Centrin/Cdc31 is a novel regulator of protein degradation

Rad23 is required for efficient protein degradation and performs an important role in nucleotide excision repair. Saccharomyces cerevisiae Rad23, and its human counterpart (hHR23), are present in a complex containing the DNA repair factor Rad4 (termed XPC, for xeroderma pigmentosum group C, in humans). XPC/hHR23 was also reported to bind centrin-2, a member of the superfamily of calcium-binding EF-hand proteins. We report here that yeast centrin, which is encoded by CDC31, is similarly present in a complex with Rad4/Rad23 (called NEF2). The interaction between Cdc31 and Rad23/Rad4 varied by growth phase and reflected oscillations in Cdc31 levels. Strikingly, a cdc31 mutant that formed a weaker interaction with Rad4 showed sensitivity to UV light. Based on the dual function of Rad23, in both DNA repair and protein degradation, we questioned if Cdc31 also participated in protein degradation. We report here that Cdc31 binds the proteasome and multiubiquitinated proteins through its carboxy-terminal EF-hand motifs. Moreover, cdc31 mutants were highly sensitive to drugs that cause protein damage, failed to efficiently degrade proteolytic substrates, and formed altered interactions with the proteasome. These findings reveal for the first time a new role for centrin/Cdc31 in protein degradation.     

Ypt31/32 GTPases and their novel F-box effector protein Rcy1 regulate protein recycling

Ypt/Rab GTPases control various aspects of vesicle formation and targeting via their diverse effectors. We report a new role for these GTPases in protein recycling through a novel effector. The F-box protein Rcy1, which mediates plasma membrane recycling, is identified here as a downstream effector of the Ypt31/32 GTPase pair because it binds active GTP-bound Ypt31/32 and colocalizes with these GTPases on late Golgi and endosomes. Furthermore, Ypt31/32 regulates the polarized localization and half-life of Rcy1. This suggests that Ypt/Rabs can regulate the protein level of their effectors, in addition to the established ways by which they control their effectors. We show that like Rcy1, Ypt31/32 regulate the coupled phosphorylation and recycling of the plasma membrane v-SNARE Snc1. Moreover, Ypt31/32 and Rcy1 regulate the recycling of the furin-homolog Kex2 to the Golgi. Therefore, Ypt31/32 and Rcy1 mediate endosome-to-Golgi transport, because this is the only step shared by Snc1 and Kex2. Finally, we show that Rcy1 physically interacts with Snc1. Based on this result and because F-box proteins serve as adaptors between specific substrates and ubiquitin ligases, we propose that Ypt31/32 GTPases regulate the function of Rcy1 in the phosphorylation and/or ubiquitination of proteins that recycle through the Golgi.     

The product of the UL31 gene of herpes simplex virus 1 is a nuclear phosphoprotein which partitions with the nuclear matrix.

The nucleotide sequence of the UL31 open reading frame is predicted to encode a basic protein with a hydrophilic amino terminus and a nuclear localization signal. To identify its gene product, we constructed a viral genome in which the thymidine kinase gene was inserted between the UL31 and UL32 open reading frames. The thymidine kinase gene was then deleted, and in the process, the 5' terminus of the UL31 open reading frame was replaced with a 64-bp sequence in frame with the complete, authentic sequence of the UL31 open reading frame. The inserted sequence encoded a hydrophilic epitope derived from glycoprotein B of human cytomegalovirus and for which a monoclonal antibody is available. We report that in infected cells, the tagged protein localized in and was dispersed throughout the nucleus. Nuclear fractionation studies revealed that the UL31 protein partitions with the nuclear matrix. The protein is phosphorylated in infected cells maintained in medium containing 32Pi.     

