Calmodulin Binding Proteins and Alzheimer's Disease: Biomarkers, Regulatory Enzymes and Receptors That Are Regulated by Calmodulin.

The integral role of calmodulin in the amyloid pathway and neurofibrillary tangle formation in Alzheimer's disease was first established leading to the "Calmodulin Hypothesis". Continued research has extended our insight into the central function of the small calcium sensor and effector calmodulin and its target proteins in a multitude of other events associated with the onset and progression of this devastating neurodegenerative disease. Calmodulin's involvement in the contrasting roles of calcium/CaM-dependent kinase II (CaMKII) and calcineurin (CaN) in long term potentiation and depression, respectively, and memory impairment and neurodegeneration are updated. The functions of the proposed neuronal biomarker neurogranin, a calmodulin binding protein also involved in long term potentiation and depression, is detailed. In addition, new discoveries into calmodulin's role in regulating glutamate receptors (mGluR, NMDAR) are overviewed. The interplay between calmodulin and amyloid beta in the regulation of PMCA and ryanodine receptors are prime examples of how the buildup of classic biomarkers can underly the signs and symptoms of Alzheimer's. The role of calmodulin in the function of stromal interaction molecule 2 (STIM2) and adenosine A2A receptor, two other proteins linked to neurodegenerative events, is discussed. Prior to concluding, an analysis of how targeting calmodulin and its binding proteins are viable routes for Alzheimer's therapy is presented. In total, calmodulin and its binding proteins are further revealed to be central to the onset and progression of Alzheimer's disease.

Calmodulin-binding transcription activator (CAMTA) genes family: Genome-wide survey and phylogenetic analysis in flax (Linum usitatissimum).

Flax (Linum usitatissimum) is a member of family linaceae with annual growth habit. It is included among those crops which were domesticated very early and has been used in development related studies as a model plant. In plants, Calmodulin-binding transcription activators (CAMTAs) comprise a unique set of Calmodulin-binding proteins. To elucidate the transport mechanism of secondary metabolites in flax, a genome-based study on these transporters was performed. The current investigation identified nine CAMTAs proteins, classified into three categories during phylogenetic analysis. Each group had significant evolutionary role as illustrated by the conservation of gene structures, protein domains and motif organizations over the distinctive phylogenetic classes. GO annotation suggested a link to sequence-specific DNA and protein binding, response to low temperature and transcription regulation by RNA polymerase II. The existence of different hormonal and stress responsive cis-regulatory elements in promotor region may directly correlate with the variation of their transcripts. MicroRNA target analysis revealed that various groups of miRNA families targeted the LuCAMTAs genes. Identification of CAMTA genes, miRNA studies and phylogenetic analysis may open avenues to uncover the underlying functional mechanism of this important family of genes in flax.

Genome-wide analysis of the Chinese cabbage IQD gene family and the response of BrIQD5 in drought resistance.

KEY MESSAGE: Thirty-five IQD genes were identified and analysed in Chinese cabbage and BrIQD5 transgenic plants enhanced the drought resistance of plants. The IQD (IQ67-domain) family plays an important role in various abiotic stress responses in plant species. However, the roles of IQD genes in the Chinese cabbage response to abiotic stress remain unclear. Here, 35 IQD genes, from BrIQD1 to BrIQD35, were identified in Chinese cabbage (Brassica rapa ssp. pekinensis). Based on the phylogenetic analysis, these genes were clustered into three subfamilies (I-III), and members within the same subfamilies shared conserved exon-intron distribution and motif composition. The 35 BrIQD genes were unevenly distributed on 9 of the 10 chromosomes with 4 segmental duplication events. Ka/Ks ratios showed that the duplicated BrIQDs had mainly experienced strong purifying selection. Quantitative real-time polymerase chain reaction of 35 BrIQDs under PEG6000 indicated that BrIQD5 was significantly induced by PEG6000. To verify BrIQD5 function, BrIQD5 was heterologously overexpressed in tobacco and was silenced in Chinese cabbage. BrIQD5-overexpressed plants showed more tolerance to drought stress than wild-type plants, while BrIQD5-silenced plants in Chinese cabbage showed decreased drought tolerance. Additionally, six BrIQD5 potential interactive proteins were isolated by the yeast two-hybrid assay, including BrCaMa, BrCaMb and four other stress-related proteins. Motif IQ1 of BrIQD5 is important for the interaction with BrCaMa and BrCaMb, and the isoleucine in motif IQ1 is an essential amino acid for calmodulin binding to BrIQD5. The identification and cloning of the new Chinese cabbage drought tolerance genes will promote the drought-resistant breeding of Chinese cabbage and help to better understand the mechanism of IQD involved in the drought tolerance of plants.

