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G-protein-coupled receptors (GPCRs) are a target for over 34% of current drugs. The calcium-sensing receptor (CaSR), a family C GPCR, regulates systemic calcium (Ca2+) homeostasis that is critical for many physiological, calciotropical, and noncalciotropical outcomes in multiple organs. However, the mechanisms by which extracellular Ca2+ (Ca2+ex) and the CaSR mediate networks of intracellular Ca2+-signaling and players involved throughout the life cycle of CaSR are largely unknown. Here we report the first CaSR protein–protein interactome with 94 novel putative and 8 previously published interactors using proteomics. Ca2+ex promotes enrichment of 66% of the identified CaSR interactors, pertaining to Ca2+ dynamics, endocytosis, degradation, trafficking, and primarily to protein processing in the endoplasmic reticulum (ER). These enhanced ER-related processes are governed by Ca2+ex-activated CaSR which directly modulates ER-Ca2+ (Ca2+ER), as monitored by a novel ER targeted Ca2+-sensor. Moreover, we validated the Ca2+ex dependent colocalizations and interactions of CaSR with ER-protein processing chaperone, 78-kDa glucose regulated protein (GRP78), and with trafficking-related protein. Live cell imaging results indicated that CaSR and vesicle-associated membrane protein-associated A (VAPA) are inter-dependent during Ca2+ex induced enhancement of near-cell membrane expression. This study significantly extends the repertoire of the CaSR interactome and reveals likely novel players and pathways of CaSR participating in Ca2+ER dynamics, agonist mediated ER-protein processing and surface expression.

Multi-scale calcium (Ca2+ ) dynamics, exhibiting wide-ranging temporal kinetics, constitutes a ubiquitous mode of signal transduction. We report a novel endoplasmic-reticulum (ER)-targeted Ca2+ indicator, R-CatchER, which showed superior kinetics in vitro (koff ≥2×103 s-1 , kon ≥7×106 M-1 s-1 ) and in multiple cell types. R-CatchER captured spatiotemporal ER Ca2+ dynamics in neurons and hotspots at dendritic branchpoints, enabled the first report of ER Ca2+ oscillations mediated by calcium sensing receptors (CaSRs), and revealed ER Ca2+ -based functional cooperativity of CaSR. We elucidate the mechanism of R-CatchER and propose a principle to rationally design genetically encoded Ca2+ indicators with a single Ca2+ -binding site and fast kinetics by tuning rapid fluorescent-protein dynamics and the electrostatic potential around the chromophore. The design principle is supported by the development of G-CatchER2, an upgrade of our previous (G-)CatchER with improved dynamic range. Our work may facilitate protein design, visualizing Ca2+ dynamics, and drug discovery.

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 αvβ3 at a novel site and induces apoptosis in integrin αvβ3-expressing cells. Because both CAPaSC and angiogenic endothelial cells express high levels of integrin αvβ3, we aimed to analyze the effects of ProAgio in PDAC tumor. Methods Expression of integrin αvβ3 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., Graphical abstract

Chronic Liver Diseases (CLD) are characterized by abnormal accumulation of collagen fibrils, neo-angiogenesis, and sinusoidal remodeling. Collagen deposition along with intrahepatic angiogenesis and sinusoidal remodeling alters sinusoid structure resulting in portal hypertension, liver failure, and other complications. Efforts were made to develop treatments for CLDs. However, the success of such treatments is limited and unpredictable. We report a strategy for CLD treatment by induction of integrin αvβ3 mediated cell apoptosis using a rationally designed protein (ProAgio). ProAgio is designed to target integrin αvβ3 at a novel site. Integrin αvβ3 is highly expressed in activated Hepatic Stellate Cells (HSC), angiogenic endothelium, and capillarized Liver Sinusoidal Endothelial Cells (LSEC). ProAgio induces apoptosis of these disease causative cells. Tests with liver fibrosis mouse models demonstrate that ProAgio reverses liver fibrosis and relieves blood flow resistance by depleting activated HSC and capillarized LSEC. Our studies demonstrate an effective approach for CLD treatment., As activated hepatic stellate cells and liver sinusoidal endothelial cells express high levels of integrin α v β3, Turaga et al. present a strategy for treatment of chronic liver disease using their rationally designed protein (ProAgio) which targets integrin α v β3. They find that ProAgio –mediated apoptosis of these disease-associated cells reverses liver fibrosis and ameliorates intrahepatic vascular resistance in a mouse model

Noninvasive detection of early-stage liver metastases from different primary cancers is a pressing unmet medical need. The lack of both molecular biomarkers and the sensitive imaging methodology makes the detection challenging. In this study, we observed the elevated expression of chemokine receptor 4 (CXCR4) in uveal melanoma (UM) patient liver tissues, and high CXCR4 expression in liver metastases of UM murine models, regardless of the expression levels in the primary tumors. Based on these findings, we identified CXCR4 as an imaging biomarker and exploited a CXCR4-targeted MRI contrast agent ProCA32.CXCR4 for molecular MRI imaging. ProCA32.CXCR4 has strong CXCR4 binding affinity, high metal selectivity, and r1 and r2 relaxivities, which enables the sensitive detection of liver micrometastases. The MRI imaging capacity for detecting liver metastases was demonstrated in three UM models and one ovarian cancer model. The imaging results were validated by histological and immunohistochemical analysis. ProCA32.CXCR4 has strong potential clinical application for non-invasive diagnosis of liver metastases.

