Rescue of growth defects of yeast cdc48 mutants by pathogenic IBMPFD-VCPs

VCP/p97/Cdc48 is a hexameric ring-shaped AAA ATPase that participates in a wide variety of cellular functions. VCP is a very abundant protein in essentially all types of cells and is highly conserved among eukaryotes. To date, 19 different single amino acid-substitutions in VCP have been reported to cause IBMPFD (inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia), an autosomal dominant inherited human disease. Moreover, several similar single amino acid substitutions have been proposed to associate with a rare subclass of familial ALS. The mechanisms by which these mutations contribute to the pathogenesis are unclear. To elucidate potential functional differences between wild-type and pathogenic VCPs, we expressed both VCPs in yeast cdc48 mutants. We observed that all tested pathogenic VCPs suppressed the temperature-sensitive phenotype of cdc48 mutants more efficiently than wild-type VCP. In addition, pathogenic VCPs, but not wild-type VCP, were able to rescue a lethal cdc48 disruption. In yeast, pathogenic VCPs, but not wild-type VCP, formed apparent cytoplasmic foci, and these foci were transported to budding sites by the Myo2/actin-mediated transport machinery. The foci formation of pathogenic VCPs appeared to be associated with their suppression of the temperature-sensitive phenotype of cdc48 mutants. These results support the idea that the pathogenic VCP mutations create dominant gain-of-functions rather than a simple loss of functional VCP. Their unique properties in yeast could provide a convenient drug-screening system for the treatment of these diseases.     

Cdc48 is required for the stability of Cut1/separase in mitotic anaphase

Separase, a large protease essential for sister chromatid separation, cleaves the cohesin subunit Scc1/Rad21 during anaphase and leads to dissociation of the link between sister chromatids. Securin, a chaperone and inhibitor of separase, is ubiquitinated by APC/cyclosome, and degraded by 26S proteasome in anaphase. Cdc48/VCP/p97, an AAA ATPase, is involved in a variety of cellular activities, many of which are implicated in the proteasome-mediated degradation. We previously reported that temperature-sensitive (ts) fission yeast Schizosaccharomyces pombe cdc48 mutants were suppressed by multicopy plasmid carrying the cut1(+)/separase gene and that the defective mitotic phenotypes of cut1 and cdc48 were similar. We here describe characterizations of Cdc48 mutant protein and the role of Cdc48 in sister chromatid separation. Mutant residue resides in the conserved D1 domain within the central hole of hexamer, while Cdc48 mutant protein possesses the ATPase activity. Consistent with the phenotypic similarity and the rescue of cdc48 mutant by overproduced Cut1/separase, the levels of Cut1 and also Cut2 are diminished in cdc48 mutant. We show that the stability of Cut1 during anaphase requires Cdc48. Cells lose viability during the traverse of anaphase in cdc48 mutant cells. Cdc48 may protect Cut1/separase and Cut2/securin against the instability during polyubiquitination and degradation in the metaphase-anaphase transition.     

Mechanisms of Cdc48/VCP-mediated cell death: from yeast apoptosis to human disease

The evolutionary conserved protein Cdc48/VCP is involved in various cellular processes, such as protein degradation, membrane fusion and chaperone activity. Increased levels of Cdc48/VCP correlate with cancer, whereas Cdc48/VCP at endogenous levels has been proposed to be a pathological effector in protein deposition diseases. Upon mutation Cdc48/VCP triggers the multisystem disorder 'inclusion body myopathy associated with Paget's disease of bone and frontotemporal dementia' (IBMPFD). The roles of Cdc48/VCP under these diverse pathological conditions, especially its function in decreased and increased incidences of cell death underlying these diseases, are poorly understood. Mutation of yeast CDC48 (cdc48(S565G)) results in yeast cells demonstrating morphological markers of apoptotic cell death. In other species it has been confirmed that mutations and depletion of Cdc48/VCP cause apoptosis, whereas increased levels of this protein provide an anti-apoptotic effect. This review critically compares mechanisms of Cdc48/VCP-mediated apoptosis observed in yeast and other species. Cdc48/VCP plays a triple role in cell death. At first, loss-of-function of Cdc48/VCP due to mutation or depletion causes ER stress and oxidative stress, triggering apoptosis. Secondly, upon exogenously applied ER stress functional Cdc48/VCP is important in the processing of caspases and plays therewith a pro-apoptotic role. Finally, Cdc48/VCP protects cells from apoptosis through mediating and activating pro-survival signaling pathways, namely Akt and NFkappaB signaling. This complex role in cell death pathways could correspond with the various pathophysiological conditions Cdc48/VCP is involved in.     

