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1.
NfeD-like proteins are widely distributed throughout prokaryotes and are frequently associated with genes encoding stomatin-like proteins (slipins). Here, we reveal that the NfeD family is ancient and comprises three major groups: NfeD1a, NfeD1b and truncated NfeD1b. Members of each group are associated with one of four conserved gene partners, three of which have eukaryotic homologues that are membrane raft associated, namely stomatin, paraslipin (previously SLP-2) and flotillin. The first NfeD group (NfeD1b), comprises proteins of approximately 460-aa long that have three functional domains: an N-terminal protease, a middle membrane-spanning region and a soluble C-terminal region rich in β-strands. The nfeD1b gene is adjacent to eoslipin in prokaryotic genomes except in Firmicutes and Deinococci, where yqfA replaces eoslipin. Proteins in the second major group (NfeD1a) are homologous to the C-terminus of NfeD1b which forms a β-barrel-like domain, and their genes are associated with paraslipin. Using OrthoMCL clustering, we show that nfeD1b genes have become truncated on many independent occasions giving rise to the third major group. These short NfeD homologues frequently remain associated with their ancestral gene neighbour, resembling NfeD1a in structure, yet are much more related to full-length NfeD1b; we term these “truncated NfeD1b”. These conserved associations suggest that NfeD proteins are dependent on gene partners for their function and that the site of interaction may lie within the C-terminal portion that is common to all NfeD homologues. Although NfeD homologues are confined to prokaryotes, this conserved association could represent an excellent system to study slipin and flotillin proteins.  相似文献   

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3.
A common way to study protein function is to deplete the protein of interest from cells and observe the response. Traditional methods involve disrupting gene expression but these techniques are only effective against newly synthesized proteins and leave previously existing and stable proteins untouched. Here, we introduce a technique that induces the rapid degradation of specific proteins in mammalian cells by shuttling the proteins to the proteasome for degradation in a ubiquitin-independent manner. We present two implementations of the system in human culture cells that can be used individually to control protein concentration. Our study presents a simple, robust, and flexible technology platform for manipulating intracellular protein levels.  相似文献   

4.
Intracellular juxtamembrane regions of transmembrane proteins play pivotal roles in cell signalling, mediated by protein-protein interactions. Disordered protein regions, and short conserved motifs within them, are emerging as key determinants of many such interactions. Here, we investigated whether disorder and conserved motifs are enriched in the juxtamembrane area of human single-pass transmembrane proteins. Conserved motifs were defined as short disordered regions that were much more conserved than the adjacent disordered residues. Human single-pass proteins had higher mean disorder in their cytoplasmic segments than their extracellular parts. Some, but not all, of this effect reflected the shorter length of the cytoplasmic tail. A peak of cytoplasmic disorder was seen at around 30 residues from the membrane. We noted a significant increase in the incidence of conserved motifs within the disordered regions at the same location, even after correcting for the extent of disorder. We conclude that elevated disorder within the cytoplasmic tail of many transmembrane proteins is likely to be associated with enrichment for signalling interactions mediated by conserved short motifs.  相似文献   

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6.
NP220s compose a family of RNA binding proteins together with matrin 3, one of major proteins of the nuclear matrix. They have repeats of RNA recognition motif (RRM; MH2) homologous to RRM in heterogeneous nuclear RNPs I/L in addition to MH1 and MH3 with unknown function. In search of additional homologous sequences, we found the reported sequence of rat matrin 3 is partially incorrect. Correction of this sequence showed that the NP220 family has a fourth homologous motif with the characteristics of a Cys2-His2 zinc finger-like motif. The sequence of this motif is perfectly conserved in human and mouse NP220s despite their 75% overall sequence homology.  相似文献   

