Glucose-induced translational control of proinsulin biosynthesis is proportional to preproinsulin mRNA levels in islet beta-cells but not regulated via a positive feedback of secreted insulin

Proinsulin biosynthesis is regulated in response to nutrients, most notably glucose. In the short term (/=10-fold). Importantly, neither exogenously added nor secreted insulin were found to play any role in regulating insulin secretion, proinsulin translation, preproinsulin mRNA levels, or total protein synthesis. The results presented here indicate that long term nutritional state sets the preproinsulin mRNA level in the beta-cell at which translation control regulates short term changes in rates of proinsulin biosynthesis in response to glucose, but this is not mediated by any autocrine effect of insulin.     

Intracellular transport and sorting of mutant human proinsulins that fail to form hexamers

Human proinsulin and insulin oligomerize to form dimers and hexamers. It has been suggested that the ability of prohormones to self associate and form aggregates may be responsible for the sorting process at the trans-Golgi. To examine whether insulin oligomerization is required for proper sorting into regulated storage granules, we have constructed point mutations in human insulin B chain that have been previously shown to prevent formation of insulin hexamers (Brange, J., U. Ribel, J. F. Hansen, G. Dodson, M. T. Hansen, S. Havelund, S. G. Melberg, F. Norris, K. Norris, L. Snel, A. R. Sorensen, and H. O. Voight. 1988. Nature [Lond.]. 333:679-682). One mutant (B10His----Asp) allows formation of dimers but not hexamers and the other (B9Ser----Asp) prevents formation of both dimers and hexamers. The mutants were transfected into the mouse pituitary AtT-20 cells, and their ability to be sorted into regulated secretory granules was compared to wild-type insulin. We found that while B10His----Asp is sorted somewhat less efficiently than wild-type insulin as reported previously (Carroll, R. J., R. E. Hammer, S. J. Chan, H. H. Swift, A. H. Rubenstein, and D. F. Steiner. 1988. Proc. Natl. Acad. Sci. USA. 85:8943-8947; Gross, D. J., P. A. Halban, C. R. Kahn, G. C. Weir, and L. Villa-Kumaroff. 1989. Proc. Natl. Acad. Sci. USA. 86:4107-4111). B9Ser----Asp is targeted to granules as efficiently as wild-type insulin. These results indicate that self association of proinsulin into hexamers is not required for its targeting to the regulated secretory pathway.     

Analyses of the stability and function of three surface mutants (R82C, K69H, and L32R) of the gene V protein from Ff phage by X-ray crystallography.

The high-resolution crystal structure of the gene V protein (GVP) from the Ff filamentous phages (M13, fl, fd) has been solved recently for the wild-type and two surface mutant (Y41F and Y41H) proteins, leading to a plausible model for the polymeric GVP-ssDNA complex (Guan Y, Zhang H, Wang AHJ, 1995, Protein Sci 4:187-197). The model of the complex shows extensive contacts between neighboring dimer GVPs involving electrostatic interactions between the K69 from one and the D79 and R82 from the next dimer. In addition, hydrophobic interactions between the amino acids L32 and L44 from one and G23 from the next dimer also contribute to the dimer-dimer interactions. Mutations at the L32, K69, and R82 amino acid sites generally destabilize the protein and many of these affect the function of the phage. We have studied the structural effects of three mutant proteins involving those sites, i.e., L32R, K69H, and R82C, by X-ray crystallographic analysis at 2.0 A resolution. In L32R GVP, the structural perturbation is localized, whereas in K69H and R82C GVPs, some long-range effects are also detected in addition to the local perturbation. We have interpreted the protein stability and the functional properties associated with those mutations in terms of the observed structural perturbations.     