P26h and dicarbonyl/L-xylulose reductase are two distinct proteins present in the hamster epididymis

We have previously identified a 34 kDa protein (P34H) on the human sperm surface covering the acrosome. Using the hamster, we have also described a sperm protein, P26h, which is acquired by spermatozoa during epididymal transit. Both P34H and P26h belong to the carbonyl reductase family. Using molecular tools derived from P34H, we searched in the hamster epididymis for another protein related to the human sperm protein. Cloning and sequencing of P31h cDNA revealed 100% homology with the kidney DCXR (Dicarbonyl/L-Xylulose reductase). Northern Blot experiments revealed a single mRNA that was more expressed in the caput than in the corpus and cauda segment of adult epididymides. In situ hybridization was performed on sexually mature hamsters showing that the mRNA was localized in the principal cells throughout the epididymis. Using an anti-P34H antibody we have identified a P34H related protein named P31h (for 31 kDa). This protein showed 2D-electrophoretic behavior different from P26h and was detectable all along the epididymis (caput, corpus, and cauda) by Western Blot analysis. Immunohistochemistry techniques showed that P31h was localized in the perinuclear region of the principal cells of the epididymal epithelium within the three sections, both in sexually mature and immature animals. Results are discussed with regards to the potential function of DCXR in the epididymis.     

Association of BAP31 with CD11b/CD18. Potential role in intracellular trafficking of CD11b/CD18 in neutrophils

The beta2 integrin CD11b/CD18 is an integral membrane protein that is present in the plasma membrane and secondary granules of neutrophils and functions as a major adhesion molecule. Upon cellular activation, there is translocation of intracellular pools of CD11b/CD18 to the plasma membrane in concert with enhanced cellular adhesion. Although much is known about the function of CD11b/CD18, how this protein is transported within the cell is less well defined. Here we report that CD11b/CD18 specifically binds to BAP31, a member of a novel class of sorting proteins regulating cellular anterograde transport. Through experiments aimed at identifying CD11b/CD18-binding proteins, we produced a monoclonal antibody termed E1B2 that recognizes a 28-kDa membrane protein that co-precipitates with CD11b/CD18. Microsequence analysis of the E1B2 antigen revealed that it is BAP31. Co-association of CD11b/CD18 and BAP31 was confirmed in co-immunoprecipitation and protein binding assays. Additional experiments revealed that the binding of BAP31 to CD11b/CD18 was not dependent on divalent cations nor mediated by the I-domain of CD11b. Using glutathione S-transferase fusion chimeras, we determined that binding of CD11b/CD18 to BAP31 is mediated through interactions with the cytoplasmic tail of BAP31. Immunolocalization studies revealed colocalization of BAP31 and CD11b/CD18 within neutrophil secondary granules. Subcellular fractionation studies in polymorphonuclear leukocytes (PMN) revealed similar patterns of redistribution of BAP31 and CD11b/CD18 from fractions enriched in secondary granules to the plasma membrane following stimulation with formylmethionylleucylphenylalanine (fMLP). Given the known sorting properties of BAP31, these findings suggest that BAP31 may play a role in regulating intracellular trafficking of CD11b/CD18 in neutrophils.     

Higher order assembling of the mycobacterial polar growth factor DivIVA/Wag31

DivIVA or Wag31, which is an essential pole organizing protein in mycobacteria, can self-assemble at the negatively curved side of the membrane at the growing pole to form a higher order structural scaffold for maintaining cellular morphology and localizing various target proteins for cell-wall biogenesis. The structural organization of polar scaffold formed by polymerization of coiled-coil rich Wag31, which is implicated in the anti-tubercular activities of amino-pyrimidine sulfonamides, remains to be determined. A single-site phosphorylation in Wag31 regulates peptidoglycan biosynthesis in mycobacteria. We report biophysical characterizations of filaments formed by mycobacterial Wag31 using circular dichroism, atomic force microscopy and small angle solution X-ray scattering. Atomic force microscopic images of the wild-type, a phospho-mimetic (T73E) and a phospho-ablative (T73A) form of Wag31 show mostly linear filament formation with occasional curving, kinking and apparent branching. Solution X-ray scattering data indicates that the phospho-mimetic forms of the Wag31 polymers are on average more compact than their phospho-ablative counterparts, which is likely due to the extent of bending/branching. Observed structural features in this first view of Wag31 filaments suggest a basis for higher order Wag31 scaffold formation at the pole.     

