C. Alexander Valencia

Scanning the human proteome for calmodulin-binding proteins

The calcium ion (Ca2+) is a ubiquitous second messenger that is crucial for the regulation of a wide variety of cellular processes. The diverse transient signals transduced by Ca2+ are mediated by intracellular Ca2+-binding proteins, also known as Ca2+ sensors. A key obstacle to studying many Ca2+-sensing proteins is the difficulty in identifying the numerous downstream target interactions that respond to Ca2+-induced conformational changes. Among a number of Ca2+ sensors in the eukaryotic cell, calmodulin (CaM) is the most widespread and the best studied. Employing the mRNA display technique, we have scanned the human proteome for CaM-binding proteins and have identified and characterized a large number of both known and previously uncharacterized proteins that interact with CaM in a Ca2+-dependent manner. The interactions of several identified proteins with Ca2+/CaM were confirmed by using pull-down assays and coimmunoprecipitation. Many of the CaM-binding proteins identified belong to protein families such as the DEAD/H box proteins, ribosomal proteins, proteasome 26S subunits, and deubiquitinating enzymes, suggesting the possible involvement of Ca2+/CaM in different signaling pathways. The selection method described herein could be used to identify the binding partners of other calcium sensors on the proteome-wide scale.

Morphing low affinity ligands into high avidity nanoparticles by thermally triggered self-assembly of a genetically encoded polymer

Multivalency is the increase in avidity resulting from the simultaneous interaction of multiple ligands with multiple receptors. This phenomenon, seen in antibody-antigen and virus-cell membrane interactions, is useful in designing bioinspired materials for targeted delivery of drugs or imaging agents. While increased avidity offered by multivalent targeting is attractive, it can also promote nonspecific receptor interaction in nontarget tissues, reducing the effectiveness of multivalent targeting. Here, we present a thermal targeting strategy--dynamic affinity modulation (DAM)--using elastin-like polypeptide diblock copolymers (ELP(BC)s) that self-assemble from a low-affinity to high-avidity state by a tunable thermal switch, thereby restricting activity to the desired site of action. We used an in vitro cell binding assay to investigate the effect of the thermally triggered self-assembly of these ELP(BC)s on their receptor-mediated binding and cellular uptake. The data presented herein show that (1) ligand presentation does not disrupt ELP(BC) self-assembly; (2) both multivalent ligand presentation and upregulated receptor expression are needed for receptor-mediated interaction; (3) increased size of the hydrophobic segment of the block copolymer promotes multivalent interaction with membrane receptors, potentially due to changes in the nanoscale architecture of the micelle; and (4) nanoscale presentation of the ligand is important, as presentation of the ligand by micrometer-sized aggregates of an ELP showed a low level of binding/uptake by receptor-positive cells compared to its presentation on the corona of a micelle. These data validate the concept of thermally triggered DAM and provide rational design parameters for future applications of this technology for targeted drug delivery.

Proteome-wide identification of family member-specific natural substrate repertoire of caspases.

Caspases are proteolytic enzymes that are essential for apoptosis. Understanding the many discrete and interacting signaling pathways mediated by caspases requires the identification of the natural substrate repertoire for each caspase of interest. Using an amplification-based protein selection technique called mRNA display, we developed a high-throughput screen platform for caspase family member specific substrates on a proteome-wide scale. A large number of both known and previously uncharacterized caspase-3 substrates were identified from the human proteome. The proteolytic features of these selected substrates, including their cleavage sites and specificities, were characterized. Substrates that were cleaved only by caspase-8 or granzyme B but not by caspase-3, were readily selected. The method can be widely applied for efficient and systematic identification of the family member specific natural substrate repertoire of any caspase in an organism of interest, in addition to that of numerous other proteases with high specificity.