7.
The 26S proteasome is a eukaryotic ATP-dependent protease, but the molecular basis of its energy requirement is largely unknown. Ornithine decarboxylase (ODC) is the only known enzyme to be degraded by the 26S proteasome without ubiquitinylation. We report here that the 26S proteasome is responsible for the irreversible inactivation coupled to sequestration of ODC, a process requiring ATP and antizyme (AZ) but not proteolytic activity. Neither the 20S proteasome (catalytic core) nor PA700 (the regulatory complex) by itself contributed to this ODC inactivation. Analysis with a C-terminal mutant ODC revealed that the 26S proteasome recognizes the C-terminal degradation signal of ODC exposed by attachment of AZ, and subsequent ATP-dependent sequestration of ODC in the 26S proteasome causes irreversible inactivation, possibly unfolding, of ODC and dissociation of AZ. These processes may be linked to the translocation of ODC into the 20S proteasomal inner cavity, centralized within the 26S proteasome, for degradation.  相似文献   

8.
Fishes are known to use chemical alarm cues from both conspecifics and heterospecifics to assess local predation risks and enhance predator detection. Yet it is unknown how recognition of heterospecific cues arises for coral reef fishes. Here, we test if naïve juvenile fish have an innate recognition of heterospecific alarm cues. We also examine if there is a relationship between the intensity of the antipredator response to these cues and the degree to which species are related to each other. Naïve juvenile anemone fish, Amphiprion percula, were tested to see if they displayed antipredator responses to chemical alarm cues from four closely related heterospecific species (family Pomacentridae), a distantly related sympatric species (Asterropteryx semipunctatus) and a saltwater (control). Juveniles displayed significant reductions in foraging rate when exposed to all four confamilial heterospecific species but they did not respond to the distantly related sympatric species or the saltwater control. There was also a strong relationship between the intensity of the antipredator response and the extent to which species were related, with responses weakening as species became more distantly related. These findings demonstrate that chemical alarm cues are conserved within the pomacentrid family, providing juveniles with an innate recognition of heterospecific alarm cues as predicted by the phylogenetic relatedness hypothesis.  相似文献   

9.
An accumulation in cells of unfolded proteins is believed to be the common signal triggering the induction of heat shock proteins (hsps). Accordingly, in Saccharomyces cerevisiae, inhibition of protein breakdown at 30°C with the proteasome inhibitor MG132 caused a coordinate induction of many heat shock proteins within 1 to 2 h. Concomitantly, MG132, at concentrations that had little or no effect on growth rate, caused a dramatic increase in the cells’ resistance to very high temperature. The magnitude of this effect depended on the extent and duration of the inhibition of proteolysis. A similar induction of hsps and thermotolerance was seen with another proteasome inhibitor, clasto-lactacystin β-lactone, but not with an inhibitor of vacuolar proteases. Surprisingly, when the reversible inhibitor MG132 was removed, thermotolerance decreased rapidly, while synthesis of hsps continued to increase. In addition, exposure to MG132 and 37°C together had synergistic effects in promoting thermotolerance but did not increase hsp expression beyond that seen with either stimulus alone. Although thermotolerance did not correlate with hsp content, another thermoprotectant trehalose accumulated upon exposure of cells to MG132, and the cellular content of this disaccharide, unlike that of hsps, quickly decreased upon removal of MG132. Also, MG132 and 37°C had additive effects in causing trehalose accumulation. Thus, the resistance to heat induced by proteasome inhibitors is not just due to induction of hsps but also requires a short-lived metabolite, probably trehalose, which accumulates when proteolysis is reduced.  相似文献   

10.
Several proteasome-associated proteins regulate degradation by the 26 S proteasome using the ubiquitin chains that mark most substrates for degradation. The proteasome-associated protein Ecm29, however, has no ubiquitin-binding or modifying activity, and its direct effect on substrate degradation is unclear. Here, we show that Ecm29 acts as a proteasome inhibitor. Besides inhibiting the proteolytic cleavage of peptide substrates in vitro, it inhibits the degradation of ubiquitin-dependent and -independent substrates in vivo. Binding of Ecm29 to the proteasome induces a closed conformation of the substrate entry channel of the core particle. Furthermore, Ecm29 inhibits proteasomal ATPase activity, suggesting that the mechanism of inhibition and gate regulation by Ecm29 is through regulation of the proteasomal ATPases. Consistent with this, we identified through chemical cross-linking that Ecm29 binds to, or in close proximity to, the proteasomal ATPase subunit Rpt5. Additionally, we show that Ecm29 preferentially associates with both mutant and nucleotide depleted proteasomes. We propose that the inhibitory ability of Ecm29 is important for its function as a proteasome quality control factor by ensuring that aberrant proteasomes recognized by Ecm29 are inactive.  相似文献   

