The endoplasmic reticulum HSP40 co‐chaperone ERdj3/DNAJB11 assembles and functions as a tetramer

ERdj3/DNAJB11 is an endoplasmic reticulum (ER)‐targeted HSP40 co‐chaperone that performs multifaceted functions involved in coordinating ER and extracellular proteostasis. Here, we show that ERdj3 assembles into a native tetramer that is distinct from the dimeric structure observed for other HSP40 co‐chaperones. An electron microscopy structural model of full‐length ERdj3 shows that these tetramers are arranged as a dimer of dimers formed by distinct inter‐subunit interactions involving ERdj3 domain II and domain III. Targeted deletion of residues 175‐190 within domain II renders ERdj3 a stable dimer that is folded and efficiently secreted from mammalian cells. This dimeric ERdj3 shows impaired substrate binding both in the ER and extracellular environments and reduced interactions with the ER HSP70 chaperone BiP. Furthermore, we show that overexpression of dimeric ERdj3 exacerbates ER stress‐dependent reductions in the secretion of a destabilized, aggregation‐prone protein and increases its accumulation as soluble oligomers in extracellular environments. These results reveal ERdj3 tetramerization as an important structural framework for ERdj3 functions involved in coordinating ER and extracellular proteostasis in the presence and absence of ER stress.     

β‐Hairpin stabilization in a 28‐residue peptide derived from the β‐subunit sequence of human chorionic gonadotropin hormone

The β‐subunit of the human chorionic gonadotropin (hCG) hormone, which is believed to be related to certain types of cancer, contains three hairpin‐like fragments. To investigate the role of β‐hairpin formation in the early stages of the hCGβ folding, a 28‐residue peptide with the sequence RDVRFESIRLPGSPRGVNPVVSYAVALS, corresponding to the H3‐β hairpin fragment (residues 60–87) of the hCGβ subunit, was studied under various conditions using three optical spectroscopic methods: Fourier transform ir spectroscopy, electronic CD, and vibrational CD. Environmental conditions are critical factors for formation of secondary structure in this peptide. TFE : H₂O mixed solvents induced helical formation. Formation of β‐structure in this peptide, which may be related to the native β‐hairpin formation in the intact hormone, was found to be induced only under conditions such as high concentration, high temperature, and the presence of nonmicellar sodium dodecyl sulfate concentrations. These findings support a protein folding mechanism for the hCGβ subunit in which an initial hydrophobic collapse, which increases intermolecular interactions in hCGβ, is needed to induce the H3‐β hairpin formation. © 1999 John Wiley & Sons, Inc. Biopoly 50: 413–423, 1999     

Protein kinase‐A affects sorting and conformation of the sodium‐dependent glucose co‐transporter SGLT1

In Chinese hamster ovary cells expressing rabbit sodium‐dependent glucose transporter (rbSGLT1) protein kinase A (PKA) activators (forskolin and 8‐Br‐cAMP) stimulated α‐methyl D ‐glucopyranoside uptake. Kinetic analysis revealed an increase in both V_max and affinity of the transport. Immunohistochemistry and biotinylation experiments showed that this stimulation was accompanied by an increased amount of SGLT1 localized into the plasma membrane, which explains the higher V_max of the transport. Cytochalasin D only partly attenuated the effect of forskolin as did deletion of the PKA phosphorylation site of SGLT1 in transient transfection studies. Experiments using an anti‐phosphopeptide antibody revealed that forskolin also increased the extent of phosphorylation of SGLT1 in the membrane fraction. These results suggested that regulation of SGLT1 mediated glucose transport involves an additional direct effect on SGLT1 by phosphorylation. To evaluate this assumption further, phosphorylation studies of recombinant human SGLT1 (hSGLT1) in vitro were performed. In the presence of the catalytic subunit PKA and [³²P] ATP 1.05 mol of phosphate were incorporated/mol of hSGLT1. Additionally, phosphorylated hSGLT1 demonstrated a reduction in tryptophan fluorescence intensity and a higher quenching by the hydrophilic Trp quencher acrylamide, particularly in the presence of D ‐glucose. These results indicate that PKA‐mediated phosphorylation of SGLT1 changes the conformation of the empty carrier and the glucose carrier complex, probably causing the increase in transport affinity. Thus, PKA‐mediated phosphorylation of the transporter represents a further mechanism in the regulation of SGLT1‐mediated glucose transport in epithelial cells, in addition to a change in surface membrane expression. J. Cell. Biochem. 106: 444–452, 2009. © 2008 Wiley‐Liss, Inc.     

