Helical assemblies: structure determinants.

Protein structural motifs such as helical assemblies and α/ß barrels combine secondary structure elements with various types of interactions. Helix-helix interfaces of assemblies - Ankyrin, ARM/HEAT, PUM, LRR, and TPR repeats - exhibit unique amino acid composition and patterns of interactions that correlate with curvature of solenoids, surface geometry and mutual orientation of the helical edges. Inner rows of ankyrin, ARM/HEAT, and PUM-HD repeats utilize edges (i-1, i) and (i+1, i+2) for the interaction of the given α-helix with preceding and following helices correspondingly, whereas outer rows of these proteins and LRR repeats invert this pattern and utilize edges (i-1, i) and (i-3, i-2). Arrangement of contacts observed in protein ligands that bind helical assemblies has to mimic the assembly pattern to provide the same curvature as a determinant of binding specificity. These characteristics are important for understanding fold recognition, specificity of protein-protein interactions, and design of new drugs and materials.

Murine isoforms of UDP-GlcNAc 2-epimerase/ManNAc kinase: Secondary structures, expression profiles, and response to ManNAc therapy.

The bifunctional enzyme UDP-GlcNAc 2-epimerase/ManNAc kinase (GNE) catalyzes the first two committed steps in sialic acid synthesis. Non-allosteric GNE gene mutations cause the muscular disorder GNE myopathy (also known as hereditary inclusion body myopathy), whose exact pathology remains unknown. Increased knowledge of GNE regulation, including isoform regulation, may help elucidate the pathology of GNE myopathy. While eight mRNA transcripts encoding human GNE isoforms are described, we only identified two mouse Gne mRNA transcripts, encoding mGne1 and mGne2, homologous to human hGNE1 and hGNE2. Orthologs of the other human isoforms were not identified in mice. mGne1 appeared as the ubiquitously expressed, major mouse isoform. The mGne2 encoding transcript is differentially expressed and may act as a tissue-specific regulator of sialylation. mGne2 expression appeared significantly increased the first 2 days of life, possibly reflecting the high sialic acid demand during this period. Tissues of the knock-in Gne p.M712T mouse model had similar mGne transcript expression levels among genotypes, indicating no effect of the mutation on mRNA expression. However, upon treatment of these mice with N-acetylmannosamine (ManNAc, a Gne substrate, sialic acid precursor, and proposed therapy for GNE myopathy), Gne transcript expression, in particular mGne2, increased significantly, likely resulting in increased Gne enzymatic activities. This dual effect of ManNAc supplementation (increased flux through the sialic acid pathway and increased Gne activity) needs to be considered when treating GNE myopathy patients with ManNAc. In addition, the existence and expression of GNE isoforms needs consideration when designing other therapeutic strategies for GNE myopathy.

Multiprotein Assemblies, Phosphorylation and Dephosphorylation in Neuronal Cytoskeleton.

Filament systems are comprised of fibrous and globular cytoskeletal proteins and are key elements regulating cell shape, rigidity, and dynamics. The cellular localization and assembly of neurofilaments depend on phosphorylation by kinases. The involvement of the BRCA1 (Breast cancer associated protein 1)/BARD1 (BRCA1-associated RING domain 1) pathways in Alzheimer disease (AD) is suggested by colocalization studies. In particular, BRCA1 accumulation within neurofibrillary tangles and colocalization with tau aggregates in the cytoplasm of AD patients implicates the involvement of mutant forms of BRCA1/BARD1 proteins in disease pathogenesis. The purpose of this study is to show that the location of mutations in the translated BARD1, specifically within ankyrin repeats, has strong correlation with the Cdk5 motifs for phosphorylation. Mapping of the mutation sites on the protein's three-dimensional structure and estimation of the backbone dihedral angles show transitions between the canonical helical and extended conformations of the tetrapeptide sequence of ankyrin repeats. Clustering of mutations in BARD1 ankyrin repeats near the N-termini of the helices with T/SXXH motifs provides a basis for conformational transitions that might be necessary to ensure the compatibility of the substrate with active site geometry and accessibility of the substrate to the kinase. Ankyrin repeats are interaction sites for phosphorylation-dependent dynamic assembly of proteins including those involved in transcription regulation and signaling, and present potential targets for the design of new drugs.

