos Moleculares , Conformação Molecular , Imagem Molecular/métodos , Neoplasias/genética , Ligação Proteica , Ratos , Receptores da Bombesina/química , Receptores da Bombesina/genética , Distribuição Tecidual
9.
Proc Natl Acad Sci U S A ; 112(21): 6607-12, 2015 May 26.
Artigo em Inglês | MEDLINE | ID: mdl-25971726

RESUMO

With available MRI techniques, primary and metastatic liver cancers that are associated with high mortality rates and poor treatment responses are only diagnosed at late stages, due to the lack of highly sensitive contrast agents without Gd(3+) toxicity. We have developed a protein contrast agent (ProCA32) that exhibits high stability for Gd(3+) and a 10(11)-fold greater selectivity for Gd(3+) over Zn(2+) compared with existing contrast agents. ProCA32, modified from parvalbumin, possesses high relaxivities (r1/r2: 66.8 mmol(-1)⋅s(-1)/89.2 mmol(-1)⋅s(-1) per particle). Using T1- and T2-weighted, as well as T2/T1 ratio imaging, we have achieved, for the first time (to our knowledge), robust MRI detection of early liver metastases as small as ∼0.24 mm in diameter, much smaller than the current detection limit of 10-20 mm. Furthermore, ProCA32 exhibits appropriate in vivo preference for liver sinusoidal spaces and pharmacokinetics for high-quality imaging. ProCA32 will be invaluable for noninvasive early detection of primary and metastatic liver cancers as well as for monitoring treatment and guiding therapeutic interventions, including drug delivery.


Assuntos
Meios de Contraste , Neoplasias Hepáticas Experimentais/diagnóstico , Neoplasias Hepáticas Experimentais/metabolismo , Imagem por Ressonância Magnética/métodos , Melanoma Experimental/diagnóstico , Melanoma Experimental/metabolismo , Parvalbuminas , Animais , Linhagem Celular Tumoral , Meios de Contraste/química , Meios de Contraste/farmacocinética , Feminino , Gadolínio , Limite de Detecção , Neoplasias Hepáticas Experimentais/secundário , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Parvalbuminas/química , Parvalbuminas/farmacocinética , Engenharia de Proteínas , Estabilidade Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacocinética
10.
J Biol Chem ; 289(40): 27571-84, 2014 Oct 03.
Artigo em Inglês | MEDLINE | ID: mdl-25143381

RESUMO

7,8-dihydroxyflavone (7,8-DHF), a newly identified small molecular TrkB receptor agonist, rapidly activates TrkB in both primary neurons and the rodent brain and mimics the physiological functions of the cognate ligand BDNF. Accumulating evidence supports that 7,8-DHF exerts neurotrophic effects in a TrkB-dependent manner. Nonetheless, the differences between 7,8-DHF and BDNF in activating TrkB remain incompletely understood. Here we show that 7,8-DHF and BDNF exhibit different TrkB activation kinetics in which TrkB maturation may be implicated. Employing two independent biophysical approaches, we confirm that 7,8-DHF interacts robustly with the TrkB extracellular domain, with a Kd of ∼10 nm. Although BDNF transiently activates TrkB, leading to receptor internalization and ubiquitination/degradation, in contrast, 7,8-DHF-triggered TrkB phosphorylation lasts for hours, and the internalized receptors are not degraded. Notably, primary neuronal maturation may be required for 7,8-DHF but not for BDNF to elicit the full spectrum of TrkB signaling cascades. Hence, 7,8-DHF interacts robustly with the TrkB receptor, and its agonistic effect may be mediated by neuronal development and maturation.


Assuntos
Fator Neurotrófico Derivado do Encéfalo/química , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Flavonas/metabolismo , Receptor trkB/metabolismo , Animais , Fenômenos Biofísicos , Fator Neurotrófico Derivado do Encéfalo/genética , Células Cultivadas , Flavonas/química , Humanos , Cinética , Neurônios/química , Neurônios/metabolismo , Ligação Proteica , Ratos , Receptor trkB/agonistas , Receptor trkB/química , Receptor trkB/genética
11.
Biochem J ; 460(2): 261-71, 2014 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-24635445