S-Adenosylhomocysteine hydrolase (AdoHcyase) deficiency: enzymatic capabilities of human AdoHcyase are highly effected by changes to codon 89 and its surrounding residues

Recently, S-adenosylhomocysteine hydrolase deficiency was confirmed for the first time in an adult. Two missense mutations in codons 89 (A>V) and 143 (Y>C) in the AdoHcyase gene were identified [N.R.M. Buist, B. Glenn, O. Vugrek, C. Wagner, S. Stabler, R.H. Allen, I. Pogribny, A. Schulze, S.H. Zeisel, I. Bari?, S.H. Mudd, S-Adenosylhomocysteine hydrolase deficiency in a 26-year-old man, J. Inh. Metab. Dis. 29 (2006) 538-545]. Accordingly, we have proven the Y143C mutation to be highly inactivating [R. Beluzi?, M. Cuk, T. Pavkov, K. Fumi?, I. Bari?, S.H. Mudd, I. Jurak, O. Vugrek, A single mutation at tyrosine 143 of human S-adenosylhomocysteine hydrolase renders the enzyme thermosensitive and effects the oxidation state of bound co-factor NAD, Biochem. J. 400 (2006) 245-253]. Now we report that the A89V exchange leads to a 70% loss of enzymatic activity, respectively. Circular dichroism analysis of recombinant p.A89V protein shows a significantly reduced unfolding temperature by 5.5 degrees C compared to wild-type. Gel filtration of mutant protein is almost identical to wild-type indicating assembly of subunits into the tetrameric complex. However, electrophoretic mobility of p.A89V is notably faster as shown by native polyacrylamide gel electrophoresis implicating changes to the overall charge of the mutant complex. 'Bioinformatics' analysis indicates that Val(89) collides with Thr(84) causing sterical incompatibility. Performing site-directed mutagenesis changing Thr(84) to 'smaller' Ser(84) but preserving similar physico-chemical properties restores most of the catalytic capabilities of the mutant p.A89V enzyme. On the other hand, substitution of Thr(84) with Lys(84) or Gln(84), thereby introducing residues with higher volume in proximity to Ala(89) results in inactivation of wild-type protein. In view of our mutational analysis, we consider changes in charge and the sterical incompatibility in mutant p.A89V protein as main reason for enzyme malfunction with AdoHcyase deficiency as consequence.     

Adrenoleukodystrophy: subcellular localization and degradation of adrenoleukodystrophy protein (ALDP/ABCD1) with naturally occurring missense mutations

Mutation in the X-chromosomal adrenoleukodystrophy gene (ALD; ABCD1) leads to X-linked adrenoleukodystrophy (X-ALD), a severe neurodegenerative disorder. The encoded adrenoleukodystrophy protein (ALDP/ABCD1) is a half-size peroxisomal ATP-binding cassette protein of 745 amino acids in humans. In this study, we chose nine arbitrary mutant human ALDP forms (R104C, G116R, Y174C, S342P, Q544R, S606P, S606L, R617H, and H667D) with naturally occurring missense mutations and examined the intracellular behavior. When expressed in X-ALD fibroblasts lacking ALDP, the expression level of mutant His-ALDPs (S606L, R617H, and H667D) was lower than that of wild type and other mutant ALDPs. Furthermore, mutant ALDP-green fluorescence proteins (S606L and H667D) stably expressed in CHO cells were not detected due to rapid degradation. Interestingly, the wild type ALDP co-expressed in these cells also disappeared. In the case of X-ALD fibroblasts from an ALD patient (R617H), the mutant ALDP was not detected in the cells, but appeared upon incubation with a proteasome inhibitor. When CHO cells expressing mutant ALDP-green fluorescence protein (H667D) were cultured in the presence of a proteasome inhibitor, both the mutant and wild type ALDP reappeared. In addition, mutant His-ALDP (Y174C), which has a mutation between transmembrane domain 2 and 3, did not exhibit peroxisomal localization by immunofluorescense study. These results suggest that mutant ALDPs, which have a mutation in the COOH-terminal half of ALDP, including S606L, R617H, and H667D, were degraded by proteasomes after dimerization. Further, the region between transmembrane domain 2 and 3 is important for the targeting of ALDP to the peroxisome.     

