Calcium-binding Domain Research Articles

Cell Wall Calcium and Hemicellulose Have a Role in the Fruit Firmness during Storage of Blueberry (Vaccinium spp.).

The firmness of blueberry is one of its most significant quality attributes. Modifications in the composition of the cell wall have been associated with changes in the fruit firmness. In this work, cell wall components and calcium concentration in two blueberry cultivars with contrasting firmness phenotypes were evaluated at harvest and 30 days cold storage (0 °C). High performance anion-exchange chromatography with pulse amperometric detector (HPAEC-PAD) analysis was performed using the “Emerald” (firmer) and “Jewel” (softer) blueberry cultivars, showing increased glucose in the firmer cultivar after cold storage. Moreover, the LM15 antibody, which recognizes xyloglucan domains, displayed an increased signal in the Emerald cultivar after 30 d cold storage. Additionally, the antibody 2F4, recognizing a homogalacturonan calcium-binding domain, showed a greater signal in the firmer Emerald blueberries, which correlates with a higher calcium concentration in the cell wall. These findings suggest that xyloglucan metabolism and a higher concentration of cell wall calcium influenced the firmness of the blueberry fruit. These results open new perspectives regarding the role of cell wall components as xyloglucans and calcium in blueberry firmness.

Calcium-binding and coiled-coil domain 2 promotes the proliferation and suppresses apoptosis of prostate cancer cells.

Prostate cancer (PCa) is considered to be one of the most common tumors in men. Calcium-binding and coiled-coil domain 2 (CALCOCO2) is a known important xenophagy receptor, which mediates intracellular bacterial degradation. To the best of the authors' knowledge, the present study is the first to demonstrate that CALCOCO2 functions as an oncogene in PCa. The results of the current study indicated that CALCOCO2 knockdown suppressed cell proliferation and colony formation, whereas it promoted apoptosis of PCa cells. In addition, knockdown of CALCOCO2 in PCa cells reduced cyclin-E1 and increased p53 protein expression. Bioinformatics analysis revealed that CALCOCO2 was associated with 'autophagosome assembly', 'nucleophagy' and 'nucleic acid metabolic process' biological processes and interacted with sequestosome-1, microtubule-associated proteins 1A/1B light chain 3 (MAP1LC3)B, γ-aminobutyric acid receptor-associated protein, IκB kinase subunit γ and MAP1LC3C. Moreover, CALCOCO2 protein levels were indicated to be significantly increased in PCa samples compared with normal prostate tissues. These results suggested that CALCOCO2 may be of value as a diagnostic and therapeutic target in PCa.

Interplay between hevin, SPARC, and MDGAs: Modulators of neurexin-neuroligin transsynaptic bridges

Summary Hevin is secreted by astrocytes and its synaptogenic effects are antagonized by the related protein, SPARC. Hevin stabilizes neurexin-neuroligin transsynaptic bridges in vivo. A third protein, membrane-tethered MDGA, blocks these bridges. Here, we reveal the molecular underpinnings of a regulatory network formed by this trio of proteins. The hevin FS-EC structure differs from SPARC, in that the EC domain appears rearranged around a conserved core. The FS domain is structurally conserved and it houses nanomolar affinity binding sites for neurexin and neuroligin. SPARC also binds neurexin and neuroligin, competing with hevin, so its antagonist action is rooted in its shortened N-terminal region. Strikingly, the hevin FS domain competes with MDGA for an overlapping binding site on neuroligin, while the hevin EC domain binds the extracellular matrix protein collagen (like SPARC), so that this trio of proteins can regulate neurexin-neuroligin transsynaptic bridges and also extracellular matrix interactions, impacting synapse formation and ultimately neural circuits.

Targeting S100B Protein as a Surrogate Biomarker and its Role in Various Neurological Disorders.