Molecular cloning, characterization, and expression of the endothelial differentiation-related factor-1 (EDF-1) in Gekko japonicus.

Full-length cDNA of the Endothelial Differentiation-related Factor-1 (EDF-1) of Gekkojaponicus was obtained by the method of rapid amplification of cDNA ends polymerase chain reaction (RACE-PCR). EDF-1 is 1025 bp in length and contains an open reading frame (ORF) corresponding to a deduced protein of 148 amino acids. The molecular mass of the predicted protein is 16.28 kDa and its theoretical isoelectric point is 9.87. Its amino acid sequence shares 57-93% similarity with EDF-1 of a number of other species. Gecko EDF-1 is expressed in most gecko tissues examined including the brain, spinal cord, liver, and kidney, with higher levels in the last two. In situ hybridization (ISH) revealed that positive hybridization signals were present in both the gray and white matter of the spinal cord.

Identification of calmodulin-binding proteins in brain of worker honeybees.

Calmodulin is a Ca(+2)-binding protein important in a variety of cell functions. The Ca(+2)/calmodulin complex interacts with and regulates various enzymes and target proteins, known as calmodulin-binding proteins (CaMBPs). In this study, we revealed a comparative identification of the CaMBPs composition in the worker honeybee (Apis mellifera) brain, considering two different honeybee behaviors in the colony. To this end, the CaMBPs of forager and nurse workers were purified by affinity chromatography, separated in 1D gel, digested and submitted to peptide mass fingerprinting (PMF). In the PMF analysis, 15 different proteins, considered behavior-specific proteins, were identified, one of them exclusively in forager workers and 10 in nurses. All the proteins were classified in terms of their function and cell localization, revealing a greater expression of metabolism-related CaMBPs in both worker subcastes. Protein sequences were then analyzed for the presence of the calmodulin-binding sites. Therefore, the honeybee brain CaMBPs profiles presented differences between worker subcastes. This is the first identification of calmodulin-binding proteins in the brain of A. mellifera upon nursing and foraging behaviors in the colony and this diversity of target proteins for Ca(+2)/CaM may be involved in terms of the function of these proteins in the nervous system.

The armadillo repeat domain of the APC tumor suppressor protein interacts with Striatin family members.

Adenomatous polyposis coli (APC) is a multifunctional tumor suppressor protein that negatively regulates the Wnt signaling pathway. The APC gene is ubiquitously expressed in various tissues, especially throughout the large intestine and central nervous system. Mutations in the gene encoding APC have been found in most colorectal cancers and in other types of cancer. The APC gene product is a large multidomain protein that interacts with a variety of proteins, many of which bind to the well conserved armadillo repeat domain of APC. Through its binding partners, APC affects a large number of important cellular processes, including cell-cell adhesion, cell migration, organization of the actin and microtubule cytoskeletons, spindle formation and chromosome segregation. The molecular mechanisms that control these diverse APC functions are only partly understood. Here we describe the identification of an additional APC armadillo repeat binding partner - the Striatin protein. The Striatin family members are multidomain molecules that are mainly neuronal and are thought to function as scaffolds. We have found that Striatin is expressed in epithelial cells and co-localizes with APC in the epithelial tight junction compartment and in neurite tips of PC12 cells. The junctional localization of APC and Striatin is actin-dependent. Depletion of APC or Striatin affected the localization of the tight junction protein ZO-1 and altered the organization of F-actin. These results raise the possibility that the contribution of APC to cell-cell adhesion may be through interaction with Striatin in the tight junction compartment of epithelial cells.

The striatin family: a new signaling platform in dendritic spines.

Proteins of the striatin family have been identified in all multicellular animals. They are multidomain molecules containing several protein-interacting motifs. In mammals, these proteins are principally expressed in neurons with a somato-dendritic localization and high concentration in dendritic spines. Recent reports suggest that the proteins of the striatin family are molecular scaffolds that act as links between signal transduction and vesicular trafficking.