Sharma et al. present a treatment strategy by simultaneously depleting breast cancer CAFs and tumor angiogenic vessels using a rationally designed protein that induces integrin αvβ3 target-specific cell apoptosis. This offers a unique opportunity for TNBC treatment, especially for patients with dense tumor stroma., Fibrotic tumor stroma plays an important role in facilitating triple-negative breast cancer (TNBC) progression and chemotherapeutic resistance. We previously reported a rationally designed protein (ProAgio) that targets integrin αvβ3 at a novel site. ProAgio induces apoptosis via the integrin. Cancer-associated fibroblasts (CAFs) and angiogenic endothelial cells (aECs) in TNBC tumor express high levels of integrin αvβ3. ProAgio effectively induces apoptosis in CAFs and aECs. The depletion of CAFs by ProAgio reduces intratumoral collagen and decreases growth factors released from CAFs in the tumor, resulting in decreased cancer cell proliferation and apoptotic resistance. ProAgio also eliminates leaky tumor angiogenic vessels, which consequently reduces tumor hypoxia and improves drug delivery. The depletion of CAFs and reduction in hypoxia by ProAgio decreases lysyl oxidase (LOX) secretion, which may play a role in the reduction of metastasis. ProAgio stand-alone or in combination with a chemotherapeutic agent provides survival benefit in TNBC murine models, highlighting the therapeutic potential of ProAgio as a treatment strategy., Graphical Abstract

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.

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.

Design a protein-based contrast agent for detecting liver metastases with magnetic resonance imaging., 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 (r1 = 30.9 mM−1 s−1, r2 = 43.2 mM−1 s−1, 1.5 T; r1 = 23.5 mM−1 s−1, r2 = 98.6 mM−1 s−1, 7.0 T), strong CXCR4 binding (Kd = 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.

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 Gd(3+) 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.

Precision molecular imaging addresses major medical gaps including the early detection of small lesion and biomarker expression, especially for high-risk patients, and monitoring the dynamic changes of biomarkers during disease progression and upon therapeutic treatment. One of the major challenges in achieving precision medicine is developing our capabilities to select patients with genetic and phenotypic heterogeneity while monitoring treatment responses. Current golden standard of histological analysis coupled with biopsy has many limitations, such as invasiveness, significant sampling errors especially for small lesions and risks of tumor peritoneal seeding [1]. Important avenues to achieve these goals include identifying biomarkers whose expression are capable of accurately reflect disease stages and treatment effects, and developing sensitive and noninvasive precision molecular imaging methodology for early responsive, predictive and prognostic biomarkers with increased levels of spatial and temporal resolution [2]. Here, we will first discuss GRPR as an attractive biomarker for molecular imaging of various types of cancers. We will then report our development of protein-based MRI contrast agent against GRPR with significantly improved sensitivity and spatial resolution. GRPR is a G-protein coupled receptor belonging to the bombesin receptor family [3]. The nature ligand for GRPR is gastrin releasing peptide (GRP). The C-terminal peptide sequence of GRP is highly similar to its amphibian counterpart, bombesin, a 14 amino acid peptide with high affinity to both GRPR and neuromedin B receptor. GRP and bombesin have strong affinity to GRPR at nanomolar range. The binding of these peptides to GRPR triggers downstream signaling cascades and activates a series of physiological and biological events, such as hormone release in endocrine organs [4]. GRPR is an attractive biomarker expressed on a series of human malignancies including prostate cancer, lung cancer, breast cancer, colon cancer, ovarian cancer, pancreatic cancer, gastrointestinal carcinoid tumor, head and neck cancer, various CNS/neural tumors, renal cell cancers and uterine cancer. As investigated by PCR, immunohistochemistry and radionuclide binding assay of clinic samples, GRPR is expressed in almost 100% on the prostate tumors. The expression level of GRPR in prostate cancer is significantly higher than that of other normal tissues. In addition, 33–72% of breast cancer, 40–50% of gastric cancer, 85% of carcinoids cancer and 29–85% of small cell lung cancer also have high GRPR expression [5]. GRPR is highly expressed in prostatic intraepithelial neoplasias, primary prostate cancer and invasive prostate carcinomas, whereas the expression level in normal prostate tissue and benign prostate hyperplasia are relatively low [6,7]. The distribution of GRPR in prostate cancer is heterogeProCA1.GRPR: a new imaging agent in cancer detection

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.