Neurological disorders (ND) are the central nervous system (CNS) related complications originated by enhanced oxidative stress, mitochondrial failure and overexpression of proteins like S100B. S100B is a helix-loop-helix protein with the calcium-binding domain associated with various neurological disorders through activation of the MAPK pathway, increased NF-kB expression resulting in cell survival, proliferation and gene up-regulation. S100B protein plays a crucial role in Alzheimer’s disease, Parkinson’s disease, multiple sclerosis, Schizophrenia and epilepsy because the high expression of this protein directly targets astrocytes and promotes neuroinflammation. Under stressful conditions, S100B produces toxic effects mediated through receptor for advanced glycation end products (AGE) binding. S100B also mediates neuroprotection, minimizes microgliosis and reduces the expression of tumor necrosis factor (TNF-alpha) but that are concentration-dependent mechanisms. Increased level of S100B is useful for assessing the release of inflammatory markers, nitric oxide and excitotoxicity dependent neuronal loss. The present review summarizes the role of S100B in various neurological disorders and potential therapeutic measures to reduce the prevalence of neurological disorders.

FRCaMP, a Red Fluorescent Genetically Encoded Calcium Indicator Based on Calmodulin from Schizosaccharomyces Pombe Fungus.

Red fluorescent genetically encoded calcium indicators (GECIs) have expanded the available pallet of colors used for the visualization of neuronal calcium activity in vivo. However, their calcium-binding domain is restricted by calmodulin from metazoans. In this study, we developed red GECI, called FRCaMP, using calmodulin (CaM) from Schizosaccharomyces pombe fungus as a calcium binding domain. Compared to the R-GECO1 indicator in vitro, the purified protein FRCaMP had similar spectral characteristics, brightness, and pH stability but a 1.3-fold lower ΔF/F calcium response and 2.6-fold tighter calcium affinity with Kd of 441 nM and 2.4–6.6-fold lower photostability. In the cytosol of cultured HeLa cells, FRCaMP visualized calcium transients with a ΔF/F dynamic range of 5.6, which was similar to that of R-GECO1. FRCaMP robustly visualized the spontaneous activity of neuronal cultures and had a similar ΔF/F dynamic range of 1.7 but 2.1-fold faster decay kinetics vs. NCaMP7. On electrically stimulated cultured neurons, FRCaMP demonstrated 1.8-fold faster decay kinetics and 1.7-fold lower ΔF/F values per one action potential of 0.23 compared to the NCaMP7 indicator. The fungus-originating CaM of the FRCaMP indicator version with a deleted M13-like peptide did not interact with the cytosolic environment of the HeLa cells in contrast to the metazoa-originating CaM of the similarly truncated version of the GCaMP6s indicator with a deleted M13-like peptide. Finally, we generated a split version of the FRCaMP indicator, which allowed the simultaneous detection of calcium transients and the heterodimerization of bJun/bFos interacting proteins in the nuclei of HeLa cells with a ΔF/F dynamic range of 9.4 and a contrast of 2.3–3.5, respectively.

The Central Region of Testican-2 Forms a Compact Core and Promotes Cell Migration.

Testicans are modular proteoglycans of the extracellular matrix of various tissues where they contribute to matrix integrity and exert cellular effects like neurite outgrowth and cell migration. Using testican-2 as a representative member of the family, we tackle the complete lack of general structural information and structure–function relationship. First, we show using isothermal titration calorimetry and modeling that extracellular calcium-binding domain (EC) has only one active calcium-binding site, while the other potential site is inactive, and that testican-2 is within extracellular matrix always in the calcium-loaded form. Next, we demonstrate using various prediction methods that N- and C-terminal regions plus interdomain connections are flexible. We support this by small-angle X-ray-scattering analysis of C-terminally truncated testican-2, which indicates that the triplet follistatin-EC-thyroglobulin domain forms a moderately compact core while the unique N-terminal is disordered. Finally, using cell exclusion zone assay, we show that it is this domain triplet that is responsible for promoting cell migration and not the N- and C-terminal regions.

New recessive mutations in SYT2 causing severe presynaptic congenital myasthenic syndromes.