Misfolded forms of glyceraldehyde-3-phosphate dehydrogenase interact with GroEL and inhibit chaperonin-assisted folding of the wild-type enzyme

We studied the interaction of chaperonin GroEL with different misfolded forms of tetrameric phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPDH): (1) GAPDH from rabbit muscles with all SH-groups modified by 5,5'-dithiobis(2-nitrobenzoate); (2) O-R-type dimers of mutant GAPDH from Bacillus stearothermophilus with amino acid substitutions Y283V, D282G, and Y283V/W84F, and (3) O-P-type dimers of mutant GAPDH from B. stearothermophilus with amino acid substitutions Y46G/S48G and Y46G/R52G. It was shown that chemically modified GAPDH and the O-R-type mutant dimers bound to GroEL with 1:1 stoichiometry and dissociation constants K(d) of 0.4 and 0.9 muM, respectively. A striking feature of the resulting complexes with GroEL was their stability in the presence of Mg-ATP. Chemically modified GAPDH and the O-R-type mutant dimers inhibited GroEL-assisted refolding of urea-denatured wild-type GAPDH from B. stearothermophilus but did not affect its spontaneous reactivation. In contrast to the O-R-dimers, the O-P-type mutant dimers neither bound nor affected GroEL-assisted refolding of the wild-type GAPDH. Thus, we suggest that interaction of GroEL with certain types of misfolded proteins can result in the formation of stable complexes and the impairment of chaperonin activity.     

Mutant proinsulin that cannot be converted is secreted efficiently from primary rat beta-cells via the regulated pathway

Prohormones are directed from the trans-Golgi network to secretory granules of the regulated secretory pathway. It has further been proposed that prohormone conversion by endoproteolysis may be necessary for subsequent retention of peptides in granules and to prevent their release by the so-called "constitutive-like" pathway. To address this directly, mutant human proinsulin (Arg/Gly(32):Lys/Thr(64)), which cannot be cleaved by conversion endoproteases, was expressed in primary rat islet cells by recombinant adenovirus. The handling of the mutant proinsulin was compared with that of wild-type human proinsulin. Infected islet cells were pulse labeled and both basal and stimulated secretion of radiolabeled products followed during a chase. Labeled products were quantified by high-performance liquid chromatography. As expected, the mutant proinsulin was not converted at any time. Basal (constitutive and constitutive-like) secretion was higher for the mutant proinsulin than for wild-type proinsulin/insulin, but amounted to <1% even during a prolonged (6-h) period of basal chase. There was no difference in stimulated (regulated) secretion of mutant and wild-type proinsulin/insulin at any time. Thus, in primary islet cells, unprocessed (mutant) proinsulin is sorted to the regulated pathway and then retained in secretory granules as efficiently as fully processed insulin.     

Novel adiponectin variants identified in type 2 diabetic patients reveal multimerization and secretion defects

ADIPOQ, encoding adiponectin, is a candidate gene for type 2 diabetes (T2D) identified by genome-wide linkage analyses with supporting evidence showing the protein function in sensitizing insulin actions. In an endeavor to characterize candidate genes causing T2D in Thai patients, we identified 10 novel ADIPOQ variations, several of which were non-synonymous variations observed only in the patients. To examine the impact of these non-synonymous variations on adiponectin structure and biochemical characteristics, we conducted a structural analysis of the wild-type and variant proteins by in silico modeling and further characterized biochemical properties of the variants with predicted structural abnormalities from the modeling by molecular and biochemical studies. The recombinant plasmids containing wild-type and variant ADIPOQ cDNAs derived from the variations identified by our study (R55H, R112H, and R131H) and previous work (G90S and R112C) were constructed and transiently expressed and co-expressed in cultured HEK293T cells to investigate their oligomerization, interaction, and secretion. We found that the novel R55H variant impaired protein multimerization but it did not exert the effect over the co-expressed wild-type protein while novel R131H variant impaired protein secretion and also affected the co-expressed wild-type protein in a dominant negative fashion. The R131H variant could traffic from the endoplasmic reticulum to the Golgi, trans-Golgi network, and early endosome but could not be secreted. The R131H variant was likely to be degraded through the lysosomal system and inhibition of its degradation rescued the variant protein from secretion defect. We have shown the possibility of using in silico modeling for predicting the effect of amino acid substitution on adiponectin oligomerization. This is also the first report that demonstrates a dominant negative effect of the R131H variant on protein secretion and the possibility of using protein degradation inhibitors as therapeutic agents in the patients carrying adiponectin variants with secretion defect.     

