Ubiquitin Fusion Degradation Protein Research Articles

Genome-wide identification and characterization of grapevine UFD1 genes during berry development and salt stress response

Grapevine (Vitis vinifera) is widely applicated in food industry, which shows high economical and nutritional values. However, growth of grapevine was usually affected by various environmental stresses, such as salt, drought and disease. Ubiquitin fusion degradation protein 1 (UFD1) is an essential ubiquitin-recognition protein facilitates regulation of stress response through ERAD pathway. Even though, a comprehensive investigation of UFD1 genes in the plant species is still lacking. Here we identified three VvUFD1 proteins from genome of grapevine, which were assigned into different subgroups. All VvUFD1 genes contain highly conserved motifs in structure. Several cis-elements that related to fruit development and stress response were found in the promoter regions of VvUFD1 genes, including bHLH, NCA, MYB, HD-ZIP, GATA and AP2. Expression analysis found VvUFD1 genes showed different expression patterns in different tissues. Most importantly, VvUFD1 genes were found to be involved in salt stress response during growth of grapevine. Transcriptomic analyses were investigated for further understanding the genes’ function. Expression of VvUFD1 were increased at late stage of berry ripening. In addition, expression of VvUFD1 were also regulated by elevated light treatment and pathogen Neofusicoccum parvum infection. Co-expression network analysis revealed several major transcription factors that co-expressed with VvUFD1 genes. These results provide a basis for investigating the function of UFD1 genes in plant species and expand understanding of the regulation of berry development and salt stress response in grapevine.

Functional Inhibition of Valosin-Containing Protein Induces Cardiac Dilation and Dysfunction in a New Dominant-Negative Transgenic Mouse Model.

Valosin-containing protein (VCP) was found to play a vital protective role against cardiac stresses. Genetic mutations of VCP are associated with human dilated cardiomyopathy. However, the essential role of VCP in the heart during the physiological condition remains unknown since the VCP knockout in mice is embryonically lethal. We generated a cardiac-specific dominant-negative VCP transgenic (DN-VCP TG) mouse to determine the effects of impaired VCP activity on the heart. Using echocardiography, we showed that cardiac-specific overexpression of DN-VCP induced a remarkable cardiac dilation and progressively declined cardiac function during the aging transition. Mechanistically, DN-VCP did not affect the endogenous VCP (EN-VCP) expression but significantly reduced cardiac ATPase activity in the DN-VCP TG mouse hearts, indicating a functional inhibition. DN-VCP significantly impaired the aging-related cytoplasmic/nuclear shuffling of EN-VCP and its co-factors in the heart tissues and interrupted the balance of the VCP-cofactors interaction between the activating co-factors, ubiquitin fusion degradation protein 1 (UFD-1)/nuclear protein localization protein 4 (NPL-4) complex, and its inhibiting co-factor P47, leading to the binding preference with the inhibitory co-factor, resulting in functional repression of VCP. This DN-VCP TG mouse provides a unique functional-inactivation model for investigating VCP in the heart in physiological and pathological conditions.

Ufd1 phosphorylation at serine 229 negatively regulates endoplasmic reticulum-associated degradation by inhibiting the interaction of Ufd1 with VCP

Misfolded proteins in the endoplasmic reticulum (ER) are removed through multistep processes termed ER-associated degradation (ERAD). Valosin-containing protein (VCP) plays a crucial role in ERAD as the interaction of ubiquitin fusion degradation protein 1 (Ufd1) with VCP via its SHP box motif (228F-S-G-S-G-N-R-L235) is required for ERAD. However, the mechanisms by which the VCP-Ufd1 interaction is regulated are not well understood. Here, we found that the serine 229 residue located in the Ufd1 SHP box is phosphorylated in vitro and in vivo by cyclic adenosine monophosphate-dependent protein kinase A (PKA), with this process being enhanced by either forskolin (an adenylyl cyclase activator) or calyculin A (a protein phosphatase inhibitor). Moreover, a phosphomimetic mutant (S229D) of Ufd1 as well as treatment by forskolin, calyculin A, or activated PKA strongly reduced Ufd1 binding affinity for VCP. Consistent with this, the Ufd1 S229D mutant significantly inhibited ERAD leading to the accumulation of ERAD substrates such as a tyrosinase mutant (C89R) and 3-hydroxy-3-methylglutaryl coenzyme A reductase. However, a non-phosphorylatable Ufd1 mutant (S229A) retained VCP-binding ability and was less effective in blocking ERAD. Collectively, our results support that Ufd1 S229 phosphorylation status mediated by PKA serves as a key regulatory point for the VCP-Ufd1 interaction and functional ERAD.