Metal ions play crucial roles in numerous biological processes, facilitating biochemical reactions by binding to various proteins. An increasing body of evidence suggests that neurotoxicity associated with exposure to nonessential metals (e.g., Pb(2+)) involves disruption of synaptic activity, and these observed effects are associated with the ability of Pb(2+) to interfere with Zn(2+) and Ca(2+)-dependent functions. However, the molecular mechanism behind Pb(2+) toxicity remains a topic of debate. In this review, we first discuss potential neuronal Ca(2+) binding protein (CaBP) targets for Pb(2+) such as calmodulin (CaM), synaptotagmin, neuronal calcium sensor-1 (NCS-1), N-methyl-d-aspartate receptor (NMDAR) and family C of G-protein coupled receptors (cGPCRs), and their involvement in Ca(2+)-signalling pathways. We then compare metal binding properties between Ca(2+) and Pb(2+) to understand the structural implications of Pb(2+) binding to CaBPs. Statistical and biophysical studies (e.g., NMR and fluorescence spectroscopy) of Pb(2+) binding are discussed to investigate the molecular mechanism behind Pb(2+) toxicity. These studies identify an opportunistic, allosteric binding of Pb(2+) to CaM, which is distinct from ionic displacement. Together, these data suggest three potential modes of Pb(2+) activity related to molecular and/or neural toxicity: (i) Pb(2+) can occupy Ca(2+)-binding sites, inhibiting the activity of the protein by structural modulation, (ii) Pb(2+) can mimic Ca(2+) in the binding sites, falsely activating the protein and perturbing downstream activities, or (iii) Pb(2+) can bind outside of the Ca(2+)-binding sites, resulting in the allosteric modulation of the protein activity. Moreover, the data further suggest that even low concentrations of Pb(2+) can interfere at multiple points within the neuronal Ca(2+) signalling pathways to cause neurotoxicity.

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.

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 β1 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. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s13311-018-0635-2) contains supplementary material, which is available to authorized users.

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 Ca(2+)/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.

Fast calcium signaling is regulated by numerous calcium channels exhibiting high spatiotemporal profiles which are currently measured by fluorescent calcium sensors. There is still a strong need to improve the kinetics of genetically encoded calcium indicators (sensors) to capture calcium dynamics in the millisecond time frame. In this review, we summarize several major fast calcium signaling pathways and discuss the recent developments and application of genetically encoded calcium indicators to detect these pathways. A new class of genetically encoded calcium indicators designed with site-directed mutagenesis on the surface of beta-barrel fluorescent proteins to form a pentagonal bipyramidal-like calcium binding domain dramatically accelerates calcium binding kinetics. Furthermore, novel genetically encoded calcium indicators with significantly increased fluorescent lifetime change are advantageous in deep-field imaging with high light-scattering and notable morphology change.

Significance 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 and robust imaging methodologies. We have developed a protein MRI contrast agent (ProCA32) by engineering high-affinity Gd 3+ -binding pockets in rat and human α-parvalbumin. ProCA32 can function as both a T 1 - and T 2 -weighted contrast agent, which enables noninvasive detection of early-stage micrometastatic liver tumors with sizes as small as 0.24 mm using T 1 - and T 2 -weighted or T 2 /T 1 ratio MRI. Our protein-based MRI contrast agents and imaging methodology are expected to provide robust results for the early detection of liver cancer as well as other liver diseases.

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.

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.

It is long known that pyruvate kinase isoform M2 (PKM2) is released into the circulation of cancer patients. The PKM2 levels in patients have been suggested as a diagnostic marker for many types of cancers. However, it is not known how PKM2 is released in the blood, and whether the circulating PKM2 has any physiological function(s) in tumor progression. In this report, we demonstrate that PKM2 in the blood facilitates tumor growth by promoting tumor angiogenesis. Our experiments show that PKM2 promotes tumor angiogenesis by increasing endothelial cell proliferation, migration, and cell-ECM adhesion. Only the dimeric PKM2 possess the activity in promoting tumor angiogenesis, which is consistent with the observations that PKM2 in circulation of cancer patients is a dimer form.

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., This paper reports the finding of a new class of Ca2+-binding domain of intracellular Ca2+ channels from muscle cells. This domain provides channels with distinctive properties that result in channel-specific modulation of the intracellular resting Ca2+ concentration.

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.

Intracellular Ca2+ activated calmodulin (CaM) inhibits gap junction channels in the low nanomolar to high micromolar range of [Ca2+]i. This regulation plays an essential role in numerous cellular processes that include hearing, lens transparency, and synchronized contractions of the heart. Previous studies have indicated that gap junction mediated cell-to-cell communication was inhibited by CaM antagonists. More recent evidence indicates a direct role of CaM in regulating several members of the connexin family. Since the intracellular loop and carboxyl termini of connexins are largely “invisible” in electron microscopy and X-ray crystallographic structures due to disorder in these domains, peptide models encompassing the putative CaM binding sites of several intracellular domains of connexins have been used to identify the Ca2+-dependent CaM binding sites of these proteins. This approach has been used to determine the CaM binding affinities of peptides derived from a number of different connexin-subfamilies.

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 ± 0.25 and 100.0 ± 0.32 mM−1 s−1 at 1.4 T, respectively, and 42.6 ± 1.0 and 217 ± 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.

