Protein MRI Contrast Agents as an Effective Approach for Precision Molecular Imaging.

ABSTRACT: Cancer and other acute and chronic diseases are results of perturbations of common molecular determinants in key biological and signaling processes. Imaging is critical for characterizing dynamic changes in tumors and metastases, the tumor microenvironment, tumor-stroma interactions, and drug targets, at multiscale levels. Magnetic resonance imaging (MRI) has emerged to be a primary imaging modality for both clinical and preclinical applications due to its advantages over other modalities, including sensitivity to soft tissues, nondepth limitations, and the use of nonionizing radiation. However, extending the application of MRI to achieve both qualitative and quantitative precise molecular imaging with the capability to quantify molecular biomarkers for early detection, staging, and monitoring therapeutic treatment requires the capacity to overcome several major challenges including the trade-off between metal-binding affinity and relaxivity, which is an issue frequently associated with small chelator contrast agents. In this review, we will introduce the criteria of ideal contrast agents for precision molecular imaging and discuss the relaxivity of current contrast agents with defined first shell coordination water molecules. We will then report our advances in creating a new class of protein-targeted MRI contrast agents (ProCAs) with contributions to relaxivity largely derived from the secondary sphere and correlation time. We will summarize our rationale, design strategy, and approaches to the development and optimization of our pioneering ProCAs with desired high relaxivity, metal stability, and molecular biomarker-targeting capability, for precision MRI. From first generation (ProCA1) to third generation (ProCA32), we have achieved dual high r1 and r2 values that are 6- to 10-fold higher than clinically approved contrast agents at magnetic fields of 1.5 T, and their relaxivity values at high field are also significantly higher, which enables high resolution during small animal imaging. Further engineering of multiple targeting moieties enables ProCA32 agents that have strong biomarker-binding affinity and specificity for an array of key molecular biomarkers associated with various chronic diseases, while maintaining relaxation and exceptional metal-binding and selectivity, serum stability, and resistance to transmetallation, which are critical in mitigating risks associated with metal toxicity. Our leading product ProCA32.collagen has enabled the first early detection of liver metastasis from multiple cancers at early stages by mapping the tumor environment and early stage of fibrosis from liver and lung in vivo, with strong translational potential to extend to precision MRI for preclinical and clinical applications for precision diagnosis and treatment.

Irisin, an exercise-induced bioactive peptide beneficial for health promotion during aging process.

Irisin is an exercise-induced myokine expressed as a bioactive peptide in multiple tissues and organs, and exercise and cold exposure are the major inducers for its secretion. Irisin presents a decreasing trend with the extension of age and is also closely associated with a wide range of aging-related diseases. Currently, many studies on irisin are being conducted with respect to physiological functions for health promotion, and the prevention, treatment and rehabilitation of chronic diseases, as well as mechanisms associated with improving energy metabolic balance, enhancing cellular homeostasis by optimizing autophagy, promoting mitochondrial quality control, reducing reactive oxygen species (ROS) production, and mitigating inflammatory responses. These diseases include: metabolic diseases (obesity, type 2 diabetes, and bone metabolism); cardiovascular diseases (hypertension, coronary heart disease, cardiomyopathy and stroke); nervous system diseases (Alzheimer's disease, Parkinson's disease, and stroke); and others (cancer and sarcopenia). Although the current studies on irisin are relatively extensive, some studies have produced unexplained experimental results. This article introduces an overview of the generation, secretion, and tissue distribution, of irisin, and its targeting of tissues or organs for the prevention and treatment of above-mentioned chronic diseases is systematically summarized, with discussion of the underlying molecular mechanisms. This study is expected to improve the understanding of irisin, which may be beneficial to identify novel and effective targets for the screening, diagnosis, or therapy of these chronic diseases, or develop promising interventional strategies, effective drug candidates, functional foods, or exercise mimetics.

Design of Calcium-Binding Proteins to Sense Calcium.

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.

Structural Mechanism of Cooperative Regulation of Calcium-Sensing Receptor-Mediated Cellular Signaling.