ObjectiveTo report the identification of 2 new homozygous recessive mutations in the synaptotagmin 2 (SYT2) gene as the genetic cause of severe and early presynaptic forms of congenital myasthenic syndromes (CMSs).MethodsNext-generation sequencing identified new homozygous intronic and frameshift mutations in the SYT2 gene as a likely cause of presynaptic CMS. We describe the clinical and electromyographic patient phenotypes, perform ex vivo splicing analyses to characterize the effect of the intronic mutation on exon splicing, and analyze the functional impact of this variation at the neuromuscular junction (NMJ).ResultsThe 2 infants presented a similar clinical phenotype evoking first a congenital myopathy characterized by muscle weakness and hypotonia. Next-generation sequencing allowed to the identification of 1 homozygous intronic mutation c.465+1G>A in patient 1 and another homozygous frameshift mutation c.328_331dup in patient 2, located respectively in the 5′ splice donor site of SYT2 intron 4 and in exon 3. Functional studies of the intronic mutation validated the abolition of the splice donor site of exon 4 leading to its skipping. In-frame skipping of exon 4 that encodes part of the C2A calcium-binding domain of SYT2 is associated with a loss-of-function effect resulting in a decrease of neurotransmitter release and severe pre- and postsynaptic NMJ defects.ConclusionsThis study identifies new homozygous recessive SYT2 mutations as the underlying cause of severe and early presynaptic form of CMS expanding the genetic spectrum of recessive SYT2-related CMS associated with defects in neurotransmitter release.

Autophagy receptor OPTN (optineurin) regulates mesenchymal stem cell fate and bone-fat balance during aging by clearing FABP3

ABSTRACT Senile osteoporosis (OP) is often concomitant with decreased autophagic activity. OPTN (optineurin), a macroautophagy/autophagy (hereinafter referred to as autophagy) receptor, is found to play a pivotal role in selective autophagy, coupling autophagy with bone metabolism. However, its role in osteogenesis is still mysterious. Herein, we identified Optn as a critical molecule of cell fate decision for bone marrow mesenchymal stem cells (MSCs), whose expression decreased in aged mice. Aged mice revealed osteoporotic bone loss, elevated senescence of MSCs, decreased osteogenesis, and enhanced adipogenesis, as well as optn– / – mice. Importantly, restoring Optn by transplanting wild-type MSCs to optn– / – mice or infecting optn– / – mice with Optn-containing lentivirus rescued bone loss. The introduction of a loss-of-function mutant of OptnK193R failed to reestablish a bone-fat balance. We further identified FABP3 (fatty acid binding protein 3, muscle and heart) as a novel selective autophagy substrate of OPTN. FABP3 promoted adipogenesis and inhibited osteogenesis of MSCs. Knockdown of FABP3 alleviated bone loss in optn– / – mice and aged mice. Our study revealed that reduced OPTN expression during aging might lead to OP due to a lack of FABP3 degradation via selective autophagy. FABP3 accumulation impaired osteogenesis of MSCs, leading to the occurrence of OP. Thus, reactivating OPTN or inhibiting FABP3 would open a new avenue to treat senile OP. Abbreviations: ADIPOQ: adiponectin, C1Q and collagen domain containing; ALPL: alkaline phosphatase, liver/bone/kidney; BGLAP/OC/osteocalcin: bone gamma carboxyglutamate protein; BFR/BS: bone formation rate/bone surface; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; CDKN1A/p21: cyclin-dependent kinase inhibitor 1A; CDKN2A/p16: cyclin dependent kinase inhibitor 2A; CDKN2B/p15: cyclin dependent kinase inhibitor 2B; CEBPA: CCAAT/enhancer binding protein (C/EBP), alpha; COL1A1: collagen, type I, alpha 1; Ct. BV/TV: cortical bone volume fraction; Ct. Th: cortical thickness; Es. Pm: endocortical perimeter; FABP4/Ap2: fatty acid binding protein 4, adipocyte; H2AX: H2A.X variant histone; HE: hematoxylin and eosin; MAP1LC3B: microtubule-associated protein 1 light chain 3 beta; MAR: mineral apposition rate; MSCs: bone marrow mesenchymal stem cells; NBR1: NBR1, autophagy cargo receptor; OP: osteoporosis; OPTN: optineurin; PDB: Paget disease of bone; PPARG: peroxisome proliferator activated receptor gamma; Ps. Pm: periosteal perimeter; qRT-PCR: quantitative real-time PCR; γH2AX: Phosphorylation of the Serine residue of H2AX; ROS: reactive oxygen species; RUNX2: runt related transcription factor 2; SA-GLB1: senescence-associated (SA)-GLB1 (galactosidase, beta 1); SP7/Osx/Osterix: Sp7 transcription factor 7; SQSTM1/p62: sequestosome 1; TAX1BP1: Tax1 (human T cell leukemia virus type I) binding protein 1; Tb. BV/TV: trabecular bone volume fraction; Tb. N: trabecular number; Tb. Sp: trabecular separation; Tb. Th: trabecular thickness; μCT: micro computed tomography.

