p97/valosin-containing protein (VCP) is highly modulated by phosphorylation and acetylation

p97/valosin-containing protein (VCP) is a member of the AAA family proteins, which plays various important roles in cells by using its ATPase activity. But mechanism of regulating its ATPase activity is mostly unknown. We report here that VCP is highly modified throughout the protein via acetylation and phosphorylation. In addition to six previously identified phosphorylation sites, we identified at least 14 serines, 14 threonines, 6 tyrosines and 22 lysines as potential modification sites. Interestingly, these sites included Lys²⁵¹ and Lys⁵²⁴, which are very critical for the ATP binding in Walker A motif of D1 and D2 domains, respectively. It is notable that 16 sites are in the N-terminal region and 16 sites are clustered in D2α domain (from Pro⁶⁴⁶ to Gly⁷⁶⁵). Indeed, amino acid substitution of Lys⁶⁹⁶ and Thr⁷⁶¹ profoundly affect VCP ATPase activities. From these results, we propose that D2α domain acts as a V CP A TPase R egulatory domain or "VAR domain". VCP modifications including those in this VAR domain may endorse adaptive and multiple functions to VCP in different cell conditions such as in the cell cycle and with abnormal protein accumulation.     

Involvement of valosin-containing protein (VCP)/p97 in the formation and clearance of abnormal protein aggregates

Abnormal protein aggregates are commonly observed in affected neurons in many neurodegenerative disorders. We have reported that valosin-containing protein (VCP) co-localizes with protein aggregates in patients' neurons and in cultured cells expressing diseased proteins. However, the significance of such co-localization remains elucidated. Here we report the involvement of VCP in the re-solubilization process of abnormal protein aggregates. VCP recognized and accumulated onto pre-formed protein aggregates created by proteasome inhibition. VCP knockdown or the expression of dominant-negative VCP both significantly delayed the elimination of ubiquitin-positive aggregates. VCP was involved in the clearance of pre-formed polyglutamine aggregates as well. Paradoxically, VCP knockdown also diminished polyglutamine aggregate formation. Furthermore, its ATPase activity was required for the re-solubilization and re-activation of heat-denatured proteins, such as luciferase, from insoluble aggregates. We thus propose that VCP functions as a mediator for both aggregate formation and clearance depending upon the concentration of soluble aggregate-prone proteins, indicating dual VCP functions as an aggregate formase and an unfoldase.     

A novel WARS mutation (p.Asp314Gly) identified in a Chinese distal hereditary motor neuropathy family

Distal hereditary motor neuropathy (dHMN) is a clinically and genetically heterogeneous group of inherited neuropathies characterized by distal limb muscle wasting and weakness with no or minimal sensory abnormalities. To investigate the clinical and genetic features of dHMN caused by WARS mutations in mainland China, we performed Sanger sequencing of the coding and untranslated region (UTR) regions of WARS in 160 unresolved dHMN and Charcot-Marie-Tooth (CMT) index patients. We detected a novel heterozygous variant c.941A>G (p.Asp314Gly) of WARS in an index patient from an autosomal dominant dHMN family including five affected members over three generations. The variant completely co-segregates with the dHMN phenotype in the family, and it was classified as likely pathogenic according to the American College of Medical Genetics and Genomics standards and guidelines. The clinical features included juvenile to adult onset (15-23 years), distal wasting and weakness, minimal sensory disturbance and length-dependent motor axonal degeneration with CMT examination score ranging from 6 to 10. Our report further confirms the role of WARS in dHMN and indicates that the variant c.941A>G (p.Asp314Gly) of WARS is related to a mild to moderate affected and later onset phenotype of dHMN.     

Immunocytochemical localization of the Menkes copper transport protein (ATP7A) to the trans-Golgi network

We have generated polyclonal antibodies against the amino-terminal third of the Menkes protein (ATP7A; MNK) by immunizing rabbits with a histidine-tagged MNK fusion construct containing metal-binding domains 1-4. The purified antibodies were used in Western analysis of cell lysates and in indirect immunofluorescence experiments on cultured cells. On Western blots, the antibodies recognized the approximately 165 kDa MNK protein in CHO cells and human fibroblasts. No MNK signal could be detected in fibroblasts from a patient with Menkes disease or in Hep3B hepatocellular carcinoma cells, confirming the specificity of the antibodies. Immunocytochemical analysis of CHO cells and human fibroblasts showed a distinct perinuclear signal corresponding to the pattern of the Golgi complex. This staining pattern was similar to that of alpha-mannosidase II which is a known resident enzyme of the Golgi complex. Using brefeldin A, a fungal inhibitor of protein secretion, we further demonstrated that the MNK protein is localized to the trans- Golgi network. This data provides direct evidence for a subcellular localization of the MNK protein which is similar to the proposed vacuolar localization of Ccc2p, the yeast homolog of MNK and WND (ATP7B), the Wilson disease gene product. In light of the proposed role of MNK both in subcellular copper trafficking and in copper efflux, these data suggest a model for how these two processes are linked and represent an important step in the functional analysis of the MNK protein.      