Calcaium sensing receptors (CaSRs) play a central role in regulating extracellular calcium (Ca2+) homeostasis and many (patho)physiological processes. This regulation is primarily orchestrated in response to extracellular stimuli via the extracellular domain (ECD). This paper first reviews the modeled structure of the CaSR ECD and the prediction and investigation of the Ca2+ and amino acid binding sites. Several recently solved X-ray structures are then compared to support a proposed CaSR activation model involving functional cooperativity. The review also discusses recent implications for drug development. These studies provide new insights into the molecular basis of diseases and the design of therapeutic agents that target CaSR and other family C G protein-coupled receptors (cGPCRs).

Structural Aspects and Prediction of Calmodulin-Binding Proteins.

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.

Defining potential roles of Pb(2+) in neurotoxicity from a calciomics approach.

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.

Design of nanomaterial based systems for novel vaccine development.

With lower cell toxicity and higher specificity, novel vaccines have been greatly developed and applied to emerging infectious and chronic diseases. However, due to problems associated with low immunogenicity and complicated processing steps, the development of novel vaccines has been limited. With the rapid development of bio-technologies and material sciences, nanomaterials are playing essential roles in novel vaccine design. Incorporation of nanomaterials is expected to improve delivery efficiency, to increase immunogenicity, and to reduce the administration dosage. The purpose of this review is to discuss the employment of nanomaterials, including polymeric nanoparticles, liposomes, virus-like particles, peptide amphiphiles micelles, peptide nanofibers and microneedle arrays, in vaccine design. Compared to traditional methods, vaccines made from nanomaterials display many appealing benefits, including precise stimulation of immune responses, effective targeting to certain tissue or cells, and desirable biocompatibility. Current research suggests that nanomaterials may improve our approach to the design and delivery of novel vaccines.

Metal toxicity and opportunistic binding of Pb(2+) in proteins.

Lead toxicity is associated with various human diseases. While Ca(2+) binding proteins such as calmodulin (CaM) are often reported to be molecular targets for Pb(2+)-binding and lead toxicity, the effect of Pb(2+) on the Ca(2+)/CaM regulated biological activities cannot be described by the primary mechanism of ionic displacement (e.g., ionic mimicry). The focus of this study was to investigate the mechanism of lead toxicity through binding differences between Ca(2+) and Pb(2+) for CaM, an essential intracellular trigger protein with two EF-Hand Ca(2+)-binding sites in each of its two domains that regulates many molecular targets via Ca(2+)-induced conformational change. Fluorescence changes in phenylalanine indicated that Pb(2+) binds with 8-fold higher affinity than Ca(2+) in the N-terminal domain. Additionally, NMR chemical shift changes and an unusual biphasic response observed in tyrosine fluorescence associated with C-terminal domain sites EF-III and EF-IV suggest a single higher affinity Pb(2+)-binding site with a 3-fold higher affinity than Ca(2+), coupled with a second site exhibiting affinity nearly equivalent to that of the N-terminal domain sites. Our results further indicate that Pb(2+) displaces Ca(2+) only in the N-terminal domain, with minimal perturbation of the C-terminal domain, however significant structural/dynamic changes are observed in the trans-domain linker region which appear to be due to Pb(2+)-binding outside of the known calcium-binding sites. These data suggest that opportunistic Pb(2+)-binding in Ca(2+)/CaM has a profound impact on the conformation and dynamics of the essential molecular recognition sites of the central helix, and provides insight into the molecular toxicity of non-essential metal ions.

Predicting Ca2+ -binding sites using refined carbon clusters.

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.

Integration of Diverse Research Methods to Analyze and Engineer Ca-Binding Proteins: From Prediction to Production.

In recent years, increasingly sophisticated computational and bioinformatics tools have evolved for the analyses of protein structure, function, ligand interactions, modeling and energetics. This includes the development of algorithms to recursively evaluate side-chain rotamer permutations, identify regions in a 3D structure that meet some set of search parameters, calculate and minimize energy values, and provide high-resolution visual tools for theoretical modeling. Here we discuss the interdependency between different areas of bioinformatics, the evolution of different algorithm design approaches, and finally the transition from theoretical models to real-world design and application as they relate to Ca(2+)-binding proteins. Within this context, it has become evident that significant pre-experimental design and calculations can be modeled through computational methods, thus eliminating potentially unproductive research and increasing our confidence in the correlation between real and theoretical models. Moving from prediction to production, it is anticipated that bioinformatics tools will play an increasingly significant role in research and development, improving our ability to both understand the physiological roles of Ca(2+) and other metals and to extend that knowledge to the design of function-specific synthetic proteins capable of fulfilling different roles in medical diagnostics and therapeutics.