Integrin αvβ3 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 αvβ3 outside the classical ligand-binding site. We show ProAgio induces apoptosis of integrin αvβ3-expressing cells by recruiting and activating caspase 8 to the cytoplasmic domain of integrin αvβ3. 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., Integrins are transmembrane proteins that have important roles in cell adhesion and signalling. Here the authors design a therapeutic protein that binds integrin αvβ3, has anti-angiogenic activity, and reduces tumour growth in xenograft models, while being seemingly well tolerated.

1 Department of Integrative Biology and Pharmacology, McGovern Medical School, The University of Texas Health Science Center at Houston, Houston, Texas 77030, USA; 2 Department of Gastroenterology, Affiliated Hospital, Zunyi Medical College, and Digestive Disease Institute of Guizhou Province, Zunyi 563003, China; 3 Department of Chemistry, Center for Diagnostics and Therapeutics, Georgia State University, Atlanta, Georgia 30303, USA

Calmodulin (CaM) binding sites were recently identified on the cytoplasmic loop (CL) of at least three α-subfamily connexins (Cx43, Cx44, Cx50), while Cx40 does not have this putative CaM binding domain. The purpose of this study was to examine the functional relevance of the putative Cx43 CaM binding site on the Ca2+-dependent regulation of gap junction proteins formed by Cx43 and Cx40. Dual whole cell patch-clamp experiments were performed on stable murine Neuro-2a cells expressing Cx43 or Cx40. Addition of ionomycin to increase external Ca2+influx reduced Cx43 gap junction conductance (Gj) by 95%, while increasing cytosolic Ca2+concentration threefold. By contrast, Cx40 Gjdeclined by 2+-induced decline in Cx43 Gjwas prevented by pretreatment with calmidazolium or reversed by the addition of 10 mM EGTA to Ca2+-free extracellular solution, if Ca2+chelation was commenced before complete uncoupling, after which gjwas only 60% recoverable. The Cx43 CL136–158mimetic peptide, but not the scrambled control peptide, or Ca2+/CaM-dependent kinase II 290–309 inhibitory peptide also prevented the Ca2+/CaM-dependent decline of Cx43 Gj. Cx43 gap junction channel open probability decreased to zero without reductions in the current amplitudes during external Ca2+/ionomycin perfusion. We conclude that Cx43 gap junctions are gated closed by a Ca2+/CaM-dependent mechanism involving the carboxyl-terminal quarter of the connexin CL domain. This study provides the first evidence of intrinsic differences in the Ca2+regulatory properties of Cx43 and Cx40.

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.

Contrasting information suggests either almost complete depletion of sarcoplasmic reticulum (SR) Ca(2+) or significant residual Ca(2+) concentration after prolonged depolarization of the skeletal muscle fiber. The primary obstacle to resolving this controversy is the lack of genetically encoded Ca(2+) indicators targeted to the SR that exhibit low-Ca(2+) affinity, a fast biosensor: Ca(2+) off-rate reaction, and can be expressed in myofibers from adult and older adult mammalian species. This work used the recently designed low-affinity Ca(2+) sensor (Kd = 1.66 mM in the myofiber) CatchER (calcium sensor for detecting high concentrations in the ER) targeted to the SR, to investigate whether prolonged skeletal muscle fiber depolarization significantly alters residual SR Ca(2+) with aging. We found CatchER a proper tool to investigate SR Ca(2+) depletion in young adult and older adult mice, consistently tracking SR luminal Ca(2+) release in response to brief and repetitive stimulation. We evoked SR Ca(2+) release in whole-cell voltage-clamped flexor digitorum brevis muscle fibers from young and old FVB mice and tested the maximal SR Ca(2+) release by directly activating the ryanodine receptor (RyR1) with 4-chloro-m-cresol in the same myofibers. Here, we report for the first time that the Ca(2+) remaining in the SR after prolonged depolarization (2 s) in myofibers from aging (~220 μM) was larger than young (~132 μM) mice. These experiments indicate that SR Ca(2+) is far from fully depleted under physiological conditions throughout life, and support the concept of excitation-contraction uncoupling in functional senescent myofibers.

Cx50 (connexin50), a member of the α-family of gap junction proteins expressed in the lens of the eye, has been shown to be essential for normal lens development. In the present study, we identified a CaMBD [CaM (calmodulin)-binding domain] (residues 141–166) in the intracellular loop of Cx50. Elevations in intracellular Ca2+ concentration effected a 95% decline in gj (junctional conductance) of Cx50 in N2a cells that is likely to be mediated by CaM, because inclusion of the CaM inhibitor calmidazolium prevented this Ca2+-dependent decrease in gj. The direct involvement of the Cx50 CaMBD in this Ca2+/CaM-dependent regulation was demonstrated further by the inclusion of a synthetic peptide encompassing the CaMBD in both whole-cell patch pipettes, which effectively prevented the intracellular Ca2+-dependent decline in gj. Biophysical studies using NMR and fluorescence spectroscopy reveal further that the peptide stoichiometrically binds to Ca2+/CaM with an affinity of ~5 nM. The binding of the peptide expanded the Ca2+-sensing range of CaM by increasing the Ca2+ affinity of the C-lobe of CaM, while decreasing the Ca2+ affinity of the N-lobe of CaM. Overall, these results demonstrate that the binding of Ca2+/CaM to the intracellular loop of Cx50 is critical for mediating the Ca2+-dependent inhibition of Cx50 gap junctions in the lens of the eye.