Effects of calnexin deletion in Saccharomyces cerevisiae on the secretion of glycosylated lysozymes.

Disruption of the calnexin gene in Saccharomyces cerevisiae did not lead to gross effects on the levels of cell growth and secretion of wild-type hen egg white lysozymes (HEWL). To investigate the function of calnexin in relation to the secretion of glycoproteins, we expressed both stable and unstable mutant glycosylated lysozymes in calnexin-disrupted S. cerevisiae. The secreted amounts of stable mutant glycosylated lysozymes (G49N and S91T/G49N) were almost the same in both wild-type and calnexin-disrupted S. cerevisiae. In contrast, the secretion of unstable mutant glycosylated lysozymes (K13D/G49N, C76A/G49N, and D66H/G49N) greatly increased in calnexin-disrupted S. cerevisiae, although their secretion was very low in the wild-type strain. This indicates that calnexin may act in the quality control of glycoproteins. We further investigated the expression level of the mRNA of the molecular chaperones BiP and PDI, which play a major role in the protein folding process in the ER, when glycosylated lysozymes were expressed in wild-type and calnexin-disrupted S. cerevisiae. The mRNA concentrations of BiP and PDI were evidently increased when the glycosylated lysozymes were expressed in calnexin-disrupted S. cerevisiae. This observation indicates that BiP and PDI may be induced by the accumulation of unfolded glycosylated lysozymes due to the deletion of calnexin.     

In vivo glycosylation suppresses the aggregation of amyloidogenic hen egg white lysozymes expressed in yeast

The mutant hen egg white lysozymes Ile55Thr and Asp66His, corresponding to human amyloidogenic mutant lysozymes Ile56Thr and Asp67His, respectively, were secreted in Saccharomyces cerevisiae. The amyloidogenic mutants (I55T and D66H) of hen egg white lysozymes were remarkably less soluble than that of the wild-type protein. To enhance the secretion of these mutants, we constructed the glycosylated amyloidogenic lysozymes (I55T/G49N and D66H/G49N) having the N-glycosylation signal sequence (Asn-X-Ser) by the substitution of glycine with asparagine at position 49. The secretion of these glycosylated mutant proteins is greatly increased in S. cerevisiae, compared with that of non-glycosylated type. Both the glycosylated mutants retained about 40% enzymatic activity when incubated at pH 7.4 for 1 h at the physiological temperature of 37 degrees C whereas the non-glycosylated proteins eventually lost all activity under these conditions. These results suggest that the glycosylated chains could mask the beta-strand of amyloidogenic lysozymes from the intermolecular cross-beta-sheet association, thus improving the solubility of amyloidogenic lysozymes.     