Transgenic expression of inclusion body myopathy associated mutant p97/VCP causes weakness and ubiquitinated protein inclusions in mice

Mutations in p97/VCP cause the autosomal-dominant, inherited syndrome inclusion body myopathy (IBM) associated with Paget's disease of the bone and frontotemporal dementia (IBMPFD) (Watts, G.D., Wymer, J., Kovach, M.J., Mehta, S.G., Mumm, S., Darvish, D., Pestronk, A., Whyte, M.P. and Kimonis, V.E. (2004) Inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia is caused by mutant valosin-containing protein. p97/VCP is a multi-functional protein with a role in the ubiquitin-proteasome system (UPS) (Wang, Q., Song, C. and Li, C.C. (2004) Molecular perspectives on p97-VCP: progress in understanding its structure and diverse biological functions. To understand how mutations in this protein lead to a myopathy, we generated several lines of transgenic mice expressing p97/VCP-WT (TgVCP-WT) or the most common IBMPFD mutant, p97/VCP R155H (TgVCP-RH), under a muscle-specific promoter. TgVCP-RH animals, but not controls, became progressively weaker in a dose-dependent manner starting at 6 months of age. Abnormal muscle pathology, which included coarse internal architecture, vacuolation and disorganized membrane morphology with reduced caveolin-3 expression at the sarcolemma developed coincident with the onset of weakness. These changes were not associated with alterations in sarcolemmal integrity as measured by muscle fiber uptake of Evan's blue dye. Even before animals displayed measurable weakness, there was an increase in ubiquitin-containing protein inclusions and high-molecular-weight ubiquitinated proteins, markers of UPS dysfunction. We suggest that this early and persistent increase in ubiquitinated proteins induced by IBMPFD mutations in p97/VCP may ultimately lead to animal weakness and the observed muscle pathology. TgVCP-RH animals will be a valuable tool for understanding the pathogenesis of IBM and the role of the UPS in skeletal muscle.     

Exclusion of 5 functional candidate genes for distal hereditary motor neuropathy type II (distal HMN II) linked to 12q24.3

Distal hereditary motor neuropathies (distal HMNs) are characterised by degeneration of anterior horn cells of the spinal cord resulting in muscle weakness and atrophy. Distal HMN type II is genetically linked to chromosome 12q24.3 and located within a 13 cM region flanked by markers D12S86 and D12S340. We previously excluded the human phospholipase A2 group 1B gene ( PLA2G1B ) as the disease causing gene. Here, we report the mutation analysis of five other candidate genes localised within the distal HMN II region: the cytoskeletal proteins paxillin ( PXN ) and restin ( RSN ); the acidic ribosomal phosphoprotein, large P0 subunit ( RPLP0 ); a nucleoside diphosphate kinase ( NME2B ); and the β 3 subunit of the voltage-gated calcium channel ( CACNB3 ). DNA sequencing of the coding regions was performed but no disease causing mutations could be identified, hence excluding these five genes for distal HMN type II.     

Autosomal dominant Charcot-Marie-Tooth axonal neuropathy mapped on chromosome 7p (CMT2D)

Clinical, electrophysiological and genetic linkage studies were performed on a large autosomal dominant family with Charcot-Marie-Tooth axonal neuropathy type 2 (CMT2) with 38 members of which 14 were affected. Onset of the disease was between 16 and 30 years of age with weakness and atrophy of the hands more severe than of the feet with slow progressive course in 12 patients. Deep tendon reflexes were absent in the upper extremities and decreased in the lower extremities. There was distal hypesthesia for touch, proprioception and vibration sense for the hands more than for the feet. Motor nerve conduction velocities showed normal values (48-53 M/s) with normal latencies (2-3 msec) and electromyography revealed signs of denervation. Genetic linkage analysis used 167 short tandem repeat markers (STRPs) spaced throughout the 22 autosomes. Linkage to the short arm of chromosome 7 at 7p14 was found using the marker D7S435 (Z = 4.83 at theta = 0). Flanking markers were D7S1808 and D7S1806 and the genetic distance between them was 6.8 cM. The multipoint linkage analysis gave a peek multipoint lod score of 6.89 between the markers D7S1808 and D7S435. Linkage analysis showed significantly negative lod scores (with values less than -2) with markers of chromosomes 1 and 3 where CMT axonal forms have been previously mapped. PFGE analysis indicated the absence of the CMT1A duplication. Our findings are consistent with a new genetic type of axonal CMT neuropathy designated by us as CMT2D. Potential candidate genes are multiple T-cell gamma receptor genes which map to the same cytogenetic interval as CMT2D neuropathy.      