Expression pattern analysis and characterization of the hereditary sensory and autonomic neuropathy 2 A (HSAN2A) gene with no lysine kinase (WNK1) in human dorsal root ganglion.

Inherited painless neuropathies arise due to genetic insults that either block the normal signaling of or destroy the sensory afferent neurons in the dorsal root ganglion (DRG) responsible for transducing noxious stimuli. Complete loss of these neurons leads to profound insensitivity to all sensory modalities including pain. Hereditary sensory and autonomic neuropathy type 2 (HSNAII) is a rare genetic neuropathy characterized by a progressive distal early onset sensory loss. This syndrome is caused by autosomal recessive mutations in the with-no-lysine protein kinase 1 (WNK1) serine-threonine kinase gene. Of interest, disease-associated mutations are found in the large exon, termed "HSN2," which encodes a 498 amino acid domain C-terminal to the kinase domain. These mutations lead to truncation of the HSN2-containing proteins through the addition of an early stop codon (nonsense mutation) leading to loss of the C-terminal domains of this large protein. The present study evaluates the transcripts, gene structure, and protein structure of HSN2-containing WNK1 splice variants in DRG and spinal cord in order to establish the basal expression patterns of WNK1 and HSN2-containing WNK1 splice variants using multiplex fluorescent situ hybridization. We hypothesized that these transcripts would be enriched in pain-sensing DRG neurons, and, potentially, that enrichment in nociceptive neurons was responsible for the painless phenotypes observed. However, our in-depth analyses revealed that the HSN2-WNK1 splice variants were ubiquitously expressed but were not enriched in tachykinin 1-expressing C-fiber neurons, a class of neurons with a highly nociceptive character. We subsequently identified other subpopulations of DRG neurons with higher levels of HSN2-WNK1 expression, including mechanosensory large fibers. These data are inconsistent with the hypothesis that this transcript is enriched in nociceptive fibers, and instead suggest it may be related to general axon maintenance, or that nociceptive fibers are more sensitive to the genetic insult. These findings clarify the molecular and cellular expression pattern of this painless neuropathy gene in human tissue.

Identification, tissue distribution, and molecular modeling of novel human isoforms of the key enzyme in sialic acid synthesis, UDP-GlcNAc 2-epimerase/ManNAc kinase.

UDP-GlcNAc 2-epimerase/ManNAc kinase (GNE) catalyzes the first two committed steps in sialic acid synthesis. In addition to the three previously described human GNE isoforms (hGNE1-hGNE3), our database and polymerase chain reaction analysis yielded five additional human isoforms (hGNE4-hGNE8). hGNE1 is the ubiquitously expressed major isoform, while the hGNE2-hGNE8 isoforms are differentially expressed and may act as tissue-specific regulators of sialylation. hGNE2 and hGNE7 display a 31-residue N-terminal extension compared to hGNE1. On the basis of similarities to kinases and helicases, this extension does not seem to hinder the epimerase enzymatic active site. hGNE3 and hGNE8 contain a 55-residue N-terminal deletion and a 50-residue N-terminal extension compared to hGNE1. The size and secondary structures of these fragments are similar, and modeling predicted that these modifications do not affect the overall fold compared to that of hGNE1. However, the epimerase enzymatic activity of GNE3 and GNE8 is likely absent, because the deleted fragment contains important substrate binding residues in homologous bacterial epimerases. hGNE5-hGNE8 have a 53-residue deletion, which was assigned a role in substrate (UDP-GlcNAc) binding. Deletion of this fragment likely eliminates epimerase enzymatic activity. Our findings imply that GNE is subject to evolutionary mechanisms to improve cellular functions, without increasing the number of genes. Our expression and modeling data contribute to elucidation of the complex functional and regulatory mechanisms of human GNE and may contribute to further elucidating the pathology and treatment strategies of the human GNE-opathies sialuria and hereditary inclusion body myopathy.