Analysis and prediction of calcium-binding pockets from apo-protein structures exhibiting calcium-induced localized conformational changes.

Calcium binding in proteins exhibits a wide range of polygonal geometries that relate directly to an equally diverse set of biological functions. The binding process stabilizes protein structures and typically results in local conformational change and/or global restructuring of the backbone. Previously, we established the MUG program, which utilized multiple geometries in the Ca(2+)-binding pockets of holoproteins to identify such pockets, ignoring possible Ca(2+)-induced conformational change. In this article, we first report our progress in the analysis of Ca(2+)-induced conformational changes followed by improved prediction of Ca(2+)-binding sites in the large group of Ca(2+)-binding proteins that exhibit only localized conformational changes. The MUG(SR) algorithm was devised to incorporate side chain torsional rotation as a predictor. The output from MUG(SR) presents groups of residues where each group, typically containing two to five residues, is a potential binding pocket. MUG(SR) was applied to both X-ray apo structures and NMR holo structures, which did not use calcium distance constraints in structure calculations. Predicted pockets were validated by comparison with homologous holo structures. Defining a "correct hit" as a group of residues containing at least two true ligand residues, the sensitivity was at least 90%; whereas for a "correct hit" defined as a group of residues containing at least three true ligand residues, the sensitivity was at least 78%. These data suggest that Ca(2+)-binding pockets are at least partially prepositioned to chelate the ion in the apo form of the protein.

Towards predicting Ca2+-binding sites with different coordination numbers in proteins with atomic resolution.

Ca(2+)-binding sites in proteins exhibit a wide range of polygonal geometries that directly relate to an equally-diverse set of biological functions. Although the highly-conserved EF-Hand motif has been studied extensively, non-EF-Hand sites exhibit much more structural diversity which has inhibited efforts to determine the precise location of Ca(2+)-binding sites, especially for sites with few coordinating ligands. Previously, we established an algorithm capable of predicting Ca(2+)-binding sites using graph theory to identify oxygen clusters comprised of four atoms lying on a sphere of specified radius, the center of which was the predicted calcium position. Here we describe a new algorithm, MUG (MUltiple Geometries), which predicts Ca(2+)-binding sites in proteins with atomic resolution. After first identifying all the possible oxygen clusters by finding maximal cliques, a calcium center (CC) for each cluster, corresponding to the potential Ca(2+) position, is located to maximally regularize the structure of the (cluster, CC) pair. The structure is then inspected by geometric filters. An unqualified (cluster, CC) pair is further handled by recursively removing oxygen atoms and relocating the CC until its structure is either qualified or contains fewer than four ligand atoms. Ligand coordination is then determined for qualified structures. This algorithm, which predicts both Ca(2+) positions and ligand groups, has been shown to successfully predict over 90% of the documented Ca(2+)-binding sites in three datasets of highly-diversified protein structures with 0.22 to 0.49 A accuracy. All multiple-binding sites (i.e. sites with a single ligand atom associated with multiple calcium ions) were predicted, as were half of the low-coordination sites (i.e. sites with less than four protein ligand atoms) and 14/16 cofactor-coordinating sites. Additionally, this algorithm has the flexibility to incorporate surface water molecules and protein cofactors to further improve the prediction for low-coordination and cofactor-coordinating Ca(2+)-binding sites.

Structural differences between Pb2+- and Ca2+-binding sites in proteins: implications with respect to toxicity.