A role for the MEGF6 gene in predisposition to osteoporosis.

Osteoporosis is a common skeletal disorder characterized by deterioration of bone tissue. The set of genetic factors contributing to osteoporosis is not completely specified. High-risk osteoporosis pedigrees were analyzed to identify genes that may confer susceptibility to disease. Candidate predisposition variants were identified initially by whole exome sequencing of affected-relative pairs, approximately cousins, from 10 pedigrees. Variants were filtered on the basis of population frequency, concordance between pairs of cousins, affecting a gene associated with osteoporosis, and likelihood to have functionally damaging, pathogenic consequences. Subsequently, variants were tested for segregation in 68 additional relatives of the index carriers. A rare variant in MEGF6 (rs755467862) showed strong evidence of segregation with the disease phenotype. Predicted protein folding indicated the variant (Cys200Tyr) may disrupt structure of an EGF-like calcium-binding domain of MEGF6. Functional analyses demonstrated that complete loss of the paralogous genes megf6a and megf6b in zebrafish resulted in significant delay of cartilage and bone formation. Segregation analyses, in silico protein structure modeling, and functional assays support a role for MEGF6 in predisposition to osteoporosis.

Jute CDPK genes and their role in stress tolerance and fiber development: A genome-wide bioinformatic investigation of Chorchorus capsularis and C. olitorius

Calcium-dependent protein kinases (CDPKs) are plant-specific calcium sensors, which regulate plant growth and development and induce protective responses to environmental stresses. To date, no information on jute CDPKs is available and their uses in the genetic improvement program is underutilized. In this study, we exploited recently available genome sequence information of two jute species: Corchorus olitorius and C. capsularis to identify, characterise, and functional analysis. Using bioinformatics tools, 16 and 18 CDPK genes with serine/threonine protein kinase along with EF-hand calcium-binding domain were identified from the genomes of C. olitorius and C. capsularis, respectively. Different chromosomes harboured different CDPK genes. Four distinct clades of CDPK genes were identified by phylogenetic analysis. Protein of each clade showed higher conservation in length, structure, and presence of motif. The most prominent transcriptional responses were detected in saline and drought conditions where more than half of the CDPKs were upregulated, suggesting their important roles in abiotic stress responses of jute. Specifically, CoCDPK6, 11, 12, and their corresponding homologs CcCDPK18, 10, 8 were highly expressed in saline and drought conditions. On the other hand, CoCDPK6, 7, 11, 12, and their homologs CcCDPK18, 17, 10, 8 were highly expressed in fiber cell indicating their involvement in fiber cell development. Our results provide an important foundation for further functional dissection of CDPKs of C. olitorius and C. capsularis for salinity and drought stress adaptation and fiber quality improvement.

The inhibitory effect of KN-93 and KN-62 as a result of CaM-directed blocking animal CaMK2 and plant CDPK activation