Backbone resonance assignments of the <Emphasis Type="Italic">Escherichia coli</Emphasis> 62 kDa protein,Hsp31

Dimeric Hsp31 protein was first characterized as a holding chaperone of Escherichia coli (E. coli), and has been suggested as having protease activity due to the presence of a potential catalytic triad, Cys185, His186, and Asp214. However, it has recently been reported that Hsp31 displays a relatively strong glyoxalase III activity that can decompose reactive carbonyl species (methylglyoxal and glyoxal) in the absence of additional cofactor. Hsp31 is a representative member of the DJ-1/ThiJ/PfpI protein superfamily, and the importance of DJ-1 protein in Parkinson’s disease has been well known. The structural flexibility of the long loop region, which encompasses from the P- to the A-domain, is important for the chaperone activity of Hsp31. The backbone chemical shifts (CSs) would be useful for studying the structural changes of Hsp31 that are critical for the holding chaperone activity, and also for deciphering the switching mechanism between the glyoxalase III and the chaperone. Here, we report the backbone CSs (H^N, N, CO, C_α, and C_β) of the deuterated Hsp31 protein (62 kDa). The CS analysis showed that the predicted regions of secondary structures are in good agreement with those observed in the previous crystal structure of Hsp31.     

An aberrant sequence in a connexin46 mutant underlies congenital cataracts

An increasing number of diseases have been mapped to genes coding for ion channel proteins, including the gap junction proteins, connexins. Here, we report on the identification of an amino acid sequence underlying the behavior of a non-functional mutant connexin46 (CX46) associated with congenital cataracts. The mutant protein, CX46fs380, is 31 amino acids longer than CX46 and contains 87 aberrant amino acids in its C terminus. When expressed in mammalian cells, the mutant CX46 was not found at gap junctional plaques, but it showed extensive co-localization with markers for ERGIC and Golgi. The severe reductions in function and formation of gap junctional plaques were transferred to other connexins by creating chimeras containing the last third (or more) of the aberrant C terminus of the CX46 mutant. This sequence also impaired trafficking of a CD8 chimera. Site-directed mutagenesis of a diphenylalanine restored appositional membrane localization and function. These results suggest a novel mechanism in which a mutation causes disease by generating a motif that leads to retention within the synthetic/secretory pathway.     

Reversible unfolding of cytochrome c upon interaction with cardiolipin bilayers. 2. Evidence from phosphorus-31 NMR measurements.

31P NMR measurements were conducted to determine the structural and chemical environment of beef heart cardiolipin when bound to cytochrome c. 31P NMR line shapes infer that the majority of lipid remains in the bilayer state and that the average conformation of the lipid phosphate is not greatly affected by binding to the protein. An analysis of the spin-lattice (T1) relaxation times of hydrated cardiolipin as a function of temperature describes a T1 minimum at around 25 degrees C which leads to a correlation time for the phosphates in the lipid headgroup of 0.71 ns. The relaxation behavior of the protein-lipid complex was markedly different, showing a pronounced enhancement in the phosphorus spin-lattice relaxation rate. This effect of the protein increased progressively with increasing temperature, giving no indication of a minimum in T1 up to 75 degrees C. The enhancement in lipid phosphorus T1 relaxation was observed with protein in both oxidation states, being somewhat less marked for the reduced form. The characteristics of the T1 effects and the influence of the protein on other relaxation processes determined for the lipid phosphorus (spin-spin relaxation and longitudinal relaxation in the rotating frame) point to a strong paramagnetic interaction from the protein. A comparison with the relaxation behavior of samples spinning at the "magic angle" was also consistent with this mechanism. The results suggest that cytochrome c reversibly denatures on complexation with cardiolipin bilayers, such that the electronic ground state prevailing in the native structure of both oxidized and reduced protein can convert to high-spin states with greater magnetic susceptibility.     