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The UL24 family of proteins is widely conserved among herpesviruses. We demonstrated previously that UL24 of herpes simplex virus 1 (HSV-1) is important for the dispersal of nucleolin from nucleolar foci throughout the nuclei of infected cells. Furthermore, the N-terminal portion of UL24 localizes to nuclei and can disperse nucleolin in the absence of any other viral proteins. In this study, we tested the hypothesis that highly conserved residues in UL24 are important for the ability of the protein to modify the nuclear distribution of nucleolin. We constructed a panel of substitution mutations in UL24 and tested their effects on nucleolin staining patterns. We found that modified UL24 proteins exhibited a range of subcellular distributions. Mutations associated with a wild-type localization pattern for UL24 correlated with high levels of nucleolin dispersal. Interestingly, mutations targeting two regions, namely, within the first homology domain and overlapping or near the previously identified PD-(D/E)XK endonuclease motif, caused the most altered UL24 localization pattern and the most drastic reduction in its ability to disperse nucleolin. Viral mutants corresponding to the substitutions G121A and E99A/K101A both exhibited a syncytial plaque phenotype at 39°C. vUL24-E99A/K101A replicated to lower titers than did vUL24-G121A or KOS. Furthermore, the E99A/K101A mutation caused the greatest impairment of HSV-1-induced dispersal of nucleolin. Our results identified residues in UL24 that are critical for the ability of UL24 to alter nucleoli and further support the notion that the endonuclease motif is important for the function of UL24 during infection.The UL24 protein is conserved throughout the Herpesviridae family, and to the best of our knowledge, a UL24 homolog has been identified in all Herpesvirales genomes sequenced to date with the exception of the channel catfish virus (9, 10, 19). UL24 of herpes simplex virus 1 (HSV-1) is required for efficient virus replication both in vitro and in vivo and for reactivation from latency in a mouse model of ocular infection (18). UL24 is one of the few HSV-1 genes, along with gB, gK, and UL20, in which mutations have been identified that cause the formation of syncytial plaques (2, 7, 34, 36, 39). The UL24-associated syncytial phenotype is only partially penetrant at 37°C but is fully penetrant at 39°C. Indications are that gK and UL20 have an inhibitory effect on the formation of syncytia (1), while certain mutations in gB entrain an uncontrolled fusogenic activity (11, 13, 15).UL24 is a highly basic protein of 269 amino acids that is expressed with leaky-late kinetics (31). Five homology domains (HDs), which consist of stretches of amino acids with a high percentage of identity between homologs, are present in the UL24 open reading frame (ORF) (19). In addition, a PD-(D/E)XK endonuclease motif has been identified that falls within the HDs (20); however, a role for this motif has yet to be demonstrated. In infected cells, UL24 is detected in the nucleus and the cytoplasm and transiently localizes to nucleoli (23). In the absence of other viral proteins, UL24 accumulates in the Golgi apparatus and in the nucleus, where it usually exhibits a diffuse staining pattern, but in a minority of cells it is detected in nucleoli (3).During infection, the formation of the viral replication compartments in the nucleus and the action of several viral proteins result in a remodeling of the nucleus. Chromatin is marginalized (29, 40), promyelocytic leukemia bodies are dispersed (26, 27), and the nuclear lamina is disrupted (33, 37). HSV-1 infection also affects the nucleolus, a prominent nuclear substructure implicated in the synthesis of rRNA, cell cycle regulation, and nucleocytoplasmic shuttling (5). Nucleoli become elongated following infection, and the synthesis of mature rRNA is reduced (4, 38, 42). Several HSV-1 proteins have been shown to localize to, or associate with, the nucleolus (12). The viral protein VP22 associates with the nucleolus and with dispersed nucleolin in HSV-1-infected cells (22), and RL1, US11, and ICP0 have also been shown to localize to nucleoli (24, 30, 35). Previously we showed that nucleolin is dispersed throughout the nucleus upon HSV-1 infection and that UL24 is involved in this nuclear modification (23). We further found that the N-terminal portion of UL24 is sufficient to induce the redistribution of nucleolin in the absence of other viral proteins (3).In this study, we sought to test the hypothesis that the endonuclease motif, which is made up of some of the most highly conserved residues in UL24, is important for the dispersal of nucleolin. A panel of substitution mutations in UL24 was generated, and the impact on the function of UL24 was assessed.  相似文献   