Ca2+/Calmodulin Directly Interacts with the Pleckstrin Homology Domain of AKT1

AKT kinase, also known as protein kinase B, is a key regulator of cell growth, proliferation, and metabolism. The activation of the AKT signaling pathway is one of the most frequent molecular alterations in a wide variety of human cancers. Dickson and coworkers recently observed that Ca2+·calmodulin (Ca2+·CaM) may be a common regulator of AKT1 activation (Deb, T. B., Coticchia, C. M., and Dickson, R. B. (2004) J. Biol. Chem. 279, 38903–38911). In our efforts to scan the mRNA-displayed proteome libraries for Ca2+·CaM-binding proteins, we found that both human and Caenorhabditis elegans AKT1 kinases bound to CaM in a Ca2+-dependent manner (Shen, X., Valencia, C. A., Szostak, J., Dong, B., and Liu, R. (2005) Proc. Natl. Acad. Sci. U. S. A. 102, 5969–5974 and Shen, X., Valencia, C. A., Gao, W., Cotten, S. W., Dong, B., Chen, M., and Liu, R. (2007) submitted for publication). Here we demonstrate that Ca2+·CaM and human AKT1 were efficiently co-immunoprecipitated, and their interaction was direct rather than mediated by other proteins. The binding is in part attributed to the first 42 residues of the pleckstrin homology (PH) domain, a region that is critical for the recognition of its lipid ligands. The PH domain of human AKT1 can disrupt the complex of the full-length AKT1 with Ca2+·CaM. In addition, Ca2+·CaM competes with phosphatidylinositol 3,4,5-trisphophate for interaction with the PH domain of human AKT1. Our findings suggest that Ca2+·CaM is directly involved in regulating the functions of AKT1, presumably by releasing the activated AKT1 from the plasma membrane and/or prohibiting it from re-association with phosphoinositides on plasma membrane.

Modulation of nucleobindin-1 and nucleobindin-2 by caspases

Nucleobindin‐1 (NUCB1) and nucleobindin‐2 (NUCB2) are multifunctional proteins that interact with Ca2+, nucleic acids and various regulatory proteins in different signaling pathways. So far, our understanding of the regulation of the biological functions of nucleobindins remains limited. In our proteome‐wide selection for downstream caspase substrates, both NUCB1 and NUCB2 are found to be the downstream substrates of caspases. We report here the detailed analyses of the cleavage of nucleobindins by caspases. Significantly, the caspase cleavage sites are located exactly at one of the Ca2+‐binding EF‐hand motifs. Our results suggest that the functions of nucleobindins could be modulated by caspase‐mediated cleavage in apoptosis.

mRNA-display-based selections for proteins with desired functions: a protease-substrate case study.

mRNA‐display is an amplification‐based, iterative rounds of in vitro protein selection technique that circumvents a number of difficulties associated with yeast two‐hybrid and phage display. Because of the covalent linkage between the genotype and the phenotype, mRNA‐display provides a powerful means for reading and amplifying a peptide or protein sequence after it has been selected from a library with a diversity in the range of 1012–1013. In this paper, we briefly review the recent progress in using mRNA‐display to identify affinity reagents, binding partners, and enzyme substrates from synthetic peptide or natural proteome libraries. To facilitate the use of mRNA‐display in research laboratories without previous experience, we provide a detailed analysis of the critical steps of an mRNA‐display‐based selection in a case study for the identification of the natural substrates of caspases, including the generation of an mRNA‐displayed proteome library, removal of abundant sequences, and selection of proteins with desired functions. The advantages and technical limitations of mRNA‐display as a general peptide or protein selection tool are also addressed.

Selection of proteins with desired properties from natural proteome libraries using mRNA display

mRNA display is a powerful yet challenging in vitro selection technique that can be used to identify proteins with desired properties from both natural proteome and combinatorial polypeptide libraries. The physical conjugation between a protein and its own RNA presents unique challenges in manipulating the displayed proteins at a low nanomolar scale in an RNase-free environment. The following protocol outlines the generation of cDNA libraries derived from natural organisms as well as the steps required for generation of mRNA-protein fusion molecules, in vitro functional selection and regeneration of the selected cDNA library. The selection procedures for the identification of protease substrates and Ca2+-dependent calmodulin-binding proteins from natural cDNA libraries are presented as examples. The method can be generally applied to the identification of protein sequences with desired properties from various natural proteome libraries. One round of mRNA display–based selection can be accomplished in ∼7 d.