Functional ATPase activity of p97/valosin-containing protein (VCP) is required for the quality control of endoplasmic reticulum in neuronally differentiated mammalian PC12 cells

Abnormal protein accumulation and cell death with cytoplasmic vacuoles are hallmarks of several neurodegenerative disorders. We previously identified p97/valosin-containing protein (VCP), an AAA ATPase with two conserved ATPase domains (D1 and D2), as an interacting partner of the Machado-Joseph disease (MJD) protein with expanded polyglutamines that causes Machado-Joseph disease. To reveal its pathophysiological roles in neuronal cells, we focused on its ATPase activity. We constructed and characterized PC12 cells expressing wild-type p97/VCP and p97(K524A), a D2 domain mutant. The expression level, localization, and complex formation of both proteins were indistinguishable, but the ATPase activity of p97(K524A) was much lower than that of the wild type. p97(K524A) induced cytoplasmic vacuoles that stained with an endoplasmic reticulum (ER) marker, and accumulation of polyubiquitinated proteins in the nuclear and membrane but not cytoplasmic fractions was observed, together with the elevation of ER stress markers. These results show that p97/VCP is essential for degrading membrane-associated ubiquitinated proteins and that profound deficits in its ATPase activity severely affect ER quality control, leading to abnormal ER expansion and cell death. Excessive accumulation of misfolded proteins may inactivate p97/VCP in several neurodegenerative disorders, eventually leading to the neurodegenerations.     

Cdc48 and cofactors Npl4-Ufd1 are important for G1 progression during heat stress by maintaining cell wall integrity in Saccharomyces cerevisiae

The ubiquitin-selective chaperone Cdc48, a member of the AAA (ATPase Associated with various cellular Activities) ATPase superfamily, is involved in many processes, including endoplasmic reticulum-associated degradation (ERAD), ubiquitin- and proteasome-mediated protein degradation, and mitosis. Although Cdc48 was originally isolated as a cell cycle mutant in the budding yeast Saccharomyces cerevisiae, its cell cycle functions have not been well appreciated. We found that temperature-sensitive cdc48-3 mutant is largely arrested at mitosis at 37°C, whereas the mutant is also delayed in G1 progression at 38.5°C. Reporter assays show that the promoter activity of G1 cyclin CLN1, but not CLN2, is reduced in cdc48-3 at 38.5°C. The cofactor npl4-1 and ufd1-2 mutants also exhibit G1 delay and reduced CLN1 promoter activity at 38.5°C, suggesting that Npl4-Ufd1 complex mediates the function of Cdc48 at G1. The G1 delay of cdc48-3 at 38.5°C is a consequence of cell wall defect that over-activates Mpk1, a MAPK family member important for cell wall integrity in response to stress conditions including heat shock. cdc48-3 is hypersensitive to cell wall perturbing agents and is synthetic-sick with mutations in the cell wall integrity signaling pathway. Our results suggest that the cell wall defect in cdc48-3 is exacerbated by heat shock, which sustains Mpk1 activity to block G1 progression. Thus, Cdc48-Npl4-Ufd1 is important for the maintenance of cell wall integrity in order for normal cell growth and division.     