13.
14.
Four cDNA clones of tobacco that could code for polypeptides with two WRKY domains were isolated. Among four NtWRKYs and other WRKY family proteins, sequence similarity was basically limited to the two WRKY domains. Glutathione S-transferase fusion proteins with the C-terminal WRKY domain of four NtWRKYs bound specifically to the W-box (TTGACC), and the N-terminal WRKY domain showed weaker binding activity with the W-box compared to the C-terminal domain. The DNA-binding activity of the WRKY domain was abolished by o-phenanthroline and this inhibition was recovered specifically by Zn2+. Substitution of the conserved cysteine and histidine residues of the plant-specific C2H2-type zinc finger-like motif in the WRKY domain abolished the DNA binding. In addition, mutations in the invariable WRKYGQK sequence at the N-terminal side of the zinc finger-like motif also significantly reduced the DNA-binding activity, suggesting that these residues are required for proper folding of the DNA-binding zinc finger.  相似文献   

15.
Thirteen point mutations targeting predicted domains conserved in homologous protein kinases were introduced into the UL97 coding region of the human cytomegalovirus. All mutagenized proteins were expressed in cells infected with recombinant vaccinia viruses (rVV). Several mutations drastically reduced ganciclovir (GCV) phosphorylation. Mutations at amino acids G340, A442, L446, and F523 resulted in a complete loss of pUL97 phosphorylation, which was strictly associated with a loss of GCV phosphorylation. Our results confirm that in rVV-infected cells pUL97 phosphorylation is due to autophosphorylation and show that several amino acids conserved within domains of protein kinases are essential for this pUL97 phosphorylation. GCV phosphorylation is dependent on pUL97 phosphorylation.  相似文献   

16.
The antibacterial peptide toxin colicin V (ColV) uses a dedicated signal sequence-independent export system for its secretion in Escherichia coli that involves the products of three genes, cvaA, cvaB, and tolC in this process. As a member of the membrane fusion protein (MFP) family, the CvaA protein has been proposed to interact with an outer membrane protein TolC via its C-terminal hydrophobic domain. The importance of this domain, which is highly conserved throughout the members of MFP family, was analyzed by use of site-directed mutagenesis of missense or nonsense mutations with suppressors. All the nonsense mutations tested resulted in the loss of ColV secretion, indicating the importance of the C-terminus of CvaA, including the last 100 residue–hydrophilic domain. The missense mutations of several conserved amino acids have no drastic effects. On the other hand, when Glu-248, Ala-262, Thr-274, Leu-285, Gly-313, Ala-322, or Val-335 of CvaA protein was mutated, the secretion of ColV was greatly reduced in certain mutants. While some mutations resulted in structural instability, Glu-248 to Lys and Ala-322 to Gly proteins were relatively stable, but were not functional in ColV secretion. The results indicate that these conserved amino acids are important for the structure and functions of CvaA in the secretion of ColV. Received: 6 February 1999 / Accepted: 26 June 1999  相似文献   