The role of the p33:p33/p92 interaction domain in RNA replication and intracellular localization of p33 and p92 proteins of Cucumber necrosis tombusvirus

Replication of plus-stranded RNA viruses is performed by the viral replicase complex, which, together with the viral RNA, must be targeted to intracellular membranes, where replication takes place in membraneous vesicles/spherules. Tombusviruses code for two overlapping replication proteins, the p33 auxiliary protein and the p92 polymerase. Using replication-competent fluorescent protein-tagged p33 of Cucumber necrosis virus (CNV), we determined that two domains affected p33 targeting to peroxisomal membranes in yeast: an N-proximal hydrophobic trans-membrane sequence and the C-proximal p33:p33/p92 interaction domain. On the contrary, only the deletion of the p33:p33/p92 interaction domain, but not the trans-membrane sequence, altered the intracellular targeting of p92 protein in the presence of wt p33 and DI-72(+) RNA. Moreover, unlike p33, p92 lacking the trans-membrane sequence was still functional in supporting the replication of a replicon RNA in yeast, whereas the p33:p33/p92 interaction domain in both p33 and p92 was essential for replication. In addition, p33 was also shown to facilitate the recruitment of the viral RNA to peroxisomal membranes and that p33 is colocalized with (+) and (-)-stranded viral RNAs. Also, FRET and pull-down analyses confirmed that p33 interacts with other p33 molecules in yeast cells. Based on these data, we propose that p33 facilitates the formation of multimolecular complexes, including p33, p92, viral RNA, and unidentified host factors, which are then targeted to the peroxisomal membranes, the sites of CNV replication.     

Distal hereditary motor neuropathy type II (distal HMN II): mapping of a locus to chromosome 12q24

The distal hereditary motor neuropathy (distal HMN) or the spinal form of Charcot-Marie-Tooth (CMT) disease is an exclusively motor disorder of the peripheral nervous system. The disorder clinically resembles the hereditary motor and sensory neuropathies (HMSN) type I and type II or CMT type 1 and type 2. Distal HMN might also be related to the spinal muscular atrophies (SMA) since, in both disorders, the lower motor neurons are affected. Electrophysiological and neuropathological examinations of peripheral nerves show the absence of sensory involvement. We performed a genome search in an extended Belgian family with autosomal dominant distal HMN type II. Significant linkage was obtained with markers located at chromosome 12q24, and the gene for distal HMN II was assigned to the 13 cM interval between D12S86 and D12S340.      

The immunohistochemical localization of synaptophysin protein (p38) in the gastro-entero-pancreatic (GEP) system of reptiles

The gastro-entero-pancreatic (GEP) system of four reptilian species: turtle (Emys orbicularis), lizards (Lacerta viridis and Lacerta agilis) and snake (Natrix natrix) has been investigated immunohistochemically for the presence and topographic distribution of synaptophysin. Among the studied reptiles, only in turtles were neural, glial and neuroendocrine elements labelled for this marker protein. Semi-quantitative evaluation of the immunolabelled neural structures distributed throughout the gastroenteric wall revealed, with two exceptions, highly significant mean differences between the successive gut segments. Significant mean differences were noted also between myenteric and submucosal neurons immunolabelled in the various gastroenteric regions. Moreover, the comparison of ganglionic perikarya groups showed, at least in the stomach, significant mean differences. The amounts of immunopositive glial cells seemed to vary similarly to those of nerve fibers along the entire gastrointestinal tract. Finally, every “closed” and “open” population of immunopositive epithelial cells showed typical fluctuations along the gut. In addition to the distribution of synaptophysin in the GEP system of turtles, the above findings furnish evidence that this marker protein, which is widespread in mammals, is only occasionally expressed in reptiles and probably in most poikilothermic vertebrates.     

