Novel keratins identified by quantitative proteomic analysis as the major cytoskeletal proteins of equine (Equus caballus) hoof lamellar tissue

The dermo-epidermal interface that connects the equine distal phalanx to the cornified hoof wall withstands great biomechanical demands, but is also a region where structural failure often ensues as a result of laminitis. The cytoskeleton in this region maintains cell structure and facilitates intercellular adhesion, making it likely to be involved in laminitis pathogenesis, although it is poorly characterized in the equine hoof lamellae. The objective of the present study was to identify and quantify the cytoskeletal proteins present in the epidermal and dermal lamellae of the equine hoof by proteomic techniques. Protein was extracted from the mid-dorsal epidermal and dermal lamellae from the front feet of 5 Standardbred geldings and 1 Thoroughbred stallion. Mass spectrometry-based spectral counting techniques, PAGE, and immunoblotting were used to identify and quantify cytoskeletal proteins, and indirect immunofluorescence was used for cellular localization of K14 and K124 (where K refers to keratin). Proteins identified by spectral counting analysis included 3 actin microfilament proteins; 30 keratin proteins along with vimentin, desmin, peripherin, internexin, and 2 lamin intermediate filament proteins; and 6 tubulin microtubule proteins. Two novel keratins, K42 and K124, were identified as the most abundant cytoskeletal proteins (22.0 ± 3.2% and 23.3 ± 4.2% of cytoskeletal proteins, respectively) in equine hoof lamellae. Immunoreactivity to K14 was localized to the basal cell layer, and that to K124 was localized to basal and suprabasal cells in the secondary epidermal lamellae. Abundant proteins K124, K42, K14, K5, and α1-actin were identified on 1- and 2-dimensional polyacrylamide gels and aligned with the results of previous studies. Results of the present study provide the first comprehensive analysis of cytoskeletal proteins present in the equine lamellae by using mass spectrometry-based techniques for protein quantification and identification.

Moderate exercise changes synaptic and cytoskeletal proteins in motor regions of the rat brain

Physical exercise is known to enhance brain function in several aspects. We evaluated the acute effects of a moderate forced exercise protocol on synaptic proteins, namely synapsin 1 (SYN) and synaptophysin (SYP), and structural proteins (neurofilaments, NFs) in rat brain regions related to motor function and often affected by neurodegenerative disorders. Immunohistochemistry, Western blotting and real-time PCR were used to analyze the expression of those proteins after 3, 7 and 15 days of exercise (EX3, EX7 and EX15). In the cerebellum, increase of SYN was observed at EX7 and EX15 and of NF68 at EX3. In the substantia nigra, increases of protein levels were observed for NF68 and NF160 at EX3. In the striatum, there was an increase of SYN at EX3 and EX7, of SYP at EX7 and of NF68 at EX3. In the cortex, decreased levels of NF68 and NF160 were observed at EX3, followed by an increase of NF68 at EX15. In the reticular formation, all NF proteins were increased at EX15. The mRNA data for each time-point and region also revealed significant exercise-related changes of SYN, SYP and NF expression. These results suggest that moderate physical exercise modulates synaptic and structural proteins in motor brain areas, which may play an important role in the exercise-dependent brain plasticity. (C) 2010 Elsevier B.V. All rights reserved.

Zyxin interacts with the SH3 domains of the cytoskeletal proteins LIM-nebulette and Lasp-1

Zyxin is a versatile component of focal adhesions in eukaryotic cells. Here we describe a novel binding partner of zyxin, which we have named LIM-nebulette. LIM-nebulette is an alternative splice variant of the sarcomeric protein nebulette, which, in contrast to nebulette, is expressed in non-muscle cells. It displays a modular structure with an N-terminal LIM domain, three nebulin-like repeats, and a C-terminal SH3 domain and shows high similarity to another cytoskeletal protein, Lasp-1 (LIM and SH3 protein-1). Co-precipitation studies and results obtained with the two-hybrid system demonstrate that LIM-nebulette and Lasp-1 interact specifically with zyxin. Moreover, the SH3 domain from LIM-nebulette is both necessary and sufficient for zyxin binding. The SH3 domains from Lasp-1 and nebulin can also interact with zyxin, but the SH3 domains from more distantly related proteins such as vinexin and sorting nexin 9 do not. On the other hand, the binding site in zyxin is situated at the extreme N terminus as shown by site-directed mutagenesis. LIM-nebulette and Lasp-1 use the same linear binding motif. This motif shows some similarity to a class II binding site but does not contain the classical PXXP sequence. LIM-nebulette reveals a subcellular distribution at focal adhesions similar to Lasp-1. Thus, LIM-nebulette, Lasp-1, and zyxin may play an important role in the organization of focal adhesions.