Structural Basis for Ovarian Tumor Domain-containing Protein 1 (OTU1) Binding to p97/Valosin-containing Protein (VCP)

Valosin-containing protein (VCP), also known as p97, is an AAA(+) ATPase that plays an essential role in a broad array of cellular processes including the endoplasmic reticulum-associated degradation (ERAD) pathway. Recently, ERAD-specific deubiquitinating enzymes have been reported to be physically associated with VCP, although the exact mechanism is not yet clear. Among these enzymes is ovarian tumor domain-containing protein 1 (OTU1). Here, we report the structural basis for interaction between VCP and OTU1. The crystal structure of the ubiquitin regulatory X-like (UBXL) domain of OTU1 (UBXLOTU1) complexed to the N-terminal domain of VCP (NVCP) at 1.8-Å resolution reveals that UBXLOTU1 adopts a ubiquitin-like fold and binds at the interface of two subdomains of NVCP using the (39)GYPP(42) loop of UBXLOTU1 with the two prolines in cis- and trans-configurations, respectively. A mutagenesis study shows that this loop is not only critical for the interaction with VCP but also for its role in the ERAD pathway. Negative staining EM shows that one molecule of OTU1 binds to one VCP hexamer, and isothermal titration calorimetry suggests that the two proteins bind with a KD of 0.71 μM. Analytical size exclusion chromatography and isothermal titration calorimetry demonstrates that OTU1 can bind VCP in both the presence and absence of a heterodimer formed by ubiquitin fusion degradation protein 1 and nuclear localization protein 4.

Concerted Action of the Ubiquitin-Fusion Degradation Protein 1 (Ufd1) and Sumo-Targeted Ubiquitin Ligases (STUbLs) in the DNA-Damage Response

In eukaryotes many players in the DNA-damage response (DDR) catalyze protein sumoylation or ubiquitylation. Emphasis has been placed on how these modifications orchestrate the sequential recruitment of repair factors to sites of DNA damage or stalled replication forks. Here, we shed light on a pathway in which sumoylated factors are eliminated through the coupled action of Sumo-targeted ubiquitin ligases (STUbLs) and the ubiquitin-fusion degradation protein 1 (Ufd1). Ufd1 is a subunit of the Cdc48-Ufd1-Npl4 complex implicated in the sorting of ubiquitylated substrates for degradation by the proteasome. We find that in fission yeast, Ufd1 interacts physically and functionally with the Sumo-targeted ubiquitin ligase (STUbL) Rfp1, homologous to human RNF4, and with the Sumo E3 ligase Pli1, homologous to human PIAS1. Deleting a C-terminal domain of Ufd1 that mediates the interaction of Ufd1 with Rfp1, Pli1, and Sumo (ufd1ΔCt 213-342) lead to an accumulation of high-molecular-weight Sumo conjugates and caused severe genomic instabilities. The spectrum of sensitivity of ufd1ΔCt 213-342 cells to genotoxins, the epistatic relationships of ufd1ΔCt 213-342 with mutations in DNA repair factors, and the localization of the repair factor Rad22 in ufd1ΔCt 213-342 cells point to ufd1ΔCt 213-342 cells accumulating aberrant structures during replication that require homologous recombination (HR) for their repair. We present evidence that HR is however often not successful in ufd1ΔCt 213-342 cells and we identify Rad22 as one of the high-molecular-weight conjugates accumulating in the ufd1ΔCt 213-342 mutant consistent with Rad22 being a STUbL/Ufd1 substrate. Suggesting a direct role of Ufd1 in the processing of Sumo-conjugates, Ufd1 formed nuclear foci colocalizing with Sumo during the DDR, and Sumo-conjugates accumulated in foci in the ufd1ΔCt 213-342 mutant. Broader functional relationships between Ufd1 and STUbLs conceivably affect numerous cellular processes beyond the DDR.