Helix-helix interfaces and ligand binding.

Helix-helix parallel interfaces can be characterized by certain combinations of amino acids, which repeatedly occur at core positions a and d (leucine zipper nomenclature) in homologous and nonhomologous proteins and influence interhelical angles. Applied for the prediction of interhelical angles in glutathione S-transferase, intracellular chloride channel and annexin molecules from various sources, correct results were achieved in 58 out of 62 proteins. Interhelical angles are found to correlate with the conformation of the glutathione S-transferase ligands glutathione, s-hexylglutathione, glutathione sulfonic acid, and glutathione-s-dinitrobenzene.

Molecular modeling of the bifunctional enzyme UDP-GlcNAc 2-epimerase/ManNAc kinase and predictions of structural effects of mutations associated with HIBM and sialuria.

The bifunctional enzyme UDP-GlcNAc 2-epimerase/ ManNAc kinase (GNE/MNK), encoded by the GNE gene, catalyzes the first two committed, rate-limiting steps in the biosynthesis of N-acetylneuraminic acid (sialic acid). GNE/MNK is feedback inhibited by binding of the downstream product, CMP-sialic acid in its allosteric site. GNE mutations can result in two human disorders, hereditary inclusion body myopathy (HIBM) or sialuria. So far, no active site geometry predictions or conformational transitions involved with function are available for mammalian GNE/MNK. The N-terminal GNE domain is homologous to various prokaryotic 2-epimerases, some of which have solved crystallographic structures. The C-terminal MNK domain belongs to the sugar kinases superfamily; its crystallographic structure is solved at 2.84 A and three-dimensional structures have also been reported for several other kinases. In this work, we employed available structural data of GNE/MNK homologs to model the active sites of human GNE/MNK and identify critical amino acid residues responsible for interactions with substrates. In addition, we modeled effects of GNE/MNK missense mutations associated with HIBM or sialuria on helix arrangement, substrate binding, and enzyme action. We found that all reported mutations are associated with the active sites or secondary structure interfaces of GNE/MNK. The Persian-Jewish HIBM founder mutation p.M712T is located at the interface alpha4alpha10 and likely affects GlcNAc, Mg2+, and ATP binding. This work contributes to further understanding of GNE/MNK function and ligand binding, which may assist future studies for therapeutic options that target misfolded GNE/MNK in HIBM and/or sialuria.

Helix-helix interactions and their impact on protein motifs and assemblies.

Protein secondary structure elements are arranged in distinct structural motifs such as four-alpha-helix bundle, 8alpha/8beta TIM-barrel, Rossmann dinucleotide binding fold, assembly of a helical rod. Each structural motif is characterized by a particular type of helix-helix interactions. A unique pattern of contacts is formed by interacting helices of the structural motif. In each type of fold, edges of the helix surface, which participate in the formation of helix-helix contacts with preceding and following helices, differ. This work shows that circular arrangements of the four, eight, and sixteen alpha-helices, which are found in the four-alpha-helical motif, TIM-barrel 8alpha/8beta fold, and helical rod of 16.3 helices per turn correspondingly, can be associated with the mutual positioning of the edges of the helix surfaces. Edges (i, i+1)-(i+1, i+2) of the helix surface are central for the interhelical contacts in a four-alpha-helix bundle. Edges (i, i+1)-(i+2, i+3) are involved in the assembly of four-alpha-helix subunits into helical rod of a tobacco mosaic virus and a three-helix fragment of a Rossmann fold. In 8alpha/8beta TIM-barrel fold, edges (i, i+1)-(i+5, i+6) are involved in the octagon arrangement. Approximation of a cross section of each motif with a polygon (n-gon, n=4, 8, 16) shows that a good correlation exists between polygon interior angles and angles formed by the edges of helix surfaces.