Structural insight into dynamic bypass of the major cisplatin‐DNA adduct by Y‐family polymerase Dpo4

Y‐family DNA polymerases bypass Pt‐GG, the cisplatin‐DNA double‐base lesion, contributing to the cisplatin resistance in tumour cells. To reveal the mechanism, we determined three structures of the Y‐family DNA polymerase, Dpo4, in complex with Pt‐GG DNA. The crystallographic snapshots show three stages of lesion bypass: the nucleotide insertions opposite the 3′G (first insertion) and 5′G (second insertion) of Pt‐GG, and the primer extension beyond the lesion site. We observed a dynamic process, in which the lesion was converted from an open and angular conformation at the first insertion to a depressed and nearly parallel conformation at the subsequent reaction stages to fit into the active site of Dpo4. The DNA translocation‐coupled conformational change may account for additional inhibition on the second insertion reaction. The structures illustrate that Pt‐GG disturbs the replicating base pair in the active site, which reduces the catalytic efficiency and fidelity. The in vivo relevance of Dpo4‐mediated Pt‐GG bypass was addressed by a dpo‐4 knockout strain of Sulfolobus solfataricus, which exhibits enhanced sensitivity to cisplatin and proteomic alterations consistent with genomic stress.     

Cloning, expression and nuclear localization of human NPM3, a member of the nucleophosmin/nucleoplasmin family of nuclear chaperones

Background

Studies suggest that the related proteins nucleoplasmin and nucleophosmin (also called B23, NO38 or numatrin) are nuclear chaperones that mediate the assembly of nucleosomes and ribosomes, respectively, and that these activities are accomplished through the binding of basic proteins via their acidic domains. Recently discovered and less well characterized members of this family of acidic phosphoproteins include mouse nucleophosmin/nucleoplasmin 3 (Npm3) and Xenopus NO29. Here we report the cloning and initial characterization of the human ortholog of Npm3.

Results

Human genomic and cDNA clones of NPM3 were isolated and sequenced. NPM3 lies 5.5 kb upstream of FGF8 and thus maps to chromosome 10q24-26. In addition to amino acid similarities, NPM3 shares many physical characteristics with the nucleophosmin/nucleoplasmin family, including an acidic domain, multiple potential phosphorylation sites and a putative nuclear localization signal. Comparative analyses of 14 members of this family from various metazoans suggest that Xenopus NO29 is a candidate ortholog of human and mouse NPM3, and they further group both proteins closer with the nucleoplasmins than with the nucleophosmins. Northern blot analysis revealed that NPM3 was strongly expressed in all 16 human tissues examined, with especially robust expression in pancreas and testis; lung displayed the lowest level of expression. An analysis of subcellular fractions of NIH3T3 cells expressing epitope-tagged NPM3 revealed that NPM3 protein was localized solely in the nucleus.

Conclusions

Human NPM3 is an abundant and widely expressed protein with primarily nuclear localization. These biological activities, together with its physical relationship to the chaparones nucleoplasmin and nucleophosmin, are consistent with the proposed function of NPM3 as a molecular chaperone functioning in the nucleus.     

Calcyon, a mammalian specific NEEP21 family member, interacts with adaptor protein complex 3 (AP-3) and regulates targeting of AP-3 cargoes

Calcyon is a neural enriched, single transmembrane protein that interacts with clathrin light chain and stimulates clathrin assembly and clathrin‐mediated endocytosis. A similar property is shared by the heterotetrameric adaptor protein (AP) complexes AP‐1, AP‐2, and AP‐3 which recruit cargoes for insertion into clathrin coated transport vesicles. Here we report that AP medium (μ) subunits interact with a YXXØ‐type tyrosine motif located at residues 133–136 in the cytoplasmic domain of calcyon. Site specific mutagenesis of the critical tyrosine and bulky hydrophobic residues tyrosine 133 and methionine 136 preferentially abrogated binding of the ubiquitous and neuronal isoforms of μ3, and also impacted μ1 and μ2 binding to a lesser degree. The relevance of these interactions was explored in vivo using mice harboring null alleles of calcyon. As seen in the mutagenesis studies, calcyon deletion in mice preferentially altered the subcellular distribution of AP‐3 suggesting that calcyon could regulate membrane‐bound pools of AP‐3 and AP‐3 function. To test this hypothesis, we focused on the hilar region of hippocampus, where levels of calcyon, AP‐3, and AP‐3 cargoes are abundant. We analyzed brain cryosections from control and calcyon null mice for zinc transporter 3 (ZnT3), and phosphatidylinositol‐4‐kinase type II alpha (PI4KIIα), two well‐defined AP‐3 cargoes. Confocal microscopy indicated that ZnT3 and PI4KIIα are significantly reduced in the hippocampal mossy fibers of calcyon knock‐out brain, a phenotype previously described in AP‐3 deficiencies. Altogether, our data suggest that calcyon directly interacts with μ3A and μ3B, and regulates the subcellular distribution of AP‐3 and the targeting of AP‐3 cargoes.     