The role of cytoskeletal proteins in the mechanism of insulin release

This thesis is concerned with the role of /3-cell cytoskeletal proteins in the mechanism of insulin release from islets of experimental animals, the Aston obese diabetic hyperglycaemic (ob/ob) mouse and their lean littermates and the cultural insulin secreting /?-cell lines, HIT-TT5 and RINm5F. Investigations were carried out into the glucose induced insulin response of the lean and obese mouse islets and HIT-TI5 cells and the D-glyceraldehyde response of RINm5F cells using a static incubation system. Colchicine was found to inhibit insulin release from both lean and obese mouse islets more significantly than cultured TTT-TI5 and RINm5F cells. (Colchicine pre-treatment also inhibited the second phase of insulin release from perifused lean mouse islets and HIT-TI5 cells). Cytocha-lasin B, used to investigate the role of the microfilamentous system in the mechanism of insulin release enhanced insulin release from both lean and obese mouse islets to a significantly greater degree than that from cultured HIT-TI5 and RINm5F cells. Pre-treatment of isolated lean and obese mouse islets and cultured /?-cells with a combination of colchicine and cytochalasin B significantly reduced the insulin response of the HIT-TI5 and RINm5F cells compared with the control values suggesting that intact microtubules are more important for the sustained release of insulin than the microfilamentous system. However, the response was not so clearly defined with the lean and obese mouse islets. Tubulin was separated from the extracts of lean mouse islets and the HIT-TI5 and RINm5F cells and actin was separated from all of the cell types including the obese mouse islets by SDS- polyacrylamide electrophoresis. A tubulin radioimmunoassay and a colchicine binding assay were developed to measure the tubulin content of lean and obese mouse islets, and the shift between the proportions of tubulin dimers and polymerized tubulin under stimulatory and non-stimulatory conditions. The assay methods developed were not prone to be accurate, sensitive and precise but gave some indication of the shift from unpolymerised to polymerised tubulin during glucose stimulated insulin release. These studies show that microtubules do play a fundamental role in the mechanism of insulin release from both islets and cultured HIT-TI5 and RINm5F cells.

Interaction of metal salts with cytoskeletal motor protein systems

Interactions of chemicals with the microtubular network of cells may lead to genotoxicity. Micronuclei (MN) might be caused by interaction of metals with tubulin and/or kinesin. The genotoxic effects of inorganic lead and mercury salts were studied using the MN assay and the CREST analysis in V79 Chinese hamster fibroblasts. Effects on the functional activity of motor protein systems were examined by measurement of tubulin assembly and kinesin-driven motility. Lead and mercury salts induced MN dose-dependently. The no-effect-concentration for MN induction was 1.1 μM PbCl2, 0.05 μM Pb(OAc)2 and 0.01 μM HgCl2. The in vitro results obtained for PbCl2 correspond to reported MN induction in workers occupationally exposed to lead, starting at 1.2 μM Hg(II) (Vaglenov et al., 2001, Environ. Health Perspect. 109, 295-298). The CREST Analysis indicate aneugenic effects of Pb(II) and aneugenic and additionally clastogenic effects of Hg(II). Lead (chloride, acetate, and nitrate) and mercury (chloride and nitrate) interfered dose-dependently with tubulin assembly in vitro. The no-effect-concentration for lead salts in this assay was 10 μM. Inhibition of tubulin assembly by mercury started at 2 μM. The gliding velocity of microtubules along immobilised kinesin molecules was affected by 25 μM Pb(NO3)2 and 0.1 μM HgCl2 in a dose-dependent manner. Our data support the hypothesis that lead and mercury genotoxicity may result, at least in part, via disturbance of chromosome segregation via interaction with cytoskeletal proteins.