Autosomal dominant sensory/motor neuropathy with Ataxia (SMNA): Linkage to chromosome 7q22-q32

The autosomal dominant (AD) spinocerebellar ataxias (SCAs) and hereditary sensory neuropathies (HSN) are heterogeneous disorders characterized by variable clinical, electrophysiological, and neuropathological profiles. The SCAs are clinically characterized by slowly progressive incoordination of gait often associated with poor coordination of hands, speech, and eyes. Peripheral neuropathy is not a frequent part of the SCA syndrome. In contrast, the HSNs are primarily characterized by progressive sensory loss. There is substantial clinical overlap between the various SCAs and the various HSNs, and they often cannot be differentiated on the basis of clinical or neuro-imaging studies. We have identified a five-generation American family of Irish ancestry with a unique neurological disorder displaying an AD pattern of inheritance. There was variable expressivity and severity of symptoms including sensory loss, ataxia, pyramidal tract signs, and muscle weakness. Nerve conduction studies were consistent with a sensory axonal neuropathy. Muscle biopsy revealed neurogenic atrophy and brain MRI showed mild cerebellar atrophy. To identify the responsible locus we pursued a whole genome linkage analysis. After analyzing 114 markers, linkage to D7S486 was detected with a two point LOD score of 4.79 at theta = 0.00. Evaluation of additional markers in the region provided a maximum LOD score of 6.36 at theta = 0.00 for marker D7S2554. Haplotype analysis delimited an approximately 14-cM region at 7q22-q32 between markers D7S2418 and D7S1804 cosegregating with the disease. Because this disorder does not easily fall into either the SCA or HSN categories, it is designated sensory/motor neuropathy with ataxia (SMNA).     

Genetic aspects of hereditary motor and sensory neuropathy (types I and II)

The genetic features of a series of 227 patients with hereditary motor and sensory neuropathy (HMSN) have been analysed. The series comprised 119 index cases from 110 families in which 108 affected relatives were identified. The cases were classified as having type I or type II HMSN on the basis of nerve conduction studies. Inheritance in the type I cases was autosomal dominant in 139 (45 families) and autosomal recessive in eight (four families) with 26 single cases. For the type II cases, 35 (17 families) were autosomal dominant and three (two families) autosomal recessive with 16 single cases. A significant excess of males was present in the combined single and recessive type I cases and in the type I index cases. No X linked pedigrees were identified.     

Immunocytochemical localization of synaptophysin (protein p38) and synapsin I in nerve terminals of rat neurohypophysis

Synaptophysin and synapsin I, the synaptic vesicle-associated proteins, were demonstrated immunocytochemically in nerve terminals of the neurohypophysis of rats. Although both are associated with microvesicles 50-60 nm in diameter, they are not localized on or around the large neurosecretory granules nor the clear vacuoles, 100-200 nm in diameter. These findings strongly suggest that the microvesicles in the nerve terminal of the neurohypophysis are, for the most part, not the structures involved in the retrieval of the limiting membranes of the released neurosecretory granules, but rather typical synaptic vesicles. The clear vacuoles, which are negative for synaptophysin and synapsin I, are considered to be related to the retrieval of the limiting membranes of the released neurosecretory granules.     

82-FIP,a novel FMRP (fragile X mental retardation protein) interacting protein,shows a cell cycle-dependent intracellular localization

FMRP is an RNA binding protein whose absence produces pathological manifestations of the fragile-X syndrome. FMRP is a component of mRNP complexes found in association with actively translating polyribosomes, RNA complexes trafficking in neurites, RNA granules in cytoplasm and, in Drosophila, with the RNAi machinery. We report here the identification and characterization of a novel FMRP-interacting protein associated to polyribosomes as a component of mRNP complexes containing FMRP. We named this protein 82-FIP (82-kD FMRP Interacting Protein). FMRP interacts with 82-FIP through a novel interaction motif located in its N-terminal region. The distribution of 82-FIP in different areas of the brain is very similar to that of FMRP. However, unlike FMRP, 82-FIP is found in both nucleus and cytoplasm in some neurons, while it appears only cytoplasmic in others. Subcellular distribution of 82-FIP is cell cycle-dependent in cultured cells, suggesting that the composition of some FMRP-containing RNP complexes may be cell cycle-modulated.     

Chromosome localization and cDNA sequence of murine and human genes forras p21 GTPase activating protein (GAP)

The cDNA coding for mouse and human rasp21 GTPase-activating protein (GAP) was isolated; the deduced amino acid sequences share over 96% homology with that previously determined for bovine brain GAP. Both the mouse and human GAP cDNAs were used as probes for the chromosomal localization of this gene. The locus designations for the gene encoding GAP in human and mouse are RASAand Rasa (for ras-activating protein), respectively. By somatic cell hybrid analysis and in situ chromosomal hybridization, we have assigned the RASAgene to human chromosome band 5q13.3. In addition, with somatic cell genetics and linkage analysis in recombinant inbred mouse strains, the murine Rasagene was localized to the distal end of mouse chromosome 13. These assignments place the gene encoding GAP in a known conserved syntenic region.     