RESUMO

Passive SR (sarcoplasmic reticulum) Ca2+ leak through the RyR (ryanodine receptor) plays a critical role in the mechanisms that regulate [Ca2+]rest (intracellular resting myoplasmic free Ca2+ concentration) in muscle. This process appears to be isoform-specific as expression of either RyR1 or RyR3 confers on myotubes different [Ca2+]rest. Using chimaeric RyR3-RyR1 receptors expressed in dyspedic myotubes, we show that isoform-dependent regulation of [Ca2+]rest is primarily defined by a small region of the receptor encompassing amino acids 3770-4007 of RyR1 (amino acids 3620-3859 of RyR3) named as the CLR (Ca2+ leak regulatory) region. [Ca2+]rest regulation by the CLR region was associated with alteration of RyRs' Ca2+-activation profile and changes in SR Ca2+-leak rates. Biochemical analysis using Tb3+-binding assays and intrinsic tryptophan fluorescence spectroscopy of purified CLR domains revealed that this determinant of RyRs holds a novel Ca2+-binding domain with conformational properties that are distinctive to each isoform. Our data suggest that the CLR region provides channels with unique functional properties that modulate the rate of passive SR Ca2+ leak and confer on RyR1 and RyR3 distinctive [Ca2+]rest regulatory properties. The identification of a new Ca2+-binding domain of RyRs with a key modulatory role in [Ca2+]rest regulation provides new insights into Ca2+-mediated regulation of RyRs.


Assuntos
Cálcio/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/fisiologia , Retículo Sarcoplasmático/metabolismo , Animais , Fibras Musculares Esqueléticas , Conformação Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo
12.
Diabetes ; 63(4): 1394-409, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24651808

RESUMO

Insulin replacement therapy is a widely adopted treatment for all patients with type 1 diabetes and some with type 2 diabetes. However, injection of insulin has suffered from problems such as tissue irritation, abscesses, discomfort, and inconvenience. The use of orally bioactive insulin mimetics thus represents an ideal treatment alternative. Here we show that a chaetochromin derivative (4548-G05) acts as a new nonpeptidyl insulin mimetic. 4548-G05 selectively activates an insulin receptor (IR) but not insulin-like growth factor receptor-I or other receptor tyrosine kinases. Through binding to the extracellular domain of the IR, 4548-G05 induces activation of the receptor and initiates the downstream Akt and extracellular signal-related kinase pathways to trigger glucose uptake in C2C12 myotubes. Moreover, it displays a potent blood glucose-lowering effect when administrated orally in normal, type 1 diabetic, and type 2 diabetic mice models. Therefore, 4548-G05 may represent a novel pharmacological agent for antidiabetes drug development.


Assuntos
Glicemia/efeitos dos fármacos , Hipoglicemiantes/uso terapêutico , Naftoquinonas/uso terapêutico , Receptor de Insulina/agonistas , Animais , Células CHO , Cricetulus , Diabetes Mellitus Experimental/tratamento farmacológico , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Receptor de Insulina/metabolismo , Transdução de Sinais/efeitos dos fármacos
13.
Med Res Rev ; 34(5): 1070-99, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24615853

RESUMO

Magnetic resonance imaging (MRI) is the leading imaging technique for disease diagnostics, providing high resolution, three-dimensional images noninvasively. MRI contrast agents are designed to improve the contrast and sensitivity of MRI. However, current clinically used MRI contrast agents have relaxivities far below the theoretical upper limit, which largely prevent advancing molecular imaging of biomarkers with desired sensitivity and specificity. This review describes current progress in the development of a new class of protein-based MRI contrast agents (ProCAs) with high relaxivity using protein design to optimize the parameters that govern relaxivity. Further, engineering with targeting moiety allows these contrast agents to be applicable for molecular imaging of prostate cancer biomarkers by MRI. The developed protein-based contrast agents also exhibit additional in vitro and in vivo advantages for molecular imaging of disease biomarkers, such as high metal-binding stability and selectivity, reduced toxicity, proper blood circulation time, and higher permeability in tumor tissue in addition to improved relaxivities.