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18.
Prior work showed that expression of acyl carrier proteins (ACPs) of a diverse set of bacteria replaced the function of Escherichia coli ACP in lipid biosynthesis. However, the AcpAs of Lactococcus lactis and Enterococcus faecalis were inactive. Both failed to support growth of an E. coli acpP mutant strain. This defect seemed likely because of the helix II sequences of the two AcpAs, which differed markedly from those of the proteins that supported growth. To test this premise, chimeric ACPs were constructed in which L. lactis helix II replaced helix II of E. coli AcpP and vice versa. Expression of the AcpP protein L. lactis AcpA helix II allowed weak growth, whereas the L. lactis AcpA-derived protein that contained E. coli AcpP helix II failed to support growth of the E. coli mutant strain. Replacement of the L. lactis AcpA helix II residues in this protein showed that substitution of valine for the phenylalanine residue four residues downstream of the phosphopanthetheine-modified serine gave robust growth and allowed modification by the endogenous AcpS phosphopantetheinyl transferase (rather than the promiscuous Sfp transferase required to modify the L. lactis AcpA and the chimera of L. lactis AcpA helix II in AcpP). Further chimera constructs showed that the lack of function of the L. lactis AcpA-derived protein containing E. coli AcpP helix II was due to incompatibility of L. lactis AcpA helix I with the downstream elements of AcpP. Therefore, the origins of ACP incompatibility can reside in either helix I or in helix II.  相似文献   

19.
The ubiquitin (Ub)/26S proteasome system (UPS) directs the turnover of numerous regulatory proteins, thereby exerting control over many aspects of plant growth, development, and survival. The UPS is directed in part by a group of Ub-like/Ub-associated (UBL/UBA) proteins that help shuttle ubiquitylated proteins to the 26S proteasome for breakdown. Here, we describe the collection of UBL/UBA proteins in Arabidopsis thaliana, including four isoforms that comprise the RADIATION SENSITIVE23 (RAD23) family. The nuclear-enriched RAD23 proteins bind Ub conjugates, especially those linked internally through Lys-48, via their UBA domains, and associate with the 26S proteasome Ub receptor RPN10 via their N-terminal UBL domains. Whereas homozygous mutants individually affecting the four RAD23 genes are without phenotypic consequences (rad23a, rad23c, and rad23d) or induce mild phyllotaxy and sterility defects (rad23b), higher-order mutant combinations generate severely dwarfed plants, with the quadruple mutant displaying reproductive lethality. Both the synergistic effects of a rad23b-1 rpn10-1 combination and the response of rad23b plants to mitomycin C suggest that RAD23b regulates cell division. Taken together, RAD23 proteins appear to play an essential role in the cell cycle, morphology, and fertility of plants through their delivery of UPS substrates to the 26S proteasome.  相似文献   

20.
Duchenne Muscular Dystrophy (DMD) is an incurable inherited disease ofchildhood, characterized by progressive muscle degeneration and weakness. Our previousfindings supported the idea that dystrophin and associated proteins, absent or greatlyreduced in DMD, are degraded in dystrophin-deficient muscle by the proteasomaldependentpathway. Indeed, treatment with the proteasome inhibitor MG-132 of skeletalmuscles from mdx mice --a spontaneous mouse model of DMD-- as well as from DMDpatients, effectively rescued the expression and correct cellular localization of dystrophinand associated proteins. These promising results led us to further explore the use ofproteasome inhibitors as a therapy for DMD. Therefore, we directed our attentiontowards two new dipeptide boronic acid inhibitors blocking the proteasomal-dependentdegradation pathway: Velcade (bortezomib or PS-341) and MLN273 (PS-273). Theexciting aspect of this development is that these drugs have already progressed to preclinicaland clinical trials, in different fields than muscular dystrophy. Indeed, Velcadehas been already FDA-approved for treatment of multiple myeloma and its side effectshad been already explored and managed. Promisingly, MLN273 is currently in thepreclinical trial phase. Here, we test the effectiveness of Velcade and MLN273 by localinjection into the gastrocnemius muscle of mdx mice. We show the rescue of expressionand membrane localization of 􀀁-dystroglycan, 􀀂-dystroglycan, 􀀁-sarcoglycan, anddystrophin after Velcade and MLN273 localized treatment, versus untreated (PBS only)mdx mice. Intriguingly, we also show that localized treatment with Velcade and MLN273reduces the activation of Nuclear Factor-kappaB (NF-kB). Because NF-kB pathway hasbeen shown to be involved in inflammation responses in myopathies and DMD, ourcurrent results may have important clinical implications. Clearly, more investigations areneeded, but our results emphasize the effectiveness of the pharmacological approach as apotential treatment for Duchenne muscular dystrophy.  相似文献   

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