P but not R-axis interface is involved in cooperative binding of NAD on tetrameric phosphorylating glyceraldehyde-3-phosphate dehydrogenase from Bacillus stearothermophilus

Homotetrameric phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Bacillus stearothermophilus can be described as a dimer of dimers with three non-equivalent P, R, and Q interfaces. In our previous study, negative cooperativity in NAD binding to wild-type GAPDH was interpreted according to the induced-fit model in terms of two independent dimers with two interacting binding sites in each dimer. Two dimeric mutant GAPDHs, i.e. Y46G/S48G and D186G/E276G, were shown to exhibit positive cooperativity in NAD binding. Based on the molecular modeling of the substitutions and the fact that the most extensive inter-subunit interactions are formed across the P-axis interface of the tetramer, it was postulated that both dimeric mutant GAPDHs were of O-P type. Therefore, the P-axis interface was assumed to play a major role in causing cooperativity in NAD binding.Here, two other mutant GAPDHs, Y46G/R52G and D282G, have been studied. Using small angle X-ray scattering, the dimeric form of the D282G mutant GAPDH is shown to be of O-R type whereas both dimeric mutant GAPDHs Y46G/R52G and Y46G/S48G are of O-P type. Similarly to dimeric Y46G/S48G mutant GAPDH, the dimeric Y46G/R52G mutant GAPDH exhibits positive cooperativity in NAD binding. On the other hand, no significant cooperativity in NAD binding to the dimeric form of the D282G mutant GAPDH is observed, whereas its tetrameric counterpart exhibits negative cooperativity, similarly to the wild-type enzyme. Altogether, the results support the view that the P-axis interface is essential in causing cooperativity in NAD binding by transmitting the structural information induced upon cofactor binding from one subunit to the other one within O-P/Q-R dimers in contrast to the R-axis interface, which does not transmit structural information within O-R/Q-P dimers. The absence of activity of O-P and O-R dimer GAPDHs is the consequence of a pertubation of the conformation of the active site, at least of the nicotinamide subsite, as evidenced by the absence of an ion pair between catalytic residues C149 and H176 and the greater accessibility of C149 to a thiol kinetic probe.     

Fluorescence studies of the binding of bacteriophage M13 gene V mutant proteins to polynucleotides.

This investigation describes how the binding characteristics of the single-stranded DNA-binding protein encoded by gene V of bacteriophage M13, are affected by single-site amino acid substitutions. The series of mutant proteins tested includes mutations in the purported monomer-monomer interaction region as well as mutations in the DNA-binding domain at positions which are thought to be functionally involved in monomer-monomer interaction or single-stranded DNA binding. The characteristics of the binding of the mutant proteins to the homopolynucleotides poly(dA), poly(dU) and poly(dT), were studied by means of fluorescence-titration experiments. The binding stoichiometry and fluorescence quenching of the mutant proteins are equal to, or lower than, the wild-type gene V protein values. In addition, all proteins measured bind a more-or-less co-operative manner to single-stranded DNA. The binding affinities for poly(dA) decrease in the following order: Y61H greater than wild-type greater than F68L and R16H greater than Y41F and Y41H greater than F73L greater than R21C greater than Y34H greater than G18D/Y56H. Possible explanations for the observed differences are discussed. The conservation of binding affinity, also for mutations in the single-stranded DNA-binding domain, suggests that the binding to homopolynucleotides is largely non-specific.     

Different effects of calnexin deletion in Saccharomyces cerevisiae on the secretion of two glycosylated amyloidogenic lysozymes

Both glycosylated amyloidogenic lysozymes I55T/G49N and D66H/G49N were expressed in wild-type and calnexin-disrupted Saccharomyces cerevisiae. The secretion amounts of mutant I55T/G49N were almost similar in both wild-type and calnexin-disrupted S. cerevisiae. In contrast, the secretion of mutant D66H/G49N greatly increased in calnexin-disrupted S. cerevisiae, while the secretion was very low in the wild-type strain. In parallel, the induction level of the molecular chaperones BiP and PDI located in the endoplasmic reticulum (ER) was investigated when these glycosylated amyloidogenic lysozymes were expressed in wild-type and calnexin-disrupted S. cerevisiae. The mRNA concentrations of BiP and PDI were evidently increased when mutant lysozyme D66H/G49N was expressed in calnexin-disrupted S. cerevisiae, while they were not so increased when I55T/G49N mutant was expressed. This observation indicates that the conformation of mutant lysozyme D66H/G49N was less stable in the ER, thus leading to the higher-level expression of ER molecular chaperones via the unfolded protein response pathway. This suggests that glycosylated amyloidogenic lysozyme I55T/G49N may have a relatively stable conformation in the ER, thus releasing it from the quality control of calnexin compared with mutant lysozyme D66H/G49N.     