The purpose of this study was to demonstrate a novel protein-based magnetic resonance imaging (MRI) contrast agent that has the capability of targeting prostate cancer and which provides high-sensitivity MR imaging in tumor cells and mouse models.A fragment of gastrin-releasing peptide (GRP) was fused into a protein-based MRI contrast agent (ProCA1) at different regions. MR imaging was obtained in both tumor cells (PC3 and H441) and a tumor mouse model administrated with ProCA1.GRP.PC3 and DU145 cells treated with ProCA1.GRPs exhibited enhanced signal in MRI. Intratumoral injection of ProCA1.GRP in a PC3 tumor model displayed enhanced MRI signal. The contrast agent was retained in the PC3 tumor up to 48 h post-injection.Protein-based MRI contrast agent with tumor targeting modality can specifically target GRPR-positive prostate cancer. Intratumoral injection of the ProCA1 agent in the prostate cancer mouse model verified the targeting capability of ProCA1.GRP and showed a prolonged retention time in tumors.

The rubella virus (RUBV) nonstructural (NS) protease domain, a Ca(2+)- and Zn(2+)-binding papain-like cysteine protease domain within the nonstructural replicase polyprotein precursor, is responsible for the self-cleavage of the precursor into two mature products, P150 and P90, that compose the replication complex that mediates viral RNA replication; the NS protease resides at the C terminus of P150. Here we report the Ca(2+)-dependent, stoichiometric association of calmodulin (CaM) with the RUBV NS protease. Co-immunoprecipitation and pulldown assays coupled with site-directed mutagenesis demonstrated that both the P150 protein and a 110-residue minidomain within NS protease interacted directly with Ca(2+)/CaM. The specific interaction was mapped to a putative CaM-binding domain. A 32-mer peptide (residues 1152-1183, denoted as RUBpep) containing the putative CaM-binding domain was used to investigate the association of RUBV NS protease with CaM or its N- and C-terminal subdomains. We found that RUBpep bound to Ca(2+)/CaM with a dissociation constant of 100-300 nm. The C-terminal subdomain of CaM preferentially bound to RUBpep with an affinity 12.5-fold stronger than the N-terminal subdomain. Fluorescence, circular dichroism and NMR spectroscopic studies revealed a "wrapping around" mode of interaction between RUBpep and Ca(2+)/CaM with substantially more helical structure in RUBpep and a global structural change in CaM upon complex formation. Using a site-directed mutagenesis approach, we further demonstrated that association of CaM with the CaM-binding domain in the RUBV NS protease was necessary for NS protease activity and infectivity.

P68 RNA helicase is a prototypical DEAD box RNA helicase. The protein plays a very important role in early organ development and maturation. In consistence with the function of the protein in transcriptional regulation and pre-mRNA splicing, p68 was found to predominately localize in the cell nucleus. However, recent experiments demonstrate a transient cytoplasmic localization of the protein. We report here that p68 shuttles between the nucleus and the cytoplasm. The nucleocytoplasmic shuttling of p68 is mediated by two nuclear localization signal (NLS) and two nuclear exporting signal (NES) sequence elements. Our experiments reveal that p68 shuttles via a classical RanGTPase dependent pathway.

Acute pancreatitis is a serious and potentially fatal disease caused by intracellular trypsinogen activation. Although protease detection has been greatly facilitated by the development of protease probes capable of monitoring protease activation and inhibition, real-time quantitative measurement of protease activity in living cells remains a challenge, and the identification of the cellular compartment for trypsinogen activation is inconclusive. Here we report a novel strategy for developing trypsin sensors by grafting an enzymatic cleavage site into a sensitive location for optical change of chromophore in a single enhanced green fluorescent protein (EGFP). Our designed trypsin sensor exhibits rapid kinetic responses for protease activation and inhibition with a large ratiometric optical signal change. In addition, it has strong specificity, as enzymatic cleavage is not observed with other proteases such as thrombin, cathepsin B, tryptase, and tissue plasminogen activator. Moreover, the developed trypsin sensor allows us for the first time to observe, in real time, trypsinogen activation by caerulein in the pancreatic cancer cell line, MIA PaCa-2 without zymogen granules. These developed protease sensors will facilitate improved understanding of mechanisms and locations of protease activation and further provide screening of protease inhibitors with therapeutic effects.