Consensus nomenclature for the human ArfGAP domain-containing proteins.

At the FASEB summer research conference on "Arf Family GTPases", held in Il Ciocco, Italy in June, 2007, it became evident to researchers that our understanding of the family of Arf GTPase activating proteins (ArfGAPs) has grown exponentially in recent years. A common nomenclature for these genes and proteins will facilitate discovery of biological functions and possible connections to pathogenesis. Nearly 100 researchers were contacted to generate a consensus nomenclature for human ArfGAPs. This article describes the resulting consensus nomenclature and provides a brief description of each of the 10 subfamilies of 31 human genes encoding proteins containing the ArfGAP domain.

Expression of SCG10 and stathmin proteins in the rat olfactory system during development and axonal regeneration.

The membrane-associated protein SCG10 is expressed specifically by neuronal cells. Recent experiments have suggested that it promotes neurite outgrowth by increasing microtubule dynamics in growth cones. SCG10 is related to the ubiquitous but neuron-enriched cytosolic protein stathmin. To better understand the role played by SCG10 and stathmin in vivo, we have analyzed the expression and localization of these proteins in both the olfactory epithelium and the olfactory bulb in developing and adult rats, as well as in adult bulbectomized rats. The olfactory epithelium is exceptional in that olfactory receptor neurons constantly regenerate and reinnervate the olfactory bulb throughout animal life-span. SCG10 and stathmin expression in the olfactory receptor neurons was found to be regulated during embryonic and postnatal development and to correlate with neuronal maturation. Whereas SCG10 expression was restricted to immature olfactory receptor neurons (GAP-43-positive, olfactory marker protein-negative), stathmin was also expressed by the basal cells. In the olfactory bulb of postnatal and adult rats, a moderate to strong SCG10 immunoreactivity was present in the olfactory nerve layer, whereas no labeling was detected in the glomerular layer. Olfactory glomeruli also showed no apparent immunoreactivity for several cytoskeletal proteins such as tubulin and microtubule-associated proteins. In unilaterally bulbectomized rats, SCG10 and stathmin were seen to be up-regulated in the regenerating olfactory epithelium at postsurgery stages corresponding to olfactory axon regeneration. Our data strongly suggest that, in vivo, both SCG10 and stathmin may play a role in axonal outgrowth during ontogenesis as well as during axonal regeneration.

Traumatic brain injury (TBI) produces cytoskeletal protein derangements within cortical neurons that can be attenuated by protease inhibition

TBI produces a consistent and extensive loss of neurofilament 68 (NF68) and neurofilament 200 (NF200), key intermediate cytoskeletal proteins found in neurons including axons and dendrites, in cortical samples from injured brain. The presence of low molecular weight NF68 breakdown products (BDPs) strongly suggest that calpain proteolysis at least in part contributes to neurofilament (NF) protein loss following injury. Furthermore, one and two-dimensional gel electrophoresis analyses of NF BDPs obtained from in situ and in vitro tissue also implicated the involvement of calpain 2 mediated proteolysis of neurofilaments following TBI. Immunohistochemical examination of derangements in cytoskeletal proteins following traumatic brain injury in rats indicated that preferential dendritic rather than axonal damage occurs within three hours post-TBI. Although proteolysis of cytoskeletal proteins occurred concurrently with early morphological alterations, evidence of proteolysis preceded the full expression of evolutionary histopathological changes. Furthermore, cytoskeletal immunofluorescence alterations were not restricted to the site of impact. Confocal microscopic investigations of NF68 and NF200 immunofluorescence within injured cortical neurons revealed alterations in neurofilament assembly in the absence of NF derangements detectable at the light microscopic level ($<$15 minutes post-TBI). Collectively immunohistochemistry studies suggest that derangements to neuronal processes are biochemical and evolutionary in nature, and not due solely to mechanical shearing. Importantly, a systemically administered calpain inhibitor (calpain inhibitor 2) significantly reduced NF200, NF68, and spectrin protein loss as well as providing marked preservation of NF proteins in neuronal somata, dendrites, and axons at 24 hours post-TBI. ^