Aim. The goal of the study was determine whether, from a molecular point of view, inhibitors KN-93 and KN-62 are capable to disrupt the functioning of plant homologs of CaMK2 and being used as tools for the experimental study of Ca2+-dependent phosphorylation in higher plants. Methods. Selected calmodulin 1 H.sapiens reference structure and reconstructed spatial structure of calcium-binding domain of CPK1 A.thaliana. We have conducted the molecular docking of calmodulin-mediated inhibitors to CaMK2: KN-93 and KN-62 for CALM1 H. sapiens and calcium-binding domain of CPK1 A. thaliana with full ligand mobility and static amino acid residues with the use of CCDC GOLD Suite. Results. We have established the presence of spatially homologous structures within CALM1 and CPK1 that might be the binding sites for KN-93 and KN-62. Performing the molecular docking we have shown the utility of those pockets from the point of binding energy for KN-93 and KN-62. We have conducted comparative analysis basing on the results of the CCDC GOLD Suite score functions (GoldScore and ASPScore). Conclusions. It has been shown that the inhibitors of animal CaMK2 - KN93 and KN62 are capable of interacting with the site of the CaM-like domain of the plant protein kinase CPK1 homologous to the similar site of animal calmodulin, which may impair its functionality.
 Keywords: CaMK2, CDPK, CPK1, protein kinases, molecular docking, KN-93, KN-62.

Abstract 1908: Kinome rewiring upon acquisition of resistance to PI3K inhibitors in ER+ breast cancer

Abstract Hyperactivation of the phosphatidylinositol-3 kinase (PI3K) pathway is a critical step in oncogenesis and plays a role in the development of treatment resistance in estrogen receptor-positive (ER+) breast cancer. Targeted inhibition of PI3K (PI3Ki) was recently approved in combination with endocrine therapy for advanced ER+ breast cancer. As most tumor-targeted therapies are encumbered by the development of secondary resistance, there remains a need to identify novel therapeutic strategies to prevent and overcome PI3Ki resistance. We treated mice bearing anti-estrogen-resistant ER+ breast cancer xenografts with the anti-estrogen fulvestrant +/- the pan-PI3Ki GDC-0941. The drug combination induced partial regression of tumors, followed by regrowth despite continued drug treatment. We harvested tumors before, during, and after acquisition of resistance to the drug combination and performed quantitative phosphoproteomic profiling. Using LC-MS/MS, we identified 203 phosphopeptides and 227 unique phosphorylation sites in the drug-resistant tumors, with clusters of 87 upregulated and 116 downregulated phosphopeptides. The NetPhorest analysis platform predicted hyperactivation of 6 kinases and hypoactivation of 16 kinases in drug-resistant tumors. Notably, two hyperactive kinases, MAPK1 and HIPK2, were predicted to target substrates MARKS (at Ser27 in the calcium binding domain) and NS1BP (at Ser322 in the homeodomain kelch repeat). MARCKS is a filamentous actin cross-linking protein, while the kelch repeats of NS1BP are known to associate with F-actin to stabilize actin filaments. In addition, the most significant predicted kinase among the cluster of 116 downregulated phosphopetides in the drug-resistant tumors was “PKCiota”, predicted to target PARD3 at Ser852. Previous work showed that PARD3 is central for the formation of tight junctions. These data collectively suggest a dynamic role of kinase-substrate interactions involved in resistance to combined anti-estrogen/PI3Ki treatment, including filamentation actin formation and stabilization, as well as the deregulation of tight junction formation. Citation Format: Jacqueline Reedy Griffin, Nicole Traphagen, Arminja Kettenbach, Todd Miller. Kinome rewiring upon acquisition of resistance to PI3K inhibitors in ER+ breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1908.

A polybasic domain in aPKC mediates Par6-dependent control of membrane targeting and kinase activity.

Mechanisms coupling the atypical PKC (aPKC) kinase activity to its subcellular localization are essential for cell polarization. Unlike other members of the PKC family, aPKC has no well-defined plasma membrane (PM) or calcium binding domains, leading to the assumption that its subcellular localization relies exclusively on protein-protein interactions. Here we show that in both Drosophila and mammalian cells, the pseudosubstrate region (PSr) of aPKC acts as a polybasic domain capable of targeting aPKC to the PM via electrostatic binding to PM PI4P and PI(4,5)P2. However, physical interaction between aPKC and Par-6 is required for the PM-targeting of aPKC, likely by allosterically exposing the PSr to bind PM. Binding of Par-6 also inhibits aPKC kinase activity, and such inhibition can be relieved through Par-6 interaction with apical polarity protein Crumbs. Our data suggest a potential mechanism in which allosteric regulation of polybasic PSr by Par-6 couples the control of both aPKC subcellular localization and spatial activation of its kinase activity.