Pseudo-T-even bacteriophage RB49 encodes CocO, a cochaperonin for GroEL, which can substitute for Escherichia coli's GroES and bacteriophage T4's Gp31

Bacteriophage T4-encoded Gp31 is a functional ortholog of the Escherichia coli GroES cochaperonin protein. Both of these proteins form transient, productive complexes with the GroEL chaperonin, required for protein folding and other related functions in the cell. However, Gp31 is specifically required, in conjunction with GroEL, for the correct folding of Gp23, the major capsid protein of T4. To better understand the interaction between GroEL and its cochaperonin cognates, we determined whether the so-called "pseudo-T-even bacteriophages" are dependent on host GroEL function and whether they also encode their own cochaperonin. Here, we report the isolation of an allele-specific mutation of bacteriophage RB49, called epsilon22, which permits growth on the E. coli groEL44 mutant but not on the isogenic wild type host. RB49 epsilon22 was used in marker rescue experiments to identify the corresponding wild type gene, which we have named cocO (cochaperonin cognate). CocO has extremely limited identity to GroES but is 34% identical and 55% similar at the protein sequence level to T4 Gp31, sharing all of the structural features of Gp31 that distinguish it from GroES. CocO can substitute for Gp31 in T4 growth and also suppresses the temperature-sensitive phenotype of the E. coli groES42 mutant. CocO's predicted mobile loop is one residue longer than that of Gp31, with the epsilon22 mutation resulting in a Q36R substitution in this extra residue. Both the CocO wild type and epsilon22 proteins have been purified and shown in vitro to assist GroEL in the refolding of denatured citrate synthase.     

Structure of a conserved dimerization domain within the F-box protein Fbxo7 and the PI31 proteasome inhibitor

F-box proteins are the substrate-recognition components of the Skp1-Cul1-F box protein (SCF) E3 ubiquitin ligases. Here we report a structural relationship between Fbxo7, a component of the SCF(Fbxo7) E3 ligase, and the proteasome inhibitor PI31. SCF(Fbxo7) is known to catalyze the ubiquitination of hepatoma-up-regulated protein (HURP) and the inhibitor of apoptosis (IAP) protein but also functions as an activator of cyclin D-Cdk6 complexes. We identify PI31 as an Fbxo7.Skp1 binding partner and show that this interaction requires an N-terminal domain present in both proteins that we term the FP (Fbxo7/PI31) domain. The crystal structure of the PI31 FP domain reveals a novel alpha/beta-fold. Biophysical and mutational analyses are used to map regions of the PI31 FP domain mediating homodimerization and required for heterodimerization with Fbxo7.Skp1. Equivalent mutations in Fbxo7 ablate interaction with PI31 and also block Fbxo7 homodimerization. Knockdown of Fbxo7 does not affect PI31 levels arguing against PI31 being a substrate for SCF(Fbxo7). We present a model for FP domain-mediated dimerization of SCF(Fbxo7) and PI31.     

Identification of tyrosine hydroxylase as a physiological substrate for Cdk5

Cyclin-dependent kinase 5 (Cdk5) is emerging as a neuronal protein kinase involved in multiple aspects of neurotransmission in both post- and presynaptic compartments. Within the reward/motor circuitry of the basal ganglia, Cdk5 regulates dopamine neurotransmission via phosphorylation of the postsynaptic signal transduction pathway integrator, DARPP-32 (dopamine- and cyclic AMP-regulated phosphoprotein, M(r) 32,000). Cdk5 has also been implicated in regulating various steps in the presynaptic vesicle cycle. Here we report that Cdk5 phosphorylates tyrosine hydroxylase (TH), the key enzyme for synthesis of dopamine. Using phosphopeptide mapping, site-directed mutagenesis, and phosphorylation state-specific antibodies, the site was identified as Ser31, a previously defined extracellular signal-regulated kinases 1/2 (ERK1/2) site. The phosphorylation of Ser31 by Cdk5 versus ERK1/2 was investigated in intact mouse striatal tissue using a pharmacological approach. The results indicated that Cdk5 phosphorylates TH directly and also regulates ERK1/2-dependent phosphorylation of TH through the phosphorylation of mitogen-activated protein kinase kinase 1 (MEK1). Finally, phospho-Ser31 TH levels were increased in dopaminergic neurons of rats trained to chronically self-administer cocaine. These results demonstrate direct and indirect regulation of the phosphorylation state of a Cdk5/ERK1/2 site on TH and suggest a role for these pathways in the neuroadaptive changes associated with chronic cocaine exposure.     