Iterative Saturation Mutagenesis of ?6 Subsite Residues in Cyclodextrin Glycosyltransferase from Paenibacillus macerans To Improve Maltodextrin Specificity for 2-O-d-Glucopyranosyl-l-Ascorbic Acid Synthesis

2-O-d-Glucopyranosyl-l-ascorbic acid (AA-2G), a stable l-ascorbic acid derivative, is usually synthesized by cyclodextrin glycosyltransferase (CGTase), which contains nine substrate-binding subsites (from +2 to −7). In this study, iterative saturation mutagenesis (ISM) was performed on the −6 subsite residues (Y167, G179, G180, and N193) in the CGTase from Paenibacillus macerans to improve its specificity for maltodextrin, which is a cheap and easily soluble glycosyl donor for AA-2G synthesis. Site saturation mutagenesis of four sites—Y167, G179, G180, and N193—was first performed and revealed that four mutants—Y167S, G179R, N193R, and G180R—produced AA-2G yields higher than those of other mutant and wild-type CGTases. ISM was then conducted with the best positive mutant as a template. Under optimal conditions, mutant Y167S/G179K/N193R/G180R produced the highest AA-2G titer of 2.12 g/liter, which was 84% higher than that (1.15 g/liter) produced by the wild-type CGTase. Kinetics analysis of AA-2G synthesis using mutant CGTases confirmed the enhanced maltodextrin specificity and showed that compared to the wild-type CGTase, the mutants had no cyclization activity but high hydrolysis and disproportionation activities. A possible mechanism for the enhanced substrate specificity was also analyzed through structure modeling of the mutant and wild-type CGTases. These results indicated that the −6 subsite played crucial roles in the substrate binding and catalytic reactions of CGTase and that the obtained CGTase mutants, especially Y167S/G179K/N193R/G180R, are promising starting points for further development through protein engineering.     

Proteasomal Degradation of Proinsulin Requires Derlin-2, HRD1 and p97

Patients with type 1 diabetes (T1D) suffer from beta-cell destruction by CD8⁺ T-cells that have preproinsulin as an important target autoantigen. It is of great importance to understand the molecular mechanism underlying the processing of preproinsulin into these CD8⁺ T-cell epitopes. We therefore studied a pathway that may contribute to the production of these antigenic peptides: degradation of proinsulin via ER associated protein degradation (ERAD). Analysis of the MHC class I peptide ligandome confirmed the presentation of the most relevant MHC class I-restricted diabetogenic epitopes in our cells: the signal peptide-derived sequence A15-A25 and the insulin B-chain epitopes H29-A38 and H34-V42. We demonstrate that specific silencing of Derlin-2, p97 and HRD1 by shRNAs increases steady state levels of proinsulin. This indicates that these ERAD constituents are critically involved in proinsulin degradation and may therefore also play a role in subsequent antigen generation. These ERAD proteins therefore represent interesting targets for novel therapies aiming at the reduction and possibly also prevention of beta-cell directed auto-immune reactions in T1D.     