A wheat PI4K gene whose product possesses threonine autophophorylation activity confers tolerance to drought and salt in Arabidopsis

Phosphoinositides are involved in regulation of recruitment and activity of signalling proteins in cell membranes. Phosphatidylinositol (PI) 4-kinases (PI4Ks) generate PI4-phosphate the precursor of regulatory phosphoinositides. No type II PI4K research on the abiotic stress response has previously been reported in plants. A stress-inducible type II PI4K gene, named TaPI4KIIγ, was obtained by de novo transcriptome sequencing of drought-treated wheat (Triticum aestivum). TaPI4KIIγ, localized on the plasma membrane, underwent threonine autophosphorylation, but had no detectable lipid kinase activity. Interaction of TaPI4KIIγ with wheat ubiquitin fusion degradation protein (TaUDF1) indicated that it might be hydrolysed by the proteinase system. Overexpression of TaPI4KIIγ revealed that it could enhance drought and salt stress tolerance during seed germination and seedling growth. A ubdkγ7 mutant, identified as an orthologue of TaPI4KIIγ in Arabidopsis, was sensitive to salt, polyethylene glycol (PEG), and abscisic acid (ABA), and overexpression of TaPI4KIIγ in the ubdkγ7 mutant compensated stress sensitivity. TaPI4KIIγ promoted root growth in Arabidopsis, suggesting that TaPI4KIIγ might enhance stress resistance by improving root growth. Overexpression of TaPI4KIIγ led to an altered expression level of stress-related genes and changes in several physiological traits that made the plants more tolerant to stress. The results provided evidence that overexpression of TaPI4KIIγ could improve drought and salt tolerance.

Alkylphenols from the Roots of <i>Ardisia brevicaulis</i> Induce G1 Arrest and Apoptosis through Endoplasmic Reticulum Stress Pathway in Human Non-small-cell Lung Cancer Cells

From the roots of Ardisia brevicaulis DIELS, two new alkylphenol derivatives, named ardisiphenol E (2) and F (3), have been isolated together with a known alkylphenol, ardisiphenol D (1). The structures of 1-3 were elucidated by chemical and spectroscopic techniques. Compounds 1 and 2 exhibited strong cytotoxicities on two human non-small-cell lung cancer cell lines (H1299 and A549). We found that compounds 1 and 2 upregulated mRNA and protein expressions of endoplasmic reticulum (ER) stress markers including C/EBP homologous protein (CHOP), binding immunoglobulin protein (Bip) and inositol-requiring enzyme 1 (IRE1) indicating 1 and 2 are novel natural ER stress inducers. Treatments with 1 and 5 µM of 1 or 2 triggered G1 arrest in H1299 and A549 cells with concomitant downregulation of ubiquitin fusion degradation protein 1 (Ufd1) and S-phase kinase-associated protein 2 (Skp2) proteins and the accumulation of p27, the key axes of ER stress-mediated G1 arrest. Compounds 1 and 2 also induced apoptosis at high concentrations (10, 20 µM) which was shown to be coupled with the upregulation of CHOP and Bim, the activation of caspase-9, caspase-3 and poly(ADP-ribose) polymerase (PARP) cleavage. These results indicate that compounds 1 and 2 induce ER stress that subsequently causes G1 arrest and apoptosis in human non-small-cell lung cancer cells and they may have potential anticancer effects.

Phenotypic characterization and genetic mapping of a new gene required for male and female gametophyte development in rice

Gametophyte development is an important process in flowering plants. Seeds are the primary organs for harvesting, and therefore spikelet fertility is especially vital for rice yield. Here, we report the characterization and genetic mapping of a novel rice male and female sterile mutant, mfs1, which was generated from a transgenic line of japonica rice variety Zhonghua 9. Anther transverse sections indicate that the microspores in these plants were abnormally degenerated at the meiotic leptotene stage, while the tapetum layer was not degenerated and remained present; therefore, no functional pollen grains formed in the mutant anther locules. In addition, the majority of mutant embryo sacs did not undergo differentiation or the differentiation was incomplete, resulting in the absence of functional embryo sac formation. Genetic analysis of three F2 populations indicated that the mutant phenotype was controlled by a single recessive gene. The mutant phenotype was confirmed by linkage analysis to be not due to a foreign T-DNA insertion. A map-based cloning strategy was employed to map the mutant gene, and, finally, the mfs1 locus was mapped to a 99-kb physical interval between simple sequence repeat markers RM12585 and D051 on rice chromosome 2. Sequence analysis indicates that 11 rice candidate genes are located in this region, including three encoded putative proteins and eight encoded known proteins, such as transcription factors having WRKY and zinc finger domains, a ubiquitin fusion degradation protein, chaperone protein clpB 1, a two-component response regulator, the ZOS2-04-C2H2 zinc finger protein, a peptidase of the T1 family and a protein kinase.