Cerato-platanin, the first member of a new fungal protein family

The ascomcete Ceratocystis fimbriata, the causal agent of “canker stain disease,” secretes a protein of 12.4 kDa that elicits phytoalexin synthesis and plant cell death. This protein, named cerato-platanin (CP), is also located in the cell walls of ascospores, hyphae, and conidia; it contains four cysteines (S-S bridged) and is moderately hydrophobic. The cp gene consists of a single exon and has 42 bp codifying for a signal peptide of 14 residues. The recombinant protein was obtained by cloning the cp gene of the mature protein in Escherichia coli (BL21), and a refolding step was needed to achieve the native active form. In the European Molecular Biology data bank, CP is reported as the first member of the CP family; this is the first example of an set of secreted fungal proteins whose primary structure is very similar. Nonetheless, the data also revealed some structural and functional features that make CP simlar to proteins of the hydrophobin family.     

Enhancing effects of the chemical adjuvant levamisole on the DNA vaccine pVIR‐P12A‐IL18‐3C

DNA‐based vaccination is an attractive alternative for overcoming the disadvantages of inactivated virus vaccines; however, DNA vaccines alone often generate only weak immune responses. In this study, the efficacy of LMS as a chemical adjuvant on a DNA vaccine (pVIR‐P12A‐IL18‐3C) encoding the P1‐2A and 3C genes of the FMDV and swine IL‐18, which provides protection against FMDV challenge, was tested. All test pigs were administered booster vaccinations 28 days after the initial inoculation, and were challenged with 1000 ID₅₀ FMDV O/NY00 20 days after the booster vaccination. Positive and negative control groups were inoculated with inactivated virus vaccine and PBS respectively. The DNA vaccine plus LMS induced greater humoral and cell‐mediated responses than the DNA vaccine alone, as evidenced by higher concentrations of neutralizing and specific anti‐FMDV antibodies, and by higher concentrations of T‐lymphocyte proliferation and IFN‐γ production, respectively. FMDV challenge revealed that the DNA vaccine plus LMS provided higher protection than the DNA vaccine alone. This study demonstrates that LMS may be useful as an adjuvant for improving the protective efficiency of DNA vaccination against FMDV in pigs.     

Tumor suppressor Tsc1 is a new Hsp90 co‐chaperone that facilitates folding of kinase and non‐kinase clients

The tumor suppressors Tsc1 and Tsc2 form the tuberous sclerosis complex (TSC), a regulator of mTOR activity. Tsc1 stabilizes Tsc2; however, the precise mechanism involved remains elusive. The molecular chaperone heat‐shock protein 90 (Hsp90) is an essential component of the cellular homeostatic machinery in eukaryotes. Here, we show that Tsc1 is a new co‐chaperone for Hsp90 that inhibits its ATPase activity. The C‐terminal domain of Tsc1 (998–1,164 aa) forms a homodimer and binds to both protomers of the Hsp90 middle domain. This ensures inhibition of both subunits of the Hsp90 dimer and prevents the activating co‐chaperone Aha1 from binding the middle domain of Hsp90. Conversely, phosphorylation of Aha1‐Y223 increases its affinity for Hsp90 and displaces Tsc1, thereby providing a mechanism for equilibrium between binding of these two co‐chaperones to Hsp90. Our findings establish an active role for Tsc1 as a facilitator of Hsp90‐mediated folding of kinase and non‐kinase clients—including Tsc2—thereby preventing their ubiquitination and proteasomal degradation.     

Intrinsic secondary structure propensities of the amino acids,using statistical ϕ–ψ matrices: Comparison with experimental scales

Today there are several different experimental scales for the intrinsic α-helix as well as β-strand, propensities of the 20 amino acids obtained from the thermodynamic analysis of various model systems. These scales do not compare well with those extracted from statistical analysis of three-dimensional structure databases. Possible explanations for this could be the limited size of the databases used, the definitions of intrinsic propensities, or the theoretical approach. Here we report a statistical determination of α-helix and β-strand propensities derived from the analysis of a database of 279 three-dimensional structures. Contrary to what has been generally done, we have considered a particular residue as in α-helix or β-strand conformation by looking only at its dihedral angles (?–ψ matrices). Neither the identity nor the conformation of the surrounding residues in the amino acid sequence has been taken into consideration. Pseudoenergy empirical scales have been calculated from the statistical propensities. These scales agree very well with the experimental ones in relative and absolute terms. Moreover, its correlation with the average of the experimental scales for α-helix or β-strand is as good as the correlations of the individual experimental scales with the average. These results show that by using a large enough database and a proper definition for the secondary structure propensities, it is possible to obtain a scale as good as any of experimental origin. Interestingly the ?–ψ analysis of the Ramachandran plot suggests that the amino acids could have different β-strand propensities in different subregions of the β-strand area. © 1994 Wiley-Liss, Inc.     