CALCOCO2 silencing represents a potential molecular therapeutic target for glioma

IntroductionGlioma is the most common primary intracranial tumour that is highly resistant to conventional therapeutic approaches including surgical resection, radiation therapy, and chemotherapy. As a promising alternative treatment, gene therapy has achieved variable degrees in both pre-clinical models and clinical trials.Material and methodsIn our present research, the role of calcium binding and coiled-coil domain 2 (CALCOCO2) in the pathogenesis and progression of glioma was investigated in human glioma U87 and U251 cell lines. In both cell lines, CALCOCO2 is highly expressed. Targeted by lentivirus vectors, the CALCOCO2 gene was successfully silenced in U87 and U251 cell lines. Both cell counting and MTT assay showed the inhibition of cell growth and cell proliferation in CALCOCO2-silenced glioma cell lines.ResultsFlow cytometry (FCM) and caspase3/7 measurements indicated that the silencing of CALCOCO2 gene could also promote cell apoptosis in both cell lines. The underlying mechanism was further explored by gene microarray and western blotting. The CALCOCO2 gene is strongly related to cancer by affecting the expression of hundreds of genes. Among which, the silencing of CALCOCO2 significantly upregulated the pro-apoptosis genes FAS and CASP1 and downregulated the autophagy-related gene BECN1. These data suggest that by regulating FAS, CASP1, and BECN1, the silencing of CALCOCO2 suppresses the growth and proliferation of U87 and U251 glioma cell lines.ConclusionsThe CALCOCO2 could be a potential target for glioma genetic therapy.

Characterization and expression of the follistatin-related protein gene in golden pompano Trachinotus ovatus larvae

The follistatin-related protein (FRSP) gene performs numerous functions through binding with cell regulatory proteins in transforming growth factor beta (TGF-β) superfamily such as activin and myostatin. The FRSP gene has been extensively studied in human and other vertebrate models such as chick and mouse to explore its role for disease control and organogenesis. However, very limited information exists on the FRSP gene and its expression during fish ontogeny. In this study, full-length cDNA sequence of the FSRP gene was characterized using next generation sequencing (Illumina HiSeq2000) and mRNA expression was evaluated using qPCR in golden pompano Trachinotus ovatus (Linnaeus 1758) larvae. The FRSP gene was composed of 1500 bp with an open reading frame of 936 bp, encoded 311 amino acids that have molecular weight of 34.55 kDa. The deduced amino acids of the FSRP gene contained a signal peptide, Kazal domain and EF-hand calcium-binding domain. The FSRP gene of T. ovatus has closest identity and similarity with Siniperca chuatsi and Larimichthys rocea. The mRNA expression of the FRSP gene gradually increased from 0 to 2 day post hatch (DPH), reached maximum at 3 DPH, and significantly decreased from 4 DPH to 18 DPH. The mRNA analysis in tissues revealed higher levels of FRSP gene expression in the liver head and eye but lower levels of expression in the kidney, muscles, and head-kidney. This study provides useful information to explore the functional mechanism of FRSP gene in fish ontogeny.

The chemokine CCL17 is associated with protection against HIV-1 acquisition

Abstract Millions are exposed to the human immunodeficiency virus type 1 (HIV-1) every year, but not all exposed individuals become infected. This suggests a key role for host genetics in resilience or susceptibility to HIV-1 infection, but a comprehensive study of the genetic mechanisms contributing to HIV-1 acquisition risk is lacking. Here, we performed LD score regression analysis using summary statistics from the largest genome-wide association study of HIV-1 acquisition, consisting of 6,334 infected patients and 7,247 population controls. We found that HIV-1 acquisition is polygenic and highly heritable (28%). Genetic correlations performed alongside UK Biobank data revealed associations between HIV-1 acquisition and smoking, prospective memory and socioeconomic traits. Gene-level enrichment analysis identified EF-hand calcium binding domain 14 (EFCAB14) as a novel risk gene for HIV-1 acquisition. We also observed that risk variants for HIV-1 acquisition were significantly enriched for genes expressed in T-cells, but also those expressed in striatal and hippocampal neurons, which could explain the behavioral parameters genetically correlated with acquisition. Finally, we modeled how inter-individual variation in risk for HIV-1 acquisition influences the pre-exposed immune profile, by testing how individualized polygenic risk scores influence blood levels of 34 inflammatory markers in 406 HIV-1-negative individuals. We showed that higher genetic risk for HIV-1 acquisition was associated with lower levels of CCL17. Our findings corroborate a complex model for HIV-1 acquisition, whereby susceptibility is moderated by specific behavioral, cellular and immunological parameters.