A stress-responsive system for mitochondrial protein degradation

We show that Ydr049 (renamed VCP/Cdc48-associated mitochondrial stress-responsive--Vms1), a member of an unstudied pan-eukaryotic protein family, translocates from the cytosol to mitochondria upon mitochondrial stress. Cells lacking Vms1 show progressive mitochondrial failure, hypersensitivity to oxidative stress, and decreased chronological life span. Both yeast and mammalian Vms1 stably interact with Cdc48/VCP/p97, a component of the ubiquitin/proteasome system with a well-defined role in endoplasmic reticulum-associated protein degradation (ERAD), wherein misfolded ER proteins are degraded in the cytosol. We show that oxidative stress triggers mitochondrial localization of Cdc48 and this is dependent on Vms1. When this system is impaired by mutation of Vms1, ubiquitin-dependent mitochondrial protein degradation, mitochondrial respiratory function, and cell viability are compromised. We demonstrate that Vms1 is a required component of an evolutionarily conserved system for mitochondrial protein degradation, which is necessary to maintain mitochondrial, cellular, and organismal viability.     

Staphylococcus aureus CymR is a new thiol-based oxidation-sensing regulator of stress resistance and oxidative response

As a human pathogen, Staphylococcus aureus must cope with oxidative stress generated by the human immune system. Here, we report that CymR utilizes its sole Cys-25 to sense oxidative stress. Oxidation followed by thiolation of this cysteine residue leads to dissociation of CymR from its cognate promoter DNA. In contrast, the DNA binding of the CymRC25S mutant was insensitive to oxidation and thiolation, suggesting that CymR senses oxidative stress through oxidation of its sole cysteine to form a mixed disulfide with low molecular weight thiols. The determined crystal structures of the reduced and oxidized forms of CymR revealed that Cys-25 is oxidized to Cys-25-SOH in the presence of H(2)O(2). Deletion of cymR reduced the resistance of S. aureus to oxidative stresses, and the resistance was restored by expressing a C25S mutant copy of cymR. In a C25S substitution mutant, the expression of two genes, tcyP and mccB, was constitutively repressed and did not respond to hydrogen peroxide stress, whereas the expression of the genes were highly induced under oxidative stress in a wild-type strain, indicating the critical role of Cys-25 in redox signaling in vivo. Thus, CymR is another master regulator that senses oxidative stress and connects stress responses to virulence regulation in S. aureus.     

Drug-stimulated ATPase activity of human P-glycoprotein is blocked by disulfide cross-linking between the nucleotide-binding sites

P-glycoprotein (P-gp) is an ATP-dependent drug pump that contains two nucleotide-binding domains (NBDs). Disulfide cross-linking analysis was done to determine if the two NBDs are close to each other. Residues within or close to the Walker A (GNSGCGKS in NDB1 and GSSGCGKS in NBD2) sequences for nucleotide binding were replaced with cysteine, and the mutant P-gps were subjected to oxidative cross-linking. Cross-linking was detected in two mutants, G427C(NBD1)/Cys-1074(NBD2) and L439C(NBD1)/Cys-1074(NBD2), because the cross-linked proteins migrated slower in SDS gels. Mutants G427C(NBD1)/Cys-1074(NBD2) and L439C(NBD1)/Cys-1074(NBD2) retained 10% and 82%, respectively, of the drug-stimulated ATPase activity relative to that of Cys-less P-gp. The cross-linking properties of the more active mutant L439C(NBD1)/Cys-1074(NBD2) were then studied. Cross-linking was reversed by addition of dithiothreitol and could be prevented by pretreatment of the mutant with N-ethylmaleimide. Cross-linking was also inhibited by MgATP, but not by the verapamil. Oxidative cross-linking of mutant L439C(NBD1)/Cys-1074(NBD2) resulted in almost complete inhibition of drug-stimulated ATPase activity. More than 60% of the drug-stimulated ATPase activity, however, was recovered after treatment with dithiothreitol. The results indicate that the two predicted nucleotide-binding sites are close to each other and that cross-linking inhibits ATP hydrolysis.     