Primary structure and tissue distribution of two novel proline-rich γ-carboxyglutamic acid proteins

Two human cDNAs that encode novel vitamin K-dependent proteins have been cloned and sequenced. The predicted amino acid sequences suggest that both are single-pass transmembrane proteins with amino-terminal γ-carboxyglutamic acid-containing domains preceded by the typical propeptide sequences required for posttranslational γ-carboxylation of glutamic acid residues. The polypeptides, with deduced molecular masses of 23 and 17 kDa, are proline-rich within their putative cytoplasmic domains and contain several copies of the sequences PPXY and PXXP, motifs found in a variety of signaling and cytoskeletal proteins. Accordingly, these two proteins have been called proline-rich Gla proteins (PRGP1 and PRGP2). Unlike the γ-carboxyglutamic acid domain-containing proteins of the blood coagulation cascade, the two PRGPs are expressed in a variety of extrahepatic tissues, with PRGP1 and PRGP2 most abundantly expressed in the spinal cord and thyroid, respectively, among those tissues tested. Thus, these observations suggest a novel physiological role for these two new members of the vitamin K-dependent family of proteins.

Transport of cytoskeletal elements in the squid giant axon.

In order to explore how cytoskeletal proteins are moved by axonal transport, we injected fluorescent microtubules and actin filaments as well as exogenous particulates into squid giant axons and observed their movements by confocal microscopy. The squid giant axon is large enough to allow even cytoskeletal assemblies to be injected without damaging the axon or its transport mechanisms. Negatively charged, 10- to 500-nm beads and large dextrans moved down the axon, whereas small (70 kDa) dextrans diffused in all directions and 1000-nm beads did not move. Only particles with negative charge were transported. Microtubules and actin filaments, which have net negative charges, made saltatory movements down the axon, resulting in a net rate approximating that previously shown for slow transport of cytoskeletal elements. The present observations suggest that particle size and charge determine which materials are transported down the axon.

Specific isoforms of actin-binding proteins on distinct populations of Golgi-derived vesicles

Golgi membranes and Golgi-derived vesicles are associated with multiple cytoskeletal proteins and motors, the diversity and distribution of which have not yet been defined. Carrier vesicles were separated from Golgi membranes, using an in vitro budding assay, and different populations of vesicles were separated using sucrose density gradients. Three main populations of vesicles labeled with beta-COP, gamma-adaptin, or p200/myosin II were separated and analyzed for the presence of actin/actin-binding proteins, beta-Actin was bound to Golgi cisternae and to all populations of newly budded vesicles. Centractin was selectively associated with vesicles co-distributing with beta-COP-vesicles, while p200/myosin II (non-muscle myosin IIA) and non-muscle myosin IIB were found on different vesicle populations. Isoforms of the Tm5 tropomyosins were found on selected Golgi-derived vesicles, while other Tm isoforms did not colocalize with Tm5 indicating the association of specialized actin filaments with Golgi-derived vesicles. Golgi-derived vesicles were shown to bind to F-actin polymerized from cytosol with Jasplakinolide. Thus, newly budded, coated vesicles derived from Golgi membranes can bind to actin and are customized for differential interactions with microfilaments by the presence of selective arrays of actin-binding proteins.

Biochemical studies of postsynaptic density signaling proteins with a focus on synaptic GTPase activating protein and PDZ domains