Characterization of a whole‐cell catalyst co‐expressing glycerol dehydrogenase and glucose dehydrogenase and its application in the synthesis of L‐glyceraldehyde

A whole‐cell catalyst using Escherichia coli BL21(DE3) as a host, co‐expressing glycerol dehydrogenase (GlyDH) from Gluconobacter oxydans and glucose dehydrogenase (GDH) from Bacillus subtilis for cofactor regeneration, has been successfully constructed and used for the reduction of aliphatic aldehydes, such as hexanal or glyceraldehyde to the corresponding alcohols. This catalyst was characterized in terms of growth conditions, temperature and pH dependency, and regarding the influence of external cofactor and permeabilization. In the case of external cofactor addition we found a 4.6‐fold increase in reaction rate caused by the addition of 1 mM NADP⁺. Due to the fact that pH and temperature are also factors which may affect the reaction rate, their effect on the whole‐cell catalyst was studied as well. Comparative studies between the whole‐cell catalyst and the cell‐free system were investigated. Furthermore, the successful application of the whole‐cell catalyst in repetitive batch conversions could be demonstrated in the present study. Since the GlyDH was recently characterized and successfully applied in the kinetic resolution of racemic glyceraldehyde, we were now able to transfer and establish the process to a whole‐cell system, which facilitated the access to L ‐glyceraldehyde in high enantioselectivity at 54% conversion. All in all, the whole‐cell catalyst shows several advantages over the cell‐free system like a higher thermal, a similar operational stability and the ability to recycle the catalyst without any loss‐of‐activity. The results obtained making the described whole‐cell catalyst an improved catalyst for a more efficient production of enantiopure L ‐glyceraldehyde. Biotechnol. Bioeng. 2010;106: 541–552. © 2010 Wiley Periodicals, Inc.     

Robust Glyoxalase activity of Hsp31, a ThiJ/DJ-1/PfpI Family Member Protein,Is Critical for Oxidative Stress Resistance in Saccharomyces cerevisiae

Methylglyoxal (MG) is a reactive metabolic intermediate generated during various cellular biochemical reactions, including glycolysis. The accumulation of MG indiscriminately modifies proteins, including important cellular antioxidant machinery, leading to severe oxidative stress, which is implicated in multiple neurodegenerative disorders, aging, and cardiac disorders. Although cells possess efficient glyoxalase systems for detoxification, their functions are largely dependent on the glutathione cofactor, the availability of which is self-limiting under oxidative stress. Thus, higher organisms require alternate modes of reducing the MG-mediated toxicity and maintaining redox balance. In this report, we demonstrate that Hsp31 protein, a member of the ThiJ/DJ-1/PfpI family in Saccharomyces cerevisiae, plays an indispensable role in regulating redox homeostasis. Our results show that Hsp31 possesses robust glutathione-independent methylglyoxalase activity and suppresses MG-mediated toxicity and ROS levels as compared with another paralog, Hsp34. On the other hand, glyoxalase-defective mutants of Hsp31 were found highly compromised in regulating the ROS levels. Additionally, Hsp31 maintains cellular glutathione and NADPH levels, thus conferring protection against oxidative stress, and Hsp31 relocalizes to mitochondria to provide cytoprotection to the organelle under oxidative stress conditions. Importantly, human DJ-1, which is implicated in the familial form of Parkinson disease, complements the function of Hsp31 by suppressing methylglyoxal and oxidative stress, thus signifying the importance of these proteins in the maintenance of ROS homeostasis across phylogeny.     

Development‐dependent expression of calreticulin in the brain and other tissues of the Japanese monkey,Macaca fuscata

Background Calreticulin (Crt) is a molecular chaperone localized to endoplasmic reticulum lumen. The protein is involved in the correct folding of glycoproteins and its cellular level changes depending on various physiologic conditions. Methods To clarify the Crt level in various tissues of the Japanese monkey during postnatal development, an enzyme‐linked immunosorbent assay system that we have established was applied. Results and Conclusions Calreticulin is distributed ubiquitously in various tissues. Its level in the heart, lung, liver, spleen, and kidney was high in newborns and decreased in juveniles and adults. In various cerebral areas, Crt was present in the gray matter but scarcely found in the white matter. The Crt levels in the cerebral areas were low in newborns and increase in juveniles and adults. These distribution and developmental changes in Crt might be correlated with the quality control of glycoproteins that are synthesized in respective tissues.