Heptameric Targeting Ligands against EGFR and HER2 with High Stability and Avidity

Multivalency of targeting ligands provides significantly increased binding strength towards their molecular targets. Here, we report the development of a novel heptameric targeting system, with general applications, constructed by fusing a target-binding domain with the heptamerization domain of the Archaeal RNA binding protein Sm1 through a flexible hinge peptide. The previously reported affibody molecules against EGFR and HER2, ZEGFR and ZHER2, were used as target binding moieties. The fusion molecules were highly expressed in E. coli as soluble proteins and efficiently self-assembled into multimeric targeting ligands with the heptamer as the predominant form. We demonstrated that the heptameric molecules were resistant to protease-mediated digestion or heat- and SDS-induced denaturation. Surface plasmon resonance (SPR) analysis showed that both heptameric ZEGFR and ZHER2 ligands have a significantly enhanced binding strength to their target receptors with a nearly 100 to 1000 fold increase relative to the monomeric ligands. Cellular binding assays showed that heptameric ligands maintained their target-binding specificities similar to the monomeric forms towards their respective receptor. The non-toxic property of each heptameric ligand was demonstrated by the cell proliferation assay. In general,, the heptamerization strategy we describe here could be applied to the facile and efficient engineering of other protein domain- or short peptide-based affinity molecules to acquire significantly improved target-binding strengths with potential applications in the targeted delivery of various imaging or therapeutic agents..

Deep Sequencing Reveals Novel Genetic Variants in Children with Acute Liver Failure and Tissue Evidence of Impaired Energy Metabolism

Background & Aims The etiology of acute liver failure (ALF) remains elusive in almost half of affected children. We hypothesized that inherited mitochondrial and fatty acid oxidation disorders were occult etiological factors in patients with idiopathic ALF and impaired energy metabolism. Methods Twelve patients with elevated blood molar lactate/pyruvate ratio and indeterminate etiology were selected from a retrospective cohort of 74 subjects with ALF because their fixed and frozen liver samples were available for histological, ultrastructural, molecular and biochemical analysis. Results A customized next-generation sequencing panel for 26 genes associated with mitochondrial and fatty acid oxidation defects revealed mutations and sequence variants in five subjects. Variants involved the genes ACAD9, POLG, POLG2, DGUOK, and RRM2B; the latter not previously reported in subjects with ALF. The explanted livers of the patients with heterozygous, truncating insertion mutations in RRM2B showed patchy micro- and macrovesicular steatosis, decreased mitochondrial DNA (mtDNA) content <30% of controls, and reduced respiratory chain complex activity; both patients had good post-transplant outcome. One infant with severe lactic acidosis was found to carry two heterozygous variants in ACAD9, which was associated with isolated complex I deficiency and diffuse hypergranular hepatocytes. The two subjects with heterozygous variants of unknown clinical significance in POLG and DGUOK developed ALF following drug exposure. Their hepatocytes displayed abnormal mitochondria by electron microscopy. Conclusion Targeted next generation sequencing and correlation with histological, ultrastructural and functional studies on liver tissue in children with elevated lactate/pyruvate ratio expand the spectrum of genes associated with pediatric ALF.

CCEPAS: the creation and validation of a fast and sensitive clinical whole exome analysis pipeline based on gene and variant ranking

Aim: Whole exome sequencing technology has permitted the discovery of genes that cause Mendelian disorders and was used in clinical laboratories. However, identifying the disease causing variant(s) for a specific disorder from thousands of variants is challenging. In this study, we describe the Cincinnati Clinical Exome Pipeline Analysis Suite (CCEPAS) that utilizes a four-level framework into one analysis procedure that rapidly identify the most likely causative gene variants to establish a clinical diagnosis. Methods: We developed and validated CCEPAS using 100 clinical exome cases. We applied this pipeline to clinical cases by first translating phenotypic information into candidate gene lists using Pheno2Gene. This list of candidate genes was given to the VarEval algorithm to guide variant filtering and prioritization. Finally, a short list of filtered variants was produced for clinical interpretation. Results: We demonstrated the development and implementation of CCEPAS to aid in the variant prioritization and filtering to produce a short list of candidate variants for clinical diagnosis. Its unique Pheno2Gene tool utilized an extensive list of resources and provided an accurate, sensitive and specific way to obtain gene lists from clinical feature keywords. In addition, VarEval narrowed down the variants from ~150,000 to the top 20 (trios) and top 50 (singleton) for further variant curation and candidate determination. Conclusion: Significantly, employment of CCEPAS rapidly provided causative variants in the top 20 and top 50 variants for single and trio cases, respectively, thus, ending the diagnostic odyssey in more than 30% of our clinical exome cases.