Memory storage in the brain involves adjustment of the strength of existing synapses and formation of new neural networks. A key process underlying memory formation is synaptic plasticity, the ability of excitatory synapses to strengthen or weaken their connections in response to patterns of activity between their connected neurons. Synaptic plasticity is governed by the precise pattern of Ca²⁺ influx through postsynaptic N-methyl-D-aspartate-type glutamate receptors (NMDARs), which can lead to the activation of the small GTPases Ras and Rap. Differential activation of Ras and Rap acts to modulate synaptic strength by promoting the insertion or removal of 2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl)propanoic acid receptors (AMPARs) from the synapse. Synaptic GTPase activating protein (synGAP) regulates AMPAR levels by catalyzing the inactivation of GTP-bound (active) Ras or Rap. synGAP is positioned in close proximity to the cytoplasmic tail regions of the NMDAR through its association with the PDZ domains of PSD-95. SynGAP’s activity is regulated by the prominent postsynaptic protein kinase, Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) and cyclin-dependent kinase 5 (CDK5), a known binding partner of CaMKII. Modulation of synGAP’s activity by phosphorylation may alter the ratio of active Ras to Rap in spines, thus pushing the spine towards the insertion or removal of AMPARs, subsequently strengthening or weakening the synapse. To date, all biochemical studies of the regulation of synGAP activity by protein kinases have utilized impure preparations of membrane bound synGAP. Here we have clarified the effects of phosphorylation of synGAP on its Ras and Rap GAP activities by preparing and utilizing purified, soluble recombinant synGAP, Ras, Rap, CaMKII, CDK5, PLK2, and CaM. Using mass spectrometry, we have confirmed the presence of previously identified CaMKII and CDK5 sites in synGAP, and have identified novel sites of phosphorylation by CaMKII, CDK5, and PLK2. We have shown that the net effect of phosphorylation of synGAP by CaMKII, CDK5, and PLK2 is an increase in its GAP activity toward HRas and Rap1. In contrast, there is no effect on its GAP activity toward Rap2. Additionally, by assaying the GAP activity of phosphomimetic synGAP mutants, we have been able to hypothesize the effects of CDK5 phosphorylation at specific sites in synGAP. In the course of this work, we also found, unexpectedly, that synGAP is itself a Ca²⁺/CaM binding protein. While Ca²⁺/CaM binding does not directly affect synGAP activity, it causes a conformational change in synGAP that increases the rate of its phosphorylation and exposes additional phosphorylation sites that are inaccessible in the absence of Ca²⁺/CaM.

The postsynaptic density (PSD) is an electron-dense region in excitatory postsynaptic neurons that contains a high concentration of glutamate receptors, cytoskeletal proteins, and associated signaling enzymes. Within the PSD, three major classes of scaffolding molecules function to organize signaling enzymes and glutamate receptors. PDZ domains present in the Shank and PSD-95 scaffolds families serve to physically link AMPARs and NMDARs to signaling molecules in the PSD. Because of the specificity and high affinity of PDZ domains for their ligands, I reasoned that these interacting pairs could provide the core components of an affinity chromatography system, including affinity resins, affinity tags, and elution agents. I show that affinity columns containing the PDZ domains of PSD-95 can be used to purify active PDZ domain-binding proteins to very high purity in a single step. Five heterologously expressed neuronal proteins containing endogenous PDZ domain ligands (NMDAR GluN2B subunit Tail, synGAP, neuronal nitric oxide synthase PDZ domain, cysteine rich interactor of PDZ three and cypin) were purified using PDZ domain resin, with synthetic peptides having the sequences of cognate PDZ domain ligands used as elution agents. I also show that conjugation of PDZ domain-related affinity tags to Proteins Of Interest (POIs) that do not contain endogenous PDZ domains or ligands does not alter protein activity and enables purification of the POIs on PDZ domain-related affinity resins.

Mutations in exon 3 of the beta-catenin gene are rare in melanoma cell lines

Mutations in exon 3 of the CTNNB1 gene encoding beta-catenin have been reported in colorectal cancer cell lines and tumours. Although one study reported mutations or deletions affecting beta-catenin in 20% of melanoma cell lines, subsequent reports detected a much lower frequency of aberrations in uncultured melanomas. To determine whether this difference in mutation frequency reflected an in vitro culturing artefact, exon 3 of CTNNB1 was screened in a panel of 62 melanoma cell lines. In addition, reverse transcription-polymerase chain reaction (RT-PCR) was performed to detect intragenic deletions affecting exon 3. One out of 62 (1.6%) cell lines was found to carry a mutation, indicating that aberration of the Wnt-1/wingless pathway through activation of beta-catenin is a rare event, even in melanoma cell lines.