Valosin‐containing protein VCP/p97 is essential for the intracellular development of Leishmania and its survival under heat stress

Valosin‐containing protein (VCP)/p97/Cdc48 is one of the best‐characterised type II cytosolic AAA+ ATPases most known for their role in ubiquitin‐dependent protein quality control. Here, we provide functional insights into the role of the Leishmania VCP/p97 homologue (LiVCP) in the parasite intracellular development. We demonstrate that although LiVCP is an essential gene, Leishmania infantum promastigotes can grow with less VCP. In contrast, growth of axenic and intracellular amastigotes is dramatically affected upon decreased LiVCP levels in heterozygous and temperature sensitive (ts) LiVCP mutants or the expression of dominant negative mutants known to specifically target the second conserved VCP ATPase domain, a major contributor of the VCP overall ATPase activity. Interestingly, these VCP mutants are also unable to survive heat stress, and a ts VCP mutant is defective in amastigote growth. Consistent with LiVCP's essential function in amastigotes, LiVCP messenger ribonucleic acid undergoes 3'Untranslated Region (UTR)‐mediated developmental regulation, resulting in higher VCP expression in amastigotes. Furthermore, we show that parasite mutant lines expressing lower VCP levels or dominant negative VCP forms exhibit high accumulation of polyubiquitinated proteins and increased sensitivity to proteotoxic stress, supporting the ubiquitin‐selective chaperone function of LiVCP. Together, these results emphasise the crucial role LiVCP plays under heat stress and during the parasite intracellular development.     

Evidence that cysteine 298 is in the active site of tryptophan indole-lyase

Escherichia coli tryptophan indole-lyase (tryptophanase) mutants, with cysteine residues 294 and 298 selectively replaced by serines, have been prepared by site-directed mutagenesis. Both mutant enzymes are highly active for beta-elimination reactions measured with both L-tryptophan and S-(o-nitrophenyl)-L-cysteine. The Cys-294----Ser mutant enzyme is virtually identical to the wild type with respect to pyridoxal phosphate binding (KCO = 2 microM), cofactor absorption spectrum (lambda max = 420 and 337 nm) and pH dependence (pK alpha = 7.3), pH profile for catalysis, and rate of bromopyruvic acid inactivation. In contrast, the Cys-298----Ser mutant enzyme exhibits a reduced affinity for pyridoxal phosphate (KCO = 6 microM), a shift in the cofactor absorption spectrum to 414 nm and an altered pK alpha = 8.5, an alkaline shift in the pH profile for catalysis, and resistance to inactivation of the apoenzyme by bromopyruvic acid. The C298S mutant enzyme (wherein cysteine 298 is altered to serine) also undergoes an isomerization to an unreactive state upon storage at 4 degrees C. These results demonstrate that the sulfhydryl groups of Cys-294 and Cys-298 are catalytically nonessential. However, these data suggest that Cys-298 is located within or very near the active site of the enzyme and is the reactive cysteine residue previously observed by others.     

Targeted deletion of p97 (VCP/CDC48) in mouse results in early embryonic lethality

The highly conserved AAA ATPase p97 (VCP/CDC48) has well-established roles in cell cycle progression, proteasome degradation and membrane dynamics. Gene disruption in Saccromyces cerevisiae, Drosophila melanogaster and Trypanosoma brucei demonstrated that p97 is essential in unicellular and multicellular organisms. To explore the requirement for p97 in mammalian cell function and embryogenesis, we disrupted the p97 locus by gene targeting. Heterozygous p97+/- mice were indistinguishable from their wild-type littermates, whereas homozygous mutants did not survive to birth and died at a peri-implantation stage. These results show that p97 is an essential gene for early mouse development.     

Phosphorylation of dis2 protein phosphatase at the C-terminal cdc2 consensus and its potential role in cell cycle regulation.