Phosphorylation, Dephosphorylation, and Multiprotein Assemblies Regulate Dynamic Behavior of Neuronal Cytoskeleton: A Mini-Review.

Cellular localization, assembly and abnormal aggregation of neurofilaments depend on phosphorylation. Pathological processes associated with neurodegeneration exhibit aberrant accumulation of microtubule associated aggregated forms of hyperphosphorylated neuronal protein tau in cell bodies. These processes are critical for the disease progression in patients suffering from Alzheimer's disease, Parkinson's disease, and Amyotrophic Lateral Sclerosis. In healthy cells, tau is localized in axons. Topographic regulation suggests that whereas the sites of synthesis of kinases and neurofilaments are the cell bodies, and sites of their functional assemblies are axons, phosphorylation/dephosphorylation are the key processes that arrange the molecules at their precise locations. Phosphorylation sites in the dynamic developmental and degenerative processes differ. Not all these processes are well understood. New advancements identify epigenetic factors involved in AD which account for the influence of age-related environment/genome interactions leading to the disease. Progress in proteomics highlights previously found major proteins and adds more to the list of those involved in AD. New key elements of specificity provide determinants of molecular recognition important for the assembly of macromolecular complexes. In this review, we discuss aberrant spatial distribution of neuronal polypeptides observed in neuropathies: aggregation, association with proteins of the neuronal cytoskeleton, and phosphorylation dependent dynamics. Particularly, we emphasize recent advancements in understanding the function and determinants of specific association of molecules involved in Alzheimer's disease with respect to the topographic regulation of phosphorylation in neuronal cytoskeleton and implications for the design of new therapies. Further, we address the role of various filament systems in maintenance of the shape, rigidity and dynamics of the cytoskeleton.

Phosphorylation of the Transient Receptor Potential Ankyrin 1 by Cyclin-dependent Kinase 5 affects Chemo-nociception.

Cyclin-dependent kinase 5 (Cdk5) is a key neuronal kinase that is upregulated during inflammation, and can subsequently modulate sensitivity to nociceptive stimuli. We conducted an in silico screen for Cdk5 phosphorylation sites within proteins whose expression was enriched in nociceptors and identified the chemo-responsive ion channel Transient Receptor Potential Ankyrin 1 (TRPA1) as a possible Cdk5 substrate. Immunoprecipitated full length TRPA1 was shown to be phosphorylated by Cdk5 and this interaction was blocked by TFP5, an inhibitor that prevents activation of Cdk5. In vitro peptide-based kinase assay revealed that four of six TRPA1 Cdk5 consensus sites acted as substrates for Cdk5, and modeling of the ankyrin repeats disclosed that phosphorylation would occur at characteristic pockets within the (T/S)PLH motifs. Calcium imaging of trigeminal ganglion neurons from genetically engineered mice overexpressing or lacking the Cdk5 activator p35 displayed increased or decreased responsiveness, respectively, to stimulation with the TRPA1 agonist allylisothiocyanate (AITC). AITC-induced chemo-nociceptive behavior was also heightened in vivo in mice overexpressing p35 while being reduced in p35 knockout mice. Our findings demonstrate that TRPA1 is a substrate of Cdk5 and that Cdk5 activity is also able to modulate TRPA1 agonist-induced calcium influx and chemo-nociceptive behavioral responses.

SH3 domains: modules of protein-protein interactions.

Src homology 3 (SH3) domains are involved in the regulation of important cellular pathways, such as cell proliferation, migration and cytoskeletal modifications. Recognition of polyproline and a number of noncanonical sequences by SH3 domains has been extensively studied by crystallography, nuclear magnetic resonance and other methods. High-affinity peptides that bind SH3 domains are used in drug development as candidates for anticancer treatment. This review summarizes the latest achievements in deciphering structural determinants of SH3 function.