Phosphatidylinositol kinases (version 2020.2) in the IUPHAR/BPS Guide to Pharmacology Database

Phosphatidylinositol may be phosphorylated at either 3- or 4- positions on the inositol ring by PI 3-kinases or PI 4-kinases, respectively.Phosphatidylinositol 3-kinasesPhosphatidylinositol 3-kinases (PI3K, provisional nomenclature) catalyse the introduction of a phosphate into the 3-position of phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PIP) or phosphatidylinositol 4,5-bisphosphate (PIP2). There is evidence that PI3K can also phosphorylate serine/threonine residues on proteins. In addition to the classes described below, further serine/threonine protein kinases, including ATM (Q13315) and mTOR (P42345), have been described to phosphorylate phosphatidylinositol and have been termed PI3K-related kinases. Structurally, PI3Ks have common motifs of at least one C2, calcium-binding domain and helical domains, alongside structurally-conserved catalytic domains. wortmannin and LY 294002 are widely-used inhibitors of PI3K activities. wortmannin is irreversible and shows modest selectivity between Class I and Class II PI3K, while LY294002 is reversible and selective for Class I compared to Class II PI3K.Class I PI3Ks (EC 2.7.1.153) phosphorylate phosphatidylinositol 4,5-bisphosphate to generate phosphatidylinositol 3,4,5-trisphosphate and are heterodimeric, matching catalytic and regulatory subunits. Class IA PI3Ks include p110α, p110β and p110δ catalytic subunits, with predominantly p85 and p55 regulatory subunits. The single catalytic subunit that forms Class IB PI3K is p110γ. Class IA PI3Ks are more associated with receptor tyrosine kinase pathways, while the Class IB PI3K is linked more with GPCR signalling.Class II PI3Ks (EC 2.7.1.154) phosphorylate phosphatidylinositol to generate phosphatidylinositol 3-phosphate (and possibly phosphatidylinositol 4-phosphate to generate phosphatidylinositol 3,4-bisphosphate). Three monomeric members exist, PI3K-C2α, β and β, and include Ras-binding, Phox homology and two C2domains.The only class III PI3K isoform (EC 2.7.1.137) is a heterodimer formed of a catalytic subunit (VPS34) and regulatory subunit (VPS15).Phosphatidylinositol 4-kinasesPhosphatidylinositol 4-kinases (EC 2.7.1.67) generate phosphatidylinositol 4-phosphate and may be divided into higher molecular weight type III and lower molecular weight type II forms.

Nasal delivery of a CRMP2-derived CBD3 adenovirus improves cognitive function and pathology in APP/PS1 transgenic mice

Calcium dysregulation is a key pathological event in Alzheimer’s disease (AD). In studying approaches to mitigate this calcium overload, we identified the collapsin response mediator protein 2 (CRMP2), an axonal guidance protein that participates in synapse dynamics by interacting with and regulating activity of N-methyl-D-aspartate receptors (NMDARs). We further identified a 15 amino acid peptide from CRMP2 (designated CBD3, for calcium-binding domain 3), that reduced NMDAR-mediated Ca2+ influx in cultured neurons and post-synaptic NMDAR-mediated currents in cortical slices. Whether targeting CRMP2 could be therapeutically beneficial in AD is unknown. Here, using CBD3, we tested the utility of this approach. Employing the APP/PS1 mouse model of AD which demonstrates robust pathophysiology including Aβ1–42 deposition, altered tau levels, and diminished cognitive functions, we asked if overexpression of CBD3 could rescue these events. CBD3 was engineered into an adeno-associated vector and nasally delivered into APP/PS1 mice and then biochemical (immunohistochemistry, immunoblotting), cellular (TUNEL apoptosis assays), and behavioral (Morris water maze test) assessments were performed. APP/PS1 mice administered adeno-associated virus (AAV, serotype 2) harboring CBD3 demonstrated: (i) reduced levels of Aβ1–42 and phosphorylated-tau (a marker of AD progression), (ii) reduced apoptosis in the hippocampus, and (iii) reduced cognitive decline compared with APP/PS1 mice or APP/PS1 administered a control virus. These results provide an instructive example of utilizing a peptide-based approach to unravel protein-protein interactions that are necessary for AD pathology and demonstrate the therapeutic potential of CRMP2 as a novel protein player in AD.