We describe the rational design of a novel class of magnetic resonance imaging contrast agents with engineered proteins (CAi.CD2, i = 1, 2, …, 9) chelated with gadolinium. The design of protein-based contrast agents involves creating high coordination Gd3+ binding sites in a stable host protein using amino acid residues and water molecules as metal coordinating ligands. Designed proteins show strong selectivity for Gd3+ over physiological metal ions such as Ca2+, Zn2+, and Mg2+. These agents exhibit a 20-fold increase in longitudinal and transverse relaxation rate values over the conventional small molecule contrast agents, e.g., Gd-DTPA (diethylene triamine pentaacetic acid), used clinically. Furthermore, they exhibit much stronger contrast enhancement and much longer blood retention time than Gd-DTPA in mice. With good biocompatibility and potential functionalities, these protein contrast agents may be used as molecular imaging probes to target disease markers, extending applications of magnetic resonance imaging (MRI).

Ca2+ regulates numerous biological processes through spatiotemporal changes in the cytosolic Ca2+ concentration and subsequent interactions with Ca2+ binding proteins. The endoplasmic reticulum (ER) serves as an intracellular Ca2+ store and plays an essential role in cytosolic Ca2+ homeostasis. There is a strong need to develop Ca2+ sensors capable of real-time quantitative Ca2+ concentration measurements in specific subcellular environments without using natural Ca2+ binding proteins such as calmodulin, which themselves participate as signaling molecules in cells. In this report, a strategy for creating such sensors by grafting a Ca2+-binding motif into chromophore sensitive locations in green fluorescence protein is described. The engineered Ca2+ sensors exhibit large ratiometric fluorescence and absorbance changes upon Ca2+ binding with affinities corresponding to the Ca2+ concentrations found in the ER (Kd values range from 0.4 to 2 mM). In addition to characterizing the optical and metal binding properties of the newly developed Ca2+ sensors with various spectroscopic methods, we also examined the kinetic properties using stopped-flow spectrofluorimetry to ensure accurate monitoring of dynamic Ca2+ changes. The developed Ca2+ sensor was successfully targeted to the ER of mammalian cell lines to monitor Ca2+ changes occurring in this compartment in response to stimulation with agonists. We envision that this class of Ca2+ sensors can be modified further to measure the Ca2+ concentration in other cellular compartments, providing tools for studying the contribution of these compartments to cellular Ca2+ signaling.

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.

Ca2+ binding is essential for the biological functions of calmodulin (CaM) as a trigger/sensor protein to regulate many biological processes in the Ca2+ -signaling cascade. A challenge in understanding the mechanism of Ca2+ signaling is to obtain site-specific information about the Ca2+ binding properties of individual Ca2+ -binding sites of EF-hand proteins, especially for CaM. In this paper, we report the first estimation of the intrinsic Ca2+ affinities of the four EF-hand loops of calmoduin (I-IV) by individually grafting into the domain 1 of CD2. Taking advantage of the Trp residues in the host protein, we first determined metal-binding affinities for Tb3+, Ca2+, and La3+ for all four grafted EF-loops using Tb3+ aromatic resonance energy transfer. EF-loop I exhibits the strongest binding affinity for Ca2+, La3+, and Tb3+, while EF-loop IV has the weakest metal-binding affinity. EF-loops I-IV of CaM have dissociation constants for Ca2+ of 34, 245, 185, and 814 microM, respectively, with the order I > III approximately equal to II > IV. These findings support a charge-ligand-balanced model in which both the number of negatively charged ligand residues and the balanced electrostatic dentate-dentate repulsion by the adjacent charged residues are two major determinants for the relative Ca2+ -binding affinities of EF-loops in CaM. Our grafting method provides a new strategy to obtain site-specific Ca2+ binding properties and a better estimation of the cooperativity and conformational change contributions of coupled EF-hand proteins.

Ca 2+ , a signal of life and death, controls numerous cellular processes through interactions with proteins. An effective approach to understanding the role of Ca 2+ is the design of a Ca 2+ -binding protein with predicted structural and functional properties. To design de novo Ca 2+ -binding sites in proteins is challenging due to the high coordination numbers and the incorporation of charged ligand residues, in addition to Ca 2+ -induced conformational change. Here, we demonstrate the successful design of a Ca 2+ -binding site in the non-Ca 2+ -binding cell adhesion protein CD2. This designed protein, Ca.CD2, exhibits selectivity for Ca 2+ versus other di- and monovalent cations. In addition, La 3+ (K d 5.0 μM) and Tb 3+ (K d 6.6 μM) bind to the designed protein somewhat more tightly than does Ca 2+ (K d 1.4 mM). More interestingly, Ca.CD2 retains the native ability to associate with the natural target molecule. The solution structure reveals that Ca.CD2 binds Ca 2+ at the intended site with the designed arrangement, which validates our general strategy for designing de novo Ca 2+ -binding proteins. The structural information also provides a close view of structural determinants that are necessary for a functional protein to accommodate the metal-binding site. This first success in designing Ca 2+ -binding proteins with desired structural and functional properties opens a new avenue in unveiling key determinants to Ca 2+ binding, the mechanism of Ca 2+ signaling, and Ca 2+ -dependent cell adhesion, while avoiding the complexities of the global conformational changes and cooperativity in natural Ca 2+ -binding proteins. It also represents a major achievement toward designing functional proteins controlled by Ca 2+ binding.