We show that the fission yeast dis2 protein phosphatase, which is highly similar to mammalian type 1 phosphatase, is a phosphoprotein containing phosphoserine (phospho-S) and threonine (phospho-T). It has several phosphorylation sites, two of which locate in the C-terminus. Phospho-T was abolished in the alanine substitution mutant at the C-terminal T316, which is conserved as a residue in the cdc2 consensus, TPPR, in a number of type 1-like phosphatases. In G2-arrested cdc2-L7 cells, the degree of T316 phosphorylation was reduced, whereas it was enhanced in metaphase-arrested nuc2-663 mutant cells. Phospho-T was produced in dis2 by fission yeast cdc2 kinase, but not in the substitution mutant A316, indicating that the T316 residue was the site for cdc2 kinase in vitro. Phosphatase activity of wild type dis2 was reduced by incubation with cdc2 kinase, but that of mutant dis2-A316 was not. Phosphorylation of T316 hence has a potential significance in cell cycle control in conjunction with cdc2 kinase activation and inactivation. Overexpression phenotypes of wild type dis2+, sds21+ and mutant dis2-A316, sds21-TPPR genes were consistent with negative regulation of dis2 by phosphorylation. This type of regulation would explain why cells harboring the dis2-11 mutation enter mitosis but fail to exit from it.     

A conformationally sensitive residue on the cytoplasmic surface of serotonin transporter

Serotonin transporter (SERT) contains a single reactive external cysteine residue at position 109 (Chen, J. G., Liu-Chen, S., and Rudnick, G. (1997) Biochemistry 36, 1479-1486) and seven predicted cytoplasmic cysteines. A mutant of rat SERT (X8C) in which those eight cysteine residues were replaced by other amino acids retained approximately 32% of wild type transport activity and approximately 56% of wild type binding activity. In contrast to wild-type SERT or the C109A mutant, X8C was resistant to inhibition of high affinity cocaine analog binding by the cysteine reagent 2-(aminoethyl)methanethiosulfonate hydrobromide (MTSEA) in membrane preparations from transfected cells. Each predicted cytoplasmic cysteine residue was reintroduced, one at a time, into the X8C template. Reintroduction of Cys-357, located in the third intracellular loop, restored MTSEA sensitivity similar to that of C109A. Replacement of only Cys-109 and Cys-357 was sufficient to prevent MTSEA sensitivity. Thus, Cys-357 was the sole cytoplasmic determinant of MTSEA sensitivity in SERT. Both serotonin and cocaine protected SERT from inactivation by MTSEA at Cys-357. This protection was apparently mediated through a conformational change following ligand binding. Although both ligands bind in the absence of Na(+) and at 4 degrees C, their ability to protect Cys-357 required Na(+) and was prevented at 4 degrees C. The accessibility of Cys-357 to MTSEA inactivation was increased by monovalent cations. The K(+) ion, which is believed to serve as a countertransport substrate for SERT, was the most effective ion for increasing Cys-357 reactivity.     

Physical and functional interaction between Dorfin and Valosin-containing protein that are colocalized in ubiquitylated inclusions in neurodegenerative disorders

Dorfin, a RING-IBR type ubiquitin ligase (E3), can ubiquitylate mutant superoxide dismutase 1, the causative gene of familial amyotrophic lateral sclerosis (ALS). Dorfin is located in ubiquitylated inclusions (UBIs) in various neurodegenerative disorders, such as ALS and Parkinson's disease (PD). Here we report that Valosin-containing protein (VCP) directly binds to Dorfin and that VCP ATPase activity profoundly contributes to the E3 activity of Dorfin. High through-put analysis using mass spectrometry identified VCP as a candidate of Dorfin-associated protein. Glycerol gradient centrifugation analysis showed that endogenous Dorfin consisted of a 400-600-kDa complex and was co-immunoprecipitated with endogenous VCP. In vitro experiments showed that Dorfin interacted directly with VCP through its C-terminal region. These two proteins were colocalized in aggresomes in HEK293 cells and UBIs in the affected neurons of ALS and PD. VCP(K524A), a dominant negative form of VCP, reduced the E3 activity of Dorfin against mutant superoxide dismutase 1, whereas it had no effect on the autoubiquitylation of Parkin. Our results indicate that VCPs functionally regulate Dorfin through direct interaction and that their functional interplay may be related to the process of UBI formation in neurodegenerative disorders, such as ALS or PD.     