The staurosporine analog, Ro-31-8220, induces apoptosis independently of its ability to inhibit protein kinase C

A series of bisindolylmaleimide (Bis) compounds were designed as analogs of the natural compound staurosporine (STS), which is a potent inducer of apoptosis. Many of the Bis analogs appear to be highly selective inhibitors of the protein kinase C (PKC) family, including PKC-alpha, -beta, -gamma, -delta, -epsilon, and -zeta, unlike STS, which is an inhibitor of a broad spectrum of protein kinases. In this report we describe the effects of the Bis analogs, Bis-I, Bis-II, Bis-III and Ro-31-8220 on the survival and proliferation of HL-60 cells, which have been widely used as a model cell system for studying the biological roles of PKC. Treatment of HL-60 cells with Bis-I, Bis-II, Bis-III, or Ro-31-8220 blocked phosphorylation of the PKC target protein Raf-1 with equal potency but did not appear to affect the general phosphorylation of proteins by other kinases. However, the biological effects of the Bis compounds were different: Bis-I and Bis-II had no observable effects on either cell survival or proliferation; Bis-III inhibited cell proliferation but not survival, whereas Ro-31-8220 induced apoptosis. These results indicated that the members of the PKC family which could be inhibited by the Bis analogs were required neither for survival nor proliferation of HL-60 cells. Analyses of cells treated with Ro-31-8220 showed that the apoptotic effect of Ro-31-8220 on HL-60 cells was mediated by a well-characterized transduction process of apoptotic signals: i.e., mitochondrial cytochrome c efflux and the activation of caspase-3 in the cytosol. Moreover, the ability of Ro-31-8220 to induce apoptotic activation was completely inhibited by the over-expression of the apoptotic suppressor gene, Bcl-2, in the cells. Interestingly, proliferation of the Bcl-2-over-expressing cells was still sensitive to the presence of Ro-31-8220, suggesting that the inhibitory effects of Ro-31-8220 on viability and cell proliferation were mediated by different mechanisms. In particular, the apoptotic effect of Ro-31-8220 on cells was not altered by the presence of an excess amount of the other Bis analogs, suggesting that this effect is mediated by a factor(s) other than PKC or by a mechanism which was not saturable by the other Bis analogs. Finally, structure-function analyses of compounds related to Ro-31-8220 revealed that a thioamidine prosthetic group in Ro-311-8220 was largely responsible for its apoptotic activity.     

Molecular cloning of a human gene (S31) encoding a novel serotonin receptor mediating inhibition of adenylyl cyclase.

We report the molecular cloning of human gene (S31) containing an open reading frame of 1095 nucleotides, which encodes a protein of 365 amino acids. The encoded protein contains seven hydrophobic putative transmembrane domains considered the hallmark of G protein-coupled receptors. The amino acid sequence shows highest homology to receptors for serotonin (5-hydroxytryptamine). Expression of this receptor in murine Ltk- cells conferred upon these cells the ability to respond to serotonin by inhibition of adenylyl cyclase. No response was observed to isoproterenol, epinephrine, histamine, dopamine or melatonin in the transfected cells. We propose that the human gene S31 encodes a novel serotonin receptor.     