Subcellular localization and axonal transport of the survival motor neuron (SMN) protein in the developing rat spinal cord

The subcellular localization of the survival motor neuron (SMN) protein, encoded by the spinal muscular atrophy determining gene, was investigated in motor neurons of the developing and adult rat spinal cord by light and electron microscopy immunocytochemistry. The experiments were carried out with a panel of anti-SMN antibodies, all recognizing an SMN-specific protein band at 39 kDa in HeLa cells and rat spinal cord protein extracts. SMN protein expression decreased during postnatal spinal cord development, but it remained unchanged in distribution and intensity in motor neurons at all ages examined. SMN protein was mainly organized in immunoreactive aggregates sparse in the nucleoplasm and cytoplasm of both mature and developing motor neurons, and it was more rarely localized within the endoplasmic reticulum and in apposition to the external mitochondrial membrane. Most strikingly, the SMN protein was found in association with cytoskeletal elements in spinal dendrites and axons, where it was particularly evident during postnatal development. The present findings suggest that SMN protein may be transported via axoplasmic flow in maturing neurons. Given the RNA-binding activity of SMN, the SMN protein could be involved in the transport of specific mRNAs in axons and dendrites of motor neurons. The reduced transport of specific mRNAs within motor neurons during development could play a role in the motoneuronal degeneration and impaired axonal sprouting observed in spinal muscular atrophy.     

Null mutation of the murine ATP7B (Wilson disease) gene results in intracellular copper accumulation and late-onset hepatic nodular transformation.

The Atp7b protein is a copper-transporting ATPase expressed predominantly in the liver and to a lesser extent in most other tissues. Mutations in the ATP7B gene lead to Wilson disease, a copper toxicity disorder characterized by dramatic build-up of intracellular hepatic copper with subsequent hepatic and neuro-logical abnormalities. Using homologous recombination to disrupt the normal translation of ATP7B, we have generated a strain of mice that are homozygous mutants (null) for the Wilson disease gene. The ATP7B null mice display a gradual accumulation of hepatic copper that increases to a level 60-fold greater than normal by 5 months of age. An increase in copper concentration was also observed in the kidney, brain, placenta and lactating mammary glands of homo-zygous mutants, although milk from the mutant glands was copper deficient. Morphological abnormalities resembling cirrhosis developed in the majority of the livers from homozygous mutants older than 7 months of age. Progeny of the homozygous mutant females demonstrated neurological abnormalities and growth retardation characteristic of copper deficiency. Copper concentration in the livers of the newborn homozygous null mutants was decreased dramatically. In summary, inactivation of the murine ATP7B gene produces a form of cirrhotic liver disease that resembles Wilson disease in humans and the 'toxic milk' phenotype in the mouse.     

C-reactive protein (CRP) peptides inactivate enolase in human neutrophils leading to depletion of intracellular ATP and inhibition of superoxide generation.

The nature and the biochemical mechanism of inhibition of neutrophil membrane-associated oxidative metabolism by two synthetic peptides p77-82 and p201-206 (amino acid sequences Val-Gly-Gly-Ser-Glu-Ile and Lys-Pro-Gln-Leu-Trp-Pro respectively, from the primary amino acid sequence of C-reactive protein) have been ascertained. Preincubating neutrophils for 15 min with 50 microM of p77-82 or p201-206 resulted in superoxide generation by opsonized zymosan stimulated neutrophils being inhibited by 34 +/- 2% (P less than 0.005) and 29 +/- 2% (P less than 0.005) respectively. With a 60-min preincubation period 6.25 microM of p77-82 or p201-206 was effective in inhibiting this superoxide generation by 12 +/- 2% (P less than 0.01) and 10 +/- 1% (P less than 0.01) respectively. Neither peptide inhibited neutrophil arachidonic acid release, transmembrane potential or transductional events preceding superoxide generation. Inhibition of neutrophil functions was found to be due to the ability of each peptide (50 microM) following a 15-min preincubation period to inhibit both neutrophil glycolysis and ATP generation by approximately 30%. The inhibition of ATP generation and glycolysis in neutrophils is attributable to the ability of these peptides to inhibit uncompetitively the glycolytic enzyme enolase. Using purified enolase the relative Ki values for p77-82 and p201-206 were 27 and 19 microM respectively. Inhibition of neutrophil function by the peptides is concluded to be due to effective interference of neutrophil energy metabolism.