Isolation and characterisation of smallminded,a Drosophila encoding a new member of the Cdc48p/VCP subfamily of AAA proteins

Smallminded (smid) encodes a new member of the cdc48p/VCP subfamily of AAA proteins in Drosophila. The gene was isolated by plasmid rescue from a GAL4 enhancer trap line which shows reporter gene expression in neuroblasts, imaginal disks and a subset of sensory neurons. Larvae homozygous for the insert arrest development as second instar larvae and die without pupating. The most obvious defect in these larvae is a significantly reduced CNS, hence the naming of the gene as smallminded. The deduced amino acid sequence of smid contains a tandem duplication of the AAA nucleotide binding domain characteristic of the cdc48p/VCP subfamily. Overall, smid shares 33% identical residues with its closest relative, yeast L0919-chrXII and 26–29% with other members of the cdc48p/VCP subfamily. The most highly conserved regions of the predicted protein structure are found in and around the nucleotide binding domains. The gene is expressed at all developmental stages.     

Two vicinal cysteines confer a peculiar redox regulation to low molecular weight protein tyrosine phosphatase in response to platelet-derived growth factor receptor stimulation.

Low molecular weight protein tyrosine phosphatase (LMW-PTP) is an enzyme involved in platelet-derived growth factor (PDGF)-induced mitogenesis and cytoskeleton rearrangement because it is able to bind and dephosphorylate the activated receptor. LMW-PTP presents two cysteines in positions 12 and 17, both belonging to the catalytic pocket; this is a unique feature of LMW-PTP among all protein tyrosine phosphatases. Our previous results demonstrated that in vitro LMW-PTP is oxidized by either H(2)O(2) or nitric oxide with the formation of a disulfide bond between Cys-12 and Cys-17. This oxidation leads to reversible enzyme inactivation because treatment with reductants permits catalytic activity rescue. In the present study we investigated the in vivo inactivation of LMW-PTP by either extracellularly or intracellularly generated H(2)O(2), evaluating its action directly on its natural substrate, PDGF receptor. LMW-PTP is oxidized and inactivated by exogenous oxidative stress and recovers its activity after oxidant removal. LMW-PTP is oxidized also during PDGF signaling, very likely upon PDGF-induced H(2)O(2) production, and recovers its activity within 40 min. Our results strongly suggest that reversibility of in vivo LMW-PTP oxidation is glutathione-dependent. In addition, we propose an intriguing and peculiar role of Cys-17 in the formation of a S-S intramolecular bond, which protects the catalytic Cys-12 from further and irreversible oxidation. On the basis of our results we propose that the presence of an additional cysteine near the catalytic cysteine could confer to LMW-PTP the ability to rapidly recover its activity and finely regulate PDGF receptor activation during both extracellularly and intracellularly generated oxidative stress.     

Crucial mitochondrial impairment upon CDC48 mutation in apoptotic yeast

Mutation in CDC48 (cdc48(S565G)), a gene essential in the endo-plasmic reticulum (ER)-associated protein degradation (ERAD) pathway, led to the discovery of apoptosis as a mechanism of cell death in the unicellular organism Saccharomyces cerevisiae. Elucidating Cdc48p-mediated apoptosis in yeast is of particular interest, because Cdc48p is the highly conserved yeast orthologue of human valosin-containing protein (VCP), a pathological effector for polyglutamine disorders and myopathies. Here we show distinct proteomic alterations in mitochondria in the cdc48(S565G) yeast strain. These observed molecular alterations can be related to functional impairment of these organelles as suggested by respiratory deficiency of cdc48(S565G) cells. Mitochondrial dysfunction in the cdc48(S565G) strain is accompanied by structural damage of mitochondria indicated by the accumulation of cytochrome c in the cytosol and mitochondrial enlargement. We demonstrate accumulation of reactive oxygen species produced predominantly by the cytochrome bc1 complex of the mitochondrial respiratory chain as suggested by the use of inhibitors of this complex. Concomitantly, emergence of caspase-like enzymatic activity occurs suggesting a role for caspases in the cell death process. These data strongly point for the first time to a mitochondrial involvement in Cdc48p/VCP-dependent apoptosis.