Dysregulation of cell polarity proteins synergize with oncogenes or the microenvironment to induce invasive behavior in epithelial cells

Changes in expression and localization of proteins that regulate cell and tissue polarity are frequently observed in carcinoma. However, the mechanisms by which changes in cell polarity proteins regulate carcinoma progression are not well understood. Here, we report that loss of polarity protein expression in epithelial cells primes them for cooperation with oncogenes or changes in tissue microenvironment to promote invasive behavior. Activation of ErbB2 in cells lacking the polarity regulators Scribble, Dlg1 or AF-6, induced invasive properties. This cooperation required the ability of ErbB2 to regulate the Par6/aPKC polarity complex. Inhibition of the ErbB2-Par6 pathway was sufficient to block ErbB2-induced invasion suggesting that two polarity hits may be needed for ErbB2 to promote invasion. Interestingly, in the absence of ErbB2 activation, either a combined loss of two polarity proteins, or exposure of cells lacking one polarity protein to cytokines IL-6 or TNFα induced invasive behavior in epithelial cells. We observed the invasive behavior only when cells were plated on a stiff matrix (Matrigel/Collagen-1) and not when plated on a soft matrix (Matrigel alone). Cells lacking two polarity proteins upregulated expression of EGFR and activated Akt. Inhibition of Akt activity blocked the invasive behavior identifying a mechanism by which loss of polarity promotes invasion of epithelial cells. Thus, we demonstrate that loss of polarity proteins confers phenotypic plasticity to epithelial cells such that they display normal behavior under normal culture conditions but display aggressive behavior in response to activation of oncogenes or exposure to cytokines.     

Purification and biochemical characterization of dog gastric lipase

A lipase was found to be present in dog stomach which appeared to be more abundant in the fundic than in the pyloric mucosa. Dog gastric lipase was extracted by soaking the gastric tissue and further purified after cation exchange, anion exchange and gel-filtration using fast protein liquid chromatography. The amino-acid composition, N-terminal amino-acid sequence, substrate specificity, interfacial and kinetic behavior and inactivation by sulfhydryl reagents were determined and compared with those of human and rabbit gastric lipases. We report for the first time that a gastric lipase is 13 times more active on long-chain than on short-chain triacylglycerols at pH 4.0, reaching a maximal specific activity of 950 U/mg on Intralipide emulsion.     

Cellular stabilization of the melatonin rhythm enzyme induced by nonhydrolyzable phosphonate incorporation

Serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase, AANAT) controls daily changes in the production and circulating levels of melatonin. Here, the significance of the phosphorylation of AANAT was studied using a semisynthetic enzyme in which a nonhydrolyzable phosphoserine/threonine mimetic, phosphonomethylenealanine (Pma), was incorporated at position 31 (AANAT-Pma31). The results of studies in which AANAT-Pma31 and related analogs were injected into cells provide the first direct evidence that Thr31 phosphorylation controls AANAT stability in the context of the intact cells by binding to 14-3-3 protein. These findings establish Thr31 phosphorylation as an essential element in the intracellular regulation of melatonin production. The application of Pma in protein semisynthesis is likely to be broadly useful in the analysis of protein serine/threonine phosphorylation.     

Molecular cloning of the 31 kDa cytosolic phospholipase A2, as an antigen recognized by the lung cancer-specific human monoclonal antibody, AE6F4

The human monoclonal antibody AE6F4 specifically reacts with human lung cancer tissues but does not with normal tissues. This monoclonal antibody recognizes a cytosolic 31 kDa antigen in the cancer cells. In a previous study, we elucidated that the 31 kDa antigen belonged to a family of proteins collectively designated as 14-3-3 proteins, which were known as protein kinase-dependent activators of tyrosine/trytophan hydroxylases, or protein kinase C inhibitor proteins. Here we report molecular cloning of the 31 kDa antigen from the human lung adenocarcinoma cell line, A549. Sequencing analysis indicates that the cloned cDNA is identical to that of previously reported human placental cytosolic phospholipase A₂ (cPLA₂), which is also a member of the 14-3-3 protein family. Western analysis demonstrated that a 31 kDa recombinant cPLA₂ expressed in monkey COS cells was recognized by the AE6F4 monoclonal antibody. Binding of the monoclonal antibody to the recombinant cPLA₂ was abolished when treated with sodium periodate, suggesting that not only are carbohydrate chains associated with the cPLA₂, but they also play a crucial role in antigen recognition by the monoclonal antibody.