Assuntos
Biomarcadores Tumorais/análise , Meios de Contraste , Gadolínio/administração & dosagem , Imagem por Ressonância Magnética/métodos , Relação Dose-Resposta a Droga , Gadolínio/química
14.
J Biol Inorg Chem ; 19(2): 259-70, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24366655

RESUMO

Epidermal growth factor receptor (EGFR) and HER2 are major prognosis biomarkers and drug targets overexpressed in various types of cancer cells. There is a pressing need to develop MRI contrast agents capable of enhancing the contrast between normal tissues and tumors with high relaxivity, capable of targeting tumors, and with high intratumoral distribution and minimal toxicity. In this review, we first discuss EGFR signaling and its role in tumor progression as a major drug target. We then report our progress in the development of protein contrast agents with significant improvement of both r1 and r2 relaxivities, pharmacokinetics, in vivo retention time, and in vivo dose efficiency. Finally, we report our effort in the development of EGFR-targeted protein contrast agents with the capability to cross the endothelial boundary and with good tissue distribution across the entire tumor mass. The noninvasive capability of MRI to visualize spatially and temporally the intratumoral distribution as well as quantify the levels of EGFR and HER2 would greatly improve our ability to track changes of the biomarkers during tumor progression, monitor treatment efficacy, aid in patient selection, and further develop novel targeted therapies for clinical application.


Assuntos
Meios de Contraste , Receptores ErbB/metabolismo , Imagem por Ressonância Magnética/métodos , Imagem Molecular/métodos , Neoplasias/diagnóstico , Neoplasias/metabolismo , Receptor ErbB-2/metabolismo , Biomarcadores Tumorais/metabolismo , Humanos , Neoplasias/patologia
15.
Artigo em Inglês | MEDLINE | ID: mdl-23335551

RESUMO

Magnetic resonance imaging (MRI) of disease biomarkers, especially cancer biomarkers, could potentially improve our understanding of the disease and drug activity during preclinical and clinical drug treatment and patient stratification. MRI contrast agents with high relaxivity and targeting capability to tumor biomarkers are highly required. Extensive work has been done to develop MRI contrast agents. However, only a few limited literatures report that protein residues can function as ligands to bind Gd(3+) with high binding affinity, selectivity, and relaxivity. In this paper, we focus on reporting our current progress on designing a novel class of protein-based Gd(3+) MRI contrast agents (ProCAs) equipped with several desirable capabilities for in vivo application of MRI of tumor biomarkers. We will first discuss our strategy for improving the relaxivity by a novel protein-based design. We then discuss the effect of increased relaxivity of ProCAs on improving the detection limits for MRI contrast agent, especially for in vivo application. We will further report our efforts to improve in vivo imaging capability and our achievement in molecular imaging of cancer biomarkers with potential preclinical and clinical applications.


Assuntos
Biomarcadores Tumorais/análise , Meios de Contraste/química , Imagem por Ressonância Magnética/métodos , Imagem Molecular/métodos , Proteínas/química , Animais , Linhagem Celular Tumoral , Humanos , Limite de Detecção
16.
Methods Mol Biol ; 963: 37-53, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23296603

RESUMO

Ca(2+) is implicated in almost every step of the life cycle of viruses, including virus entry into host cells, virus replication, virion assembly, maturation, and release. However, due to the lack of prediction algorithms and rigorous validation methods, only limited cases of viral Ca(2+)-binding sites are reported. Here, we introduce a method to predict continuous EF-hand or EF-hand-like motifs in the viral genomes based on their primary sequences. We then introduce a grafting approach, and the use of luminescence resonance energy transfer and Ca(2+) competition assay for experimental verification of predicted Ca(2+)-binding sites. This protocol will be valuable for the prediction and identification of unknown Ca(2+)-binding sites in virus.


Assuntos
Cálcio/metabolismo , Biologia Computacional/métodos , Motivos EF Hand , Proteínas Virais/química , Proteínas Virais/metabolismo , Sítios de Ligação , Genoma Viral/genética , Medições Luminescentes , Modelos Moleculares , Reconhecimento Automatizado de Padrão , Ligação Proteica , Engenharia de Proteínas , Estrutura Terciária de Proteína , Togaviridae/genética , Proteínas Virais/genética , Proteínas Virais/isolamento & purificação
17.
Metallomics ; 5(1): 29-42, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23235533

RESUMO

Calcium ion (Ca(2+)), the fifth most common chemical element in the earth's crust, represents the most abundant mineral in the human body. By binding to a myriad of proteins distributed in different cellular organelles, Ca(2+) impacts nearly every aspect of cellular life. In prokaryotes, Ca(2+) plays an important role in bacterial movement, chemotaxis, survival reactions and sporulation. In eukaryotes, Ca(2+) has been chosen through evolution to function as a universal and versatile intracellular signal. Viruses, as obligate intracellular parasites, also develop smart strategies to manipulate the host Ca(2+) signaling machinery to benefit their own life cycles. This review focuses on recent advances in applying both bioinformatic and experimental approaches to predict and validate Ca(2+)-binding proteins and their interactomes in biological systems on a genome-wide scale (termed "calciomics"). Calmodulin is used as an example of Ca(2+)-binding protein (CaBP) to demonstrate the role of CaBPs on the regulation of biological functions. This review is anticipated to rekindle interest in investigating Ca(2+)-binding proteins and Ca(2+)-modulated functions at the systems level in the post-genomic era.


Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Cálcio/metabolismo , Proteômica/métodos , Sequência de Aminoácidos , Animais , Proteínas de Ligação ao Cálcio/química , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Mapas de Interação de Proteínas
18.
Biochemistry ; 51(8): 1586-97, 2012 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-22320333

RESUMO

MauG is a diheme enzyme possessing a five-coordinate high-spin heme with an axial His ligand and a six-coordinate low-spin heme with His-Tyr axial ligation. A Ca(2+) ion is linked to the two hemes via hydrogen bond networks, and the enzyme activity depends on its presence. Removal of Ca(2+) altered the electron paramagnetic resonance (EPR) signals of each ferric heme such that the intensity of the high-spin heme was decreased and the low-spin heme was significantly broadened. Addition of Ca(2+) back to the sample restored the original EPR signals and enzyme activity. The molecular basis for this Ca(2+)-dependent behavior was studied by magnetic resonance and Mössbauer spectroscopy. The results show that in the Ca(2+)-depleted MauG the high-spin heme was converted to a low-spin heme and the original low-spin heme exhibited a change in the relative orientations of its two axial ligands. The properties of these two hemes are each different than those of the heme in native MauG and are now similar to each other. The EPR spectrum of Ca(2+)-free MauG appears to describe one set of low-spin ferric heme signals with a large g(max) and g anisotropy and a greatly altered spin relaxation property. Both EPR and Mössbauer spectroscopic results show that the two hemes are present as unusual highly rhombic low-spin hemes in Ca(2+)-depleted MauG, with a smaller orientation angle between the two axial ligand planes. These findings provide insight into the correlation of enzyme activity with the orientation of axial heme ligands and describe a role for the calcium ion in maintaining this structural orientation that is required for activity.


Assuntos
Cálcio/metabolismo , Heme/química , Hemeproteínas/química , Catálise , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Heme/metabolismo , Hemeproteínas/metabolismo , Ligantes , Modelos Moleculares , Espectroscopia de Mossbauer
19.
J Inorg Biochem ; 107(1): 111-8, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22178673

RESUMO

Magnetic resonance imaging (MRI) has emerged as a leading diagnostic technique in clinical and preclinical settings. However, the application of MRI to assess specific disease markers for diagnosis and monitoring drug effect has been severely hampered by the lack of desired contrast agents with high relaxivities, and optimized in vivo retention time. We have reported the development of protein-based MRI contrast agents (ProCA1) by rational design of Gd(3+) binding sites into a stable protein resulting in significantly increased longitudinal (r(1)) and transverse (r(2)) relaxivities compared to Gd-DTPA. Here, we report a further improvement of protein contrast agents ProCA1 for in vivo imaging by protein modification with various sizes of polyethylene glycol (PEG) chain. PEGylation results in significant increases of both r(1) and r(2) relaxivities (up to 200%), and these high relaxivities persist even at field strengths up to 9.4 T. In addition, our experimental results demonstrate that modified contrast agents have significant improvement of in vivo MR imaging and biocompatibilities including dose efficiency, protein solubility, blood retention time and decreased immunogenicity. Such improvement can be important to the animal imaging and pre-clinical research at high or ultra-high field where there is an urgent need for molecular imaging probes and optimized contrast agent.


Assuntos
Proteínas de Transporte/química , Meios de Contraste/síntese química , Complexos de Coordenação/química , Polietilenoglicóis/química , Animais , Sítios de Ligação , Bioengenharia , Proteínas de Transporte/efeitos adversos , Proteínas de Transporte/farmacocinética , Linhagem Celular Tumoral , Meios de Contraste/efeitos adversos , Meios de Contraste/farmacocinética , Complexos de Coordenação/efeitos adversos , Complexos de Coordenação/farmacocinética , Gadolínio/química , Gadolínio DTPA/química , Humanos , Imagem por Ressonância Magnética , Teste de Materiais , Camundongos , Modelos Moleculares , Polietilenoglicóis/efeitos adversos , Polietilenoglicóis/farmacocinética , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacocinética , Solubilidade
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