Pb(2+) is known to displace physiologically-relevant metal ions in proteins. To investigate potential relationships between Pb(2+)/protein complexes and toxicity, data from the protein data bank were analyzed to compare structural properties of Pb(2+)- and Ca(2+)-binding sites. Results of this analysis reveal that the majority of Pb(2+) sites (77.1%) involve 2-5 binding ligands, compared with 6+/-2 for non-EF-Hand and 7+/-1 for EF-Hand Ca(2+)-binding sites. The mean net negative charge by site (1.7) fell between values noted for non-EF-Hand (1+/-1) and EF-Hand (3+/-1). Oxygen is the dominant ligand for both Pb(2+) and Ca(2+), but Pb(2+) binds predominantly with sidechain Glu (38.4%), which is less prevalent in both non-EF-Hand (10.4%) and EF-Hand (26.6%) Ca(2+)-binding sites. A comparison of binding geometries where Pb(2+) has replaced Ca(2+) in calmodulin (CaM) and Zn(2+) in 5-aminolaevulinic acid dehydratase (ALAD) revealed protein structural changes that appear to be unrelated to ionic displacement. Structural changes observed with CaM may be related to opportunistic binding of Pb(2+) in regions of high electrostatic charge, whereas ALAD may bind multiple Pb(2+) ions in the active site. These results suggest that Pb(2+) adapts to structurally-diverse binding geometries and that opportunistic binding may play an active role in molecular metal toxicity.

Statistical analysis of structural characteristics of protein Ca2+-binding sites.

To better understand the biological significance of Ca(2+), we report a comprehensive statistical analysis of calcium-binding proteins from the Protein Data Bank to identify structural parameters associated with EF-hand and non-EF-hand Ca(2+)-binding sites. Comparatively, non-EF-hand sites utilize lower coordination numbers (6 +/- 2 vs. 7 +/- 1), fewer protein ligands (4 +/- 2 vs. 6 +/- 1), and more water ligands (2 +/- 2 vs. 1 +/- 0) than EF-hand sites. The orders of ligand preference for non-EF-hand and EF-hand sites, respectively, were H(2)O (33.1%) > side-chain Asp (24.5%) > main-chain carbonyl (23.9%) > side-chain Glu (10.4%), and side-chain Asp (29.7%) > side-chain Glu (26.6%) > main-chain carbonyl (21.4%) > H(2)O (13.3%). Less formal negative charge was observed in the non-EF-hand than in the EF-hand binding sites (1 +/- 1 vs. 3 +/- 1). Additionally, over 20% of non-EF-hand sites had formal charge values of zero due to increased utilization of water and carbonyl oxygen ligands. Moreover, the EF-hand sites presented a narrower range of ligand distances and bond angles than non-EF-hand sites, possibly owing to the highly conserved helix-loop-helix motif. Significant differences between ligand types (carbonyl, side chain, bidentate) demonstrated that angles associated with each type must be classified separately, and the EF-hand side-chain Ca-O-C angles exhibited an unusual bimodal quality consistent with an Asp distribution that differed from the Gaussian model observed for non-EF-hand proteins. The results of this survey more accurately describe differences between EF-hand and non-EF-hand proteins and provide new parameters for the prediction and design of different classes of Ca(2+)-binding proteins.

Prediction of EF-hand calcium-binding proteins and analysis of bacterial EF-hand proteins.

The EF-hand protein with a helix-loop-helix Ca(2+) binding motif constitutes one of the largest protein families and is involved in numerous biological processes. To facilitate the understanding of the role of Ca(2+) in biological systems using genomic information, we report, herein, our improvement on the pattern search method for the identification of EF-hand and EF-like Ca(2+)-binding proteins. The canonical EF-hand patterns are modified to cater to different flanking structural elements. In addition, on the basis of the conserved sequence of both the N- and C-terminal EF-hands within S100 and S100-like proteins, a new signature profile has been established to allow for the identification of pseudo EF-hand and S100 proteins from genomic information. The new patterns have a positive predictive value of 99% and a sensitivity of 96% for pseudo EF-hands. Furthermore, using the developed patterns, we have identified zero pseudo EF-hand motif and 467 canonical EF-hand Ca(2+) binding motifs with diverse cellular functions in the bacteria genome. The prediction results imply that pseudo EF-hand motifs are phylogenetically younger than canonical EF-hand motifs. Our prediction of Ca(2+) binding motifs provides not only an insight into the role of Ca(2+) and Ca(2+)-binding proteins in bacterial systems, but also a way to explore and define the role of Ca(2+) in other biological systems (calciomics).