WRKY group IId transcription factors interact with calmodulin.

Calmodulin (CaM) is a ubiquitous Ca(2+)-binding protein known to regulate diverse cellular functions by modulating the activity of various target proteins. We isolated a cDNA encoding AtWRKY7, a novel CaM-binding transcription factor, from an Arabidopsis expression library with horseradish peroxidase-conjugated CaM. CaM binds specifically to the Ca(2+)-dependent CaM-binding domain (CaMBD) of AtWRKY7, as shown by site-directed mutagenesis, a gel mobility shift assay, a split-ubiquitin assay, and a competition assay using a Ca2+/CaM-dependent enzyme. Furthermore, we show that the CaMBD of AtWRKY7 is a conserved structural motif (C-motif) found in group IId of the WRKY protein family.

Calmodulin-binding proteins in bryophytes: identification of abscisic acid-, cold-, and osmotic stress-induced genes encoding novel membrane-bound transporter-like proteins.

Plant responses to environmental stresses are mediated in part by signaling processes involving cytosolic Ca2+ and a Ca(2+)-binding protein, calmodulin. Screening with radiolabeled calmodulin of a cDNA library of the moss Physcomitrella patens resulted in identification of genes encoding novel membrane transporter-like proteins, MCamb1 and MCamb2. These proteins each had a central hydrophobic domain with two putative membrane spans and N- and C-terminal hydrophilic domains, and showed sequence similarity to mammalian inward rectifier potassium channels. Calmodulin binds to MCamb1 and MCamb2 via interaction with basic amphiphilic amino acids in the C-terminal domain. Levels of MCamb1 and MCamb2 transcripts increased dramatically following treatment with low temperature, hyperosmotic solutes, and the stress hormone abscisic acid, all of which were previously shown to increase cellular tolerance to freezing stress. These results suggest that calmodulin participates in cellular signaling events leading to enhancement of stress resistance through regulation of novel transporter-like proteins.

New nuclear functions for calmodulin.

The data reported here summarize a series of results which reveal new functions for nuclear calmodulin (CaM). The addition of CaM inhibitors to cultures of proliferating NRK cells blocked the activity of the cyclin-dependent protein kinases 4 (cdk4) and 2 (cdk2), which are enzymes implicated in the progression of G1 and in the onset of DNA replication, respectively. CaM modulates the activity of cdk4 by regulating the nuclear location of both cdk4 and cyclin D, its associated regulatory subunit. By using CaM-affinity chromatography, we have recently identified two new nuclear CaM-binding proteins: (i) the protein La/SSB, which is an autoantigen implicated in several autoimmune diseases such as lupus erythematosus and Sjögren's syndrome (since La/SSB participates in the process of transcription mediated by RNA polymerase III, CaM could be involved in the regulation of this process); and (ii) the protein SAP145, a member of the spliceosome-associated proteins (SAPs) which is a subunit of the splicing factor SF3(b). This finding suggests the involvement of CaM in pre-mRNA splicing. Finally, a screening for new CaM-binding proteins in the fission yeast performed by using the phage display analysis, revealed that several nucleolar-ribosomal proteins associate to CaM, suggesting that CaM modulates ribosomal assembly and/or function.

Structural organization of a gene encoding a novel calmodulin-binding kinesin-like protein from Arabidopsis.

Kinesin-like calmodulin-binding protein (KCBP) is a recently identified microtubule motor protein that appears to be unique to plants. Here we report isolation and sequence analysis of a gene encoding Arabidopsis KCBP. KCBP gene contains 21 exons and 20 introns. All exons except exon 3 are short (94-272 nt). Exons 1-9 code for the globular tail region whereas the coiled-coil region is coded by exons 10-15. The conserved motor domain is coded by exons 16-20. Calmodulin-binding domain that is present in the C-terminal region of the protein and unique to KCBP is coded by the last exon. The size of introns ranged from 71 (intron 17) to 320 (intron 19) nucleotides. As in most plant introns, the content of AT is very high in all introns (up to 76%). Phylogenetic analysis of KCBP using motor domain sequence grouped KCBP with other known C-terminal microtubule motor proteins. However, Arabidopsis KCBP together with its homologs from potato and tobacco constitute a distinct group within the C-terminal subfamily of motors which is consistent with structural and functional features of KCBP.