Evolutionary divergence of valosin‐containing protein/cell division cycle protein 48 binding interactions among endoplasmic reticulum‐associated degradation proteins

Endoplasmic reticulum (ER)-associated degradation (ERAD) is a cell-autonomous process that eliminates large quantities of misfolded, newly synthesized protein, and is thus essential for the survival of any basic eukaryotic cell. Accordingly, the proteins involved and their interaction partners are well conserved from yeast to mammals, and Saccharomyces cerevisiae is widely used as a model system with which to investigate this fundamental cellular process. For example, valosin-containing protein (VCP) and its yeast homologue cell division cycle protein 48 (Cdc48p), which help to direct polyubiquitinated proteins for proteasome-mediated degradation, interact with an equivalent group of ubiquitin ligases in mouse and in S. cerevisiae. A conserved structural motif for cofactor binding would therefore be expected. We report a VCP-binding motif (VBM) shared by mammalian ubiquitin ligase E4b (Ube4b)-ubiquitin fusion degradation protein 2a (Ufd2a), hydroxymethylglutaryl reductase degradation protein 1 (Hrd1)-synoviolin and ataxin 3, and a related sequence in M(r) 78,000 glycoprotein-Amfr with slightly different binding properties, and show that Ube4b and Hrd1 compete for binding to the N-terminal domain of VCP. Each of these proteins is involved in ERAD, but none has an S. cerevisiae homologue containing the VBM. Some other invertebrate model organisms also lack the VBM in one or more of these proteins, in contrast to vertebrates, where the VBM is widely conserved. Thus, consistent with their importance in ERAD, evolution has developed at least two ways to bring these proteins together with VCP-Cdc48p. However, the differing molecular architecture of VCP-Cdc48p complexes indicates a key point of divergence in the molecular details of ERAD mechanisms.

Ubiquitin Fusion Degradation Protein 1 as a Blood Marker for the Early Diagnosis of Ischemic Stroke

Background Efficacy of thrombolysis in acute ischemic stroke is strongly related to physician's ability to make an accurate diagnosis and to intervene within 3–6 h after event onset. In this context, the discovery and validation of very early blood markers have recently become an urgent, yet unmet, goal of stroke research. Ubiquitin fusion degradation protein 1 is increased in human postmortem CSF, a model of global brain insult, suggesting that its measurement in blood may prove useful as a biomarker of stroke. Methods Enzyme-linked immunosorbent assay (ELISA) was used to measure UFD1 in plasma and sera in three independent cohorts, European (Swiss and Spanish) and North-American retrospective analysis encompassing a total of 123 consecutive stroke and 90 control subjects. Results Highly significant increase of ubiquitin fusion degradation protein 1 (UFD1) was found in Swiss stroke patients with 71% sensitivity (95% CI, 52–85.8%), and 90% specificity (95% CI, 74.2–98%) ( N = 31, p < 0.0001). Significantly elevated concentration of this marker was then validated in Spanish ( N = 39, p < 0.0001, 95% sensitivity (95% CI, 82.7– 99.4%)), 76% specificity (95% CI, 56.5–89.7%)) and North-American stroke patients ( N = 53, 62% sensitivity (95% CI, 47.9–75.2%), 90% specificity (95% CI, 73.5–97.9%), p < 0.0001). Its concentration was increased within 3 h of stroke onset, on both the Swiss ( p < 0.0001) and Spanish ( p = 0.0004) cohorts. Conclusions UFD1 emerges as a reliable plasma biomarker for the early diagnosis of stroke, and in the future, might be used in conjunction with clinical assessments, neuroimaging and other blood markers.

Identification of components of the murine histone deacetylase 6 complex: link between acetylation and ubiquitination signaling pathways.

The immunopurification of the endogenous cytoplasmic murine histone deacetylase 6 (mHDAC6), a member of the class II HDACs, from mouse testis cytosolic extracts allowed the identification of two associated proteins. Both were mammalian homologues of yeast proteins known to interact with each other and involved in the ubiquitin signaling pathway: p97/VCP/Cdc48p, a homologue of yeast Cdc48p, and phospholipase A2-activating protein, a homologue of yeast UFD3 (ubiquitin fusion degradation protein 3). Moreover, in the C-terminal region of mHDAC6, a conserved zinc finger-containing domain named ZnF-UBP, also present in several ubiquitin-specific proteases, was discovered and was shown to mediate the specific binding of ubiquitin by mHDAC6. By using a ubiquitin pull-down approach, nine major ubiquitin-binding proteins were identified in mouse testis cytosolic extracts, and mHDAC6 was found to be one of them. All of these findings strongly suggest that mHDAC6 could be involved in the control of protein ubiquitination. The investigation of biochemical properties of the mHDAC6 complex in vitro further supported this hypothesis and clearly established a link between protein acetylation and protein ubiquitination.