Characterization of EfhX, a calcium sensor protein in Xanthomonas required for elicitation of the hypersensitive response in Solanum species

We have identified a gene in several strains of Xanthomonas that, when expressed, can elicit a hypersensitive response (hr) in tomato plants. Hr elicitation is normally limited to specific bacterial‐host pairings, but introduction and expression of this gene by bacterial species that do not normally infect tomato serves to elicit hr, indicating that the encoded 170 amino acid protein is crucial to the infectious process. Bioinformatic analysis of the protein, which we have named EfhX ( EF‐ hand containing protein from Xanthomonas) reveals that the protein is predicted to contain a domain of unknown function spanning the amino terminal region, a single transmembrane alpha‐helix spanning amino acids 60 to 81 and two putative EF‐hand calcium binding domains in the carboxy‐terminal domain of the protein. In order to better understand the function of this novel protein, we have cloned the EfhX gene from Xanthomonas aurantifolia and expressed it in Escherichia coli. We have successfully purified the protein to homogeneity as determined by SDS‐PAGE, anti‐hexahistidine Western Blot and MALDI‐TOF mass spectrometry. Using the purified protein, we have used isothermal titration calorimetry to confirm that the protein has two calcium binding sites, with one site having an affinity (Kd) for calcium of 6.75 mM and the second site having an affinity of 1 uM. We are currently performing circular dichroism spectropolarimetry and gel filtration studies to determine if the quaternary state of the protein changes upon calcium binding. Simultaneously, we have initiated crystallization trials of the purified protein to identify solution conditions suitable for growth of crystals for x‐ray diffraction experiments so that the structure of the protein can be determined.Support or Funding InformationThis work is supported by grants from the National Center for Research Resources (5 P20 RR16461) and the National Institute of General Medical Sciences (8 P20 GM103499) from the National Institutes of Health.

Engineering a Self-Assembling Leucine Zipper Hydrogel System with Function-Specific Motifs for Tissue Regeneration.

Protein-based self-assembling hydrogels can exhibit remarkably tunable properties as a scaffold for regenerative medicine applications. In this study, we sought to develop a leucine zipper (LZ) based self-assembling hydrogel with function-specific motifs for tissue-specific regeneration. As a proof-of-concept approach, we incorporated (a) calcium-binding domains ESQES and QESQSEQS derived from dentin matrix protein 1 (DMP1) and (b) an heparin-binding domain adjacent preceded by an MMP2 (matrix metalloprotease 2) cleavage site to facilitate loading of heparin binding growth factors, such as BMP-2, VEGF, and TGF-β1, and their release in vivo by endogenous MMP2 proteolytic cleavage. These scaffolds were characterized and evaluated in vitro and in vivo. In vivo studies highlighted the potential of the engineered LZ hydrogel with respect to osteogenic differentiation of stem cells. The premineralized LZ scaffold loaded with HMSCs showed an enhanced osteoinductive property when compared with the control nonmineralized scaffold. The LZ backbone with heparin-binding domain containing an MMP2 cleavage site facilitated tethering of heparin-binding growth factors, such as VEGF, TGF-β1 and BMP2 and demonstrated controlled release of these active growth factor both in vitro and in vivo and demonstrated growth factor specific activity in vivo (BMP-2 and TGF-β1). Overall, we present a versatile protein based self-assembling system with tunable properties for tissue regeneration.