Assigning proteins with functions based on the 3-D structure requires high-speed techniques to make a systematic survey of protein structures. Calcium regulates many biological systems by binding numerous proteins in different biological environments. Despite the great diversity in the composition of ligand residues and bond angles and lengths of calcium-binding sites, our structural analysis of 11 calcium-binding sites in different classes of proteins has shown that common local structural parameters can be used to identify and design calcium-binding proteins. Natural calcium-binding sites in both EF-hand proteins and non-EF-hand proteins can be described with the smallest deviation from the geometry of an ideal pentagonal bipyramid. Further, two different magnesium-binding sites in parvalbumin and calbindin(D9K) can also be identified using an octahedral geometry. Using the established method, we have designed de novo calcium-binding sites into the scaffold of non-calcium-binding proteins CD2 and Rop. Our results suggest that it is possible to identify calcium- and magnesium-binding sites in proteins and design de novo metal-binding sites.

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.

Cluster of differentiation 2 (CD2) is a cell surface glycoprotein expressed on most human T cells and natural killer (NK) cells and plays an important role in mediating cell adhesion in both T-lymphocytes and in signal transduction. The understanding of the biochemical basis of molecular recognition by the cell adhesion molecule CD2 has been advanced greatly through the determination of structures and the dynamic properties of the complexes and their individual components and through site-directed mutagenesis. A number of general principles can be derived from the structural and functional studies of the extracellular domains of CD2 and CD58 and their complex. Significant electrostatic interactions within the protein-protein interfaces contribute directly to the formation of macromolecular complexes of CD2 and CD58. Also, residues located on the protein-protein interface demonstrate a certain degree of conformational change upon the formation of a complex. Structural analysis of CD2 has revealed that this adhesion molecule exhibits strong conformational flexibility with a partial non-native helical conformation at high temperatures and in the presence of an organic solvent. In addition, it can be converted into a domain swapped dimer, or trimer and tetramer through hinge deletion. Thus, the conformational status of the adhesive proteins contributes to the regulation of cell adhesion and the folding of CD2.

The rubella virus (RUB) nonstructural (NS) protein (NSP) ORF encodes a protease that cleaves the NSP precursor (240 kDa) at a single site to produce two products. A cleavage site mutation was introduced into a RUB infectious cDNA clone and found to be lethal, demonstrating that cleavage of the NSP precursor is necessary for RUB replication. Based on computer alignments, the RUB NS protease was predicted to be a papain-like cysteine protease (PCP) with the residues Cys1152 and His1273 as the catalytic dyad; however, the RUB NS protease was recently found to require divalent cations such as Zn, Co, and Cd for activity (X. Liu, S. L. Ropp, R. J. Jackson, and T. K. Frey, J. Virol. 72:4463–4466, 1998). To analyze the function of metal cation binding in protease activity, Zn binding studies were performed using the minimal NS protease domain within the NSP ORF. When expressed as a maltose binding protein (MBP) fusion protein by bacteria, the NS protease exhibited activity both in the bacteria and in vitro following purification when denatured and refolded in the presence of Zn. Atomic absorption analysis detected 1.6 mol of Zn bound per mol of protein refolded in this manner. Expression of individual domains within the protease as MBP fusions and analysis by a Zn 65 binding assay revealed two Zn binding domains: one located at a predicted metal binding motif beginning at Cys1175 and the other one close to the cleavage site. Mutagenesis studies showed that Cys1175 and Cys1178 in the first domain and Cys1227 and His1273, the His in the predicted catalytic site, in the second domain are essential for zinc binding. All of these residues are also necessary for the protease activity, as were several other Cys residues not involved in Zn binding. Far-UV circular dichroism (CD) analysis of the MBP-NS protease fusion protein showed that the protease domain contained a large amount of alpha-helical structure, which is consistent with the results of secondary-structural prediction. Both far-UV–CD and fluorescence studies suggested that Zn did not exert a major effect on the overall structure of the fusion protein. Finally, protease inhibitor assays found that the protease activity can be blocked by both metal ion chelators and the metalloprotease inhibitor captopril. In conjunction with the finding that the previously predicted catalytic site, His1273, is essential for zinc binding, this suggests that the RUB NS protease is actually a novel virus metalloprotease rather than a PCP.