Cloning and characterization of the gene encoding human NPL4, a protein interacting with the ubiquitin fusion-degradation protein (UFD1L)

The ubiquitin fusion-degradation gene ( UFD1L) encodes the human homologue of the yeast ubiquitin fusion-degradation 1 protein, an essential component of the ubiquitin-dependent proteolytic turnover and mRNA processing. Although the UFD1L gene has been mapped in the region commonly deleted in patients with DiGeorge syndrome (DGS)/velocardiofacial syndrome (VCFS), correlation between its haploinsufficiency and the phenotype has not yet been established. The only functional data available about mammalian Ufd1p is the ability to form a complex with the rat Npl4 protein, a component of the nuclear pore complex. In this paper we report the cloning and molecular characterization of the human NPL4 gene. This gene encodes for a protein 96% homologous to the rat Npl4, and 44 and 34% homologous to the C. elegans and S. cerevisiae Npl4 gene products, respectively. Fluorescence in situ hybridization experiments on human metaphases localized the NPL4 gene on the most telomeric region of chromosome 17q. Northern blots analysis on foetal and adult human tissues revealed a major ∼4.5 kb transcript most abundant in heart, brain, kidney and skeletal muscle. In order to test a potential relationship between nuclear transport defects and some aspect of the DGS/VCFS phenotype, we also exclude the presence of mutations in the NPL4 coding sequence in a subset of patients with DGS/VCFS and no detectable 22q11 deletion or mutations at the UFD1L locus.

Absence of mutations in human ubiquitin fusion-degradation protein gene in tetralogy of Fallot.

Congenital defects in human chromosome 22q11 deletion syndromes are associated with the 3rd and 4th pharyngeal pouch during fetal development. In the cardiovascular system, these disorders are usually apparent as conotruncal heart defects and aortic arch anomalies. UFD1L, a gene that is downregulated in dHAND-deficient mice, expressed in the mouse embryo at the branchial arch and mapped to human chromosome 22q11, has recently been strongly suspected to be responsible for the phenotypes expressed in 22q11 deletion syndromes. Its putative causal role in relevant congenital cardiovascular malformations was studied by gene dosage analysis, mutation screening and sequence analyses. Sixty cases of tetralogy of Fallot with no detectable chromosome deletion at 22q11 or 10p13 were examined, including 51 cases of simple tetralogy of Fallot, and 9 cases of tetralogy of Fallot with pulmonary atresia. None of these patients revealed deletion limited to a portion of the UFD1L gene. Although mobility shift was found by heteroduplex analysis in 24 cases at exon 4 and flanking sequences, further sequence analysis demonstrated only two silent nucleotide variations and a single nucleotide polymorphism in intron 4. Our data suggest that, although the UFD1L gene is mapped to 22q11 and is expressed during early murine development at both cardiac and cranial neural crests, it is not responsible for the majority of tetralogy of Fallot cases in humans.

A Ufd2/D4Cole1e chimeric protein and overexpression of Rbp7 in the slow Wallerian degeneration (WldS) mouse.

Exons of three genes were identified within the 85-kilobase tandem triplication unit of the slow Wallerian degeneration mutant mouse, C57BL/Wld(S). Ubiquitin fusion degradation protein 2 (Ufd2) and a previously undescribed gene, D4Cole1e, span the proximal and distal boundaries of the repeat unit, respectively. They have the same chromosomal orientation and form a chimeric gene when brought together at the boundaries between adjacent repeat units in Wld(S). The chimeric mRNA is abundantly expressed in the nervous system and encodes an in-frame fusion protein consisting of the N-terminal 70 amino acids of Ufd2, the C-terminal 302 amino acids of D4Cole1e, and an aspartic acid formed at the junction. Antisera raised against synthetic peptides detect the expected 43-kDa protein specifically in Wld(S) brain. This expression pattern, together with the previously established role of ubiquitination in axon degeneration, makes the chimeric gene a promising candidate for Wld. The third gene altered by the triplication, Rbp7, is a novel member of the cellular retinoid-binding protein family and is highly expressed in white adipose tissue and mammary gland. The whole gene lies within the repeat unit leading to overexpression of the normal transcript in Wld(S) mice. However, it is undetectable on Northern blots of Wld(S) brain and seems unlikely to be the Wld gene. These data reveal both a candidate gene for Wld and the potential of the Wld(S) mutant for studies of ubiquitin and retinoid metabolism.