Cell adhesion molecule, CD2, from the immunoglobulin superfamily, is comprised of antibodies and Ig-like domains and plays a fundamental role, not only in the immune system, but also in the interactions between cells, specifically in cell-cell adhesion. This study examines the N-terminal domain 1 of CD2 (CD2-1) at different pHs, and in 2,2,2-trifluoroethanol (TFE), using nears- and far-UV circular dichroism (CD), fluorescence, and 1H nuclear magnetic resonance to elucidate factors contributing to the Ig beta-structure. Contrary to the complete unfolding induced by guanidinehydrochloride, CD2-1 retains its native tertiary structure at pHs from 1.0 to 10.0. Like the effects of high temperatures that have previously been observed, TFE reduces the integrity of the tertiary structure, while reorganizing the secondary structure from a native all-beta-sheet to a significantly alpha-helical conformation. The induced helicity of CD2-1 correlates with the helicity inherent in its primary sequence. Our results suggest that electrostatic interactions are less important for the formation of the native secondary and tertiary structure of CD2-1, although they are crucial for CD2's adhesion function. Interference with the protein's hydrophobic interactions and hydrogen-bonding networks, however, causes significant changes in its conformation. Residues of CD2-1, with high conformational flexibility, may contribute for the formation of a metastable dimer by domain-swapping.

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.

Four peptides encompassing the entire amino acid sequence of hen lysozyme were examined in aqueous solution and in 50% (v/v) 2,2,2-trifluoroethanol (TFE) by far-UV CD. Two peptides, 1-40 and 84-129, correspond to regions which are helical in the native protein, and together represent the alpha-domain. The beta-domain of the native enzyme was also synthesized as two peptides, one (41-60) containing the residues in the triple stranded antiparallel beta-sheet and the other (61-82) corresponding to a region lacking regular secondary structure. In water at pH 2.0 and 25 degrees C, the monomeric peptides 1-40, 41-60 and 61-82 appear to be predominantly unstructured. By contrast, the peptide 84-129 has considerable, presumably helical structure, corresponding to approximately 19%, or nine residues, on average, which can be unfolded by the addition of 8 M urea or 6 M guanidine hydrochloride. In 50% TFE the conformational properties of the four peptides are again distinct. Although little helical structure is induced in the peptides 41-60 and 61-82, and a native-like extent of helical structure is induced in the peptide 1-40, the peptide 84-29 converts almost entirely to helical structure in 50% TFE. The far-UV CD spectrum of a stoichiometric mixture of the four peptides in water resembles closely that of a denatured state of the intact protein formed by reductive methylation of its four disulphide bonds, but differs significantly from that of the native protein. The far-UV CD spectrum of the peptide mixture in TFE is indistinguishable from that of the intact protein in this solvent, both in the presence and in the absence of its four disulphide bonds. The conformational preferences of the peptides are not predicted using standard assessments of helical propensity or hydrophobicity, but correlate instead with the number of local contacts made in the native protein. On the basis of these results, we suggest that the region 84-129 could play an important role in determining the nature of the early folding events in the folding pathway of the intact polypeptide chain.

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.

p68 RNA helicase is a prototypical RNA helicase. Here we present evidence to show that, by interacting with Ca-calmodulin, p68 has a role in cancer metastasis and cell migration. A peptide fragment that spans the IQ motif of p68 strongly inhibits cancer metastasis in two different animal models. The peptide interrupts p68 and Ca-calmodulin interaction and inhibits cell migration. Our results demonstrate that the p68-Ca-calmodulin interaction is essential for the formation of lamellipodia and filopodia in migrating cells. p68 interacts with microtubules in the presence of Ca-calmodulin. Our experiments show that interaction with microtubules stimulates p68 ATPase activity. Further, microtubule gliding assays demonstrate that p68, in the presence of Ca-calmodulin, can function as a microtubule motor. This motor activity may allow p68 to transport Ca-calmodulin to the leading edge of migrating cells.

Identifying Ca(2+) -binding sites in proteins is the first step toward understanding the molecular basis of diseases related to Ca(2+) -binding proteins. Currently, these sites are identified in structures either through X-ray crystallography or NMR analysis. However, Ca(2+) -binding sites are not always visible in X-ray structures due to flexibility in the binding region or low occupancy in a Ca(2+) -binding site. Similarly, both Ca(2+) and its ligand oxygens are not directly observed in NMR structures. To improve our ability to predict Ca(2+) -binding sites in both X-ray and NMR structures, we report a new graph theory algorithm (MUG(C) ) to predict Ca(2+) -binding sites. Using carbon atoms covalently bonded to the chelating oxygen atoms, and without explicit reference to side-chain oxygen ligand co-ordinates, MUG(C) is able to achieve 94% sensitivity with 76% selectivity on a dataset of X-ray structures composed of 43 Ca(2+) -binding proteins. Additionally, prediction of Ca(2+) -binding sites in NMR structures was obtained by MUG(C) using a different set of parameters, which were determined by the analysis of both Ca(2+) -constrained and unconstrained Ca(2+) -loaded structures derived from NMR data. MUG(C) identified 20 of 21 Ca(2+) -binding sites in NMR structures inferred without the use of Ca(2+) constraints. MUG(C) predictions are also highly selective for Ca(2+) -binding sites as analyses of binding sites for Mg(2+) , Zn(2+) , and Pb(2+) were not identified as Ca(2+) -binding sites. These results indicate that the geometric arrangement of the second-shell carbon cluster is sufficient not only for accurate identification of Ca(2+) -binding sites in NMR and X-ray structures but also for selective differentiation between Ca(2+) and other relevant divalent cations.