Reactive amino acid residues involved in glutamate-binding of human glutamate dehydrogenase isozymes

In the present study, the cassette mutagenesis at several putative positions (K94, G96, K118, K130, or D172) was performed to examine the residues involved in the glutamate-binding of the human glutamate dehydrogenase isozymes (hGDH1 and hGDH2). None of the mutations tested affected the expression or stability of the proteins. There was dramatic reduction in the catalytic efficiency in mutant proteins at K94, G96, K118, or K130 site, but not at D172 site. The K(M) values for glutamate were 4-10-fold greater for the mutants at K94, G96, or K118 site than for the wild-type hGDH1 and hGDH2, whereas no differences in the K(M) values for NAD(+) were detected between the mutant and wild-type enzymes. For K130Y mutant, the K(M) value for glutamate increased 1.6-fold, whereas the catalytic efficiency (k(cat)/K(M)) showed only 2-3% of the wild-type. Therefore, the decreased catalytic efficiency of the K130 mutant mainly results from the reduced k(cat) value, suggesting a possibility that the K130Y residue may be involved in the catalysis rather than in the glutamate-binding. The D172Y mutant did not show any changes in k(cat) value and K(M) values for glutamate and NAD(+), indicating that D172Y is not directly involved in catalysis and substrates binding of the hGDH isozymes. For sensitivity to ADP activation, only the D172Y mutant showed a reduced sensitivity to ADP activation. The reduction of ADP activation in D172Y mutant was more profoundly observed in hGDH2 than in hGDH1. There were no differences in their sensitivities to GTP inhibition between the wild-type and mutant GDHs at all positions tested. Our results suggest that K94, G96, and K118 residues play an important role, although at different degrees, in the binding of glutamate to hGDH isozymes.     

Cdk5 phosphorylates PLD2 to mediate EGF-dependent insulin secretion

Insulin secretion from pancreatic beta-cells is an important process that affects the regulation of glucose level in the blood. In our previous study, we suggested that epidermal growth factor (EGF) stimulates insulin secretion by activating phospholipase D2 (PLD2) [H.Y. Lee, K. Yea, J. Kim, B.D. Lee, Y.C. Chae, H.S. Kim, D.W. Lee, S.H. Kim, J.H. Cho, C.J. Jin, D.S. Koh, K.S. Park, P.G. Suh, S.H. Ryu, 2007. Epidermal Growth Factor Increases Insulin Secretion and Lowers Blood Glucose in Diabetic Mice. J. Cell. Mol. Med. 5:5]. However, the specific mechanism by which PLD2 activation leads to insulin secretion was not determined. In this study, we suggest that the phosphorylation and activation of PLD2 by cyclin-dependent kinase 5 (Cdk5) is critical for EGF-dependent insulin secretion. We found that a Cdk5 inhibitor, roscovitine, and dominant-negative Cdk5 inhibited EGF-dependent PLD2 activation and insulin secretion. EGF stimulation activated Cdk5 activity in rat insulinoma RINm5F cells, and PLD2 phosphorylation by Cdk5 was observed in vitro and in cells. The kinetics of PLD2 phosphorylation correlates with the interaction between PLD2 and Cdk5 and its effect on EGF signaling. We determined that the phosphorylation site of PLD2 was located at Ser(134). PLD2-S134A did not show EGF-dependent phosphorylation and activation by Cdk5. Furthermore, this mutant was unable to mediate EGF-dependent insulin secretion in pancreatic beta cell lines, suggesting that the phosphorylation of PLD2 at Ser(134) by Cdk5 is critical for this process. The study results suggest that PLD2 is a new substrate of Cdk5 and that the phosphorylation of PLD2 by Cdk5 is involved in EGF-dependent insulin secretion.     

Impairment of hepatitis B virus virion secretion by single-amino-acid substitutions in the small envelope protein and rescue by a novel glycosylation site

Mutations in the S region of the hepatitis B virus (HBV) envelope gene are associated with immune escape, occult infection, and resistance to therapy. We previously identified naturally occurring mutations in the S gene that alter HBV virion secretion. Here we used transcomplementation assay to confirm that the I110M, G119E, and R169P mutations in the S domain of viral envelope proteins impair virion secretion and that an M133T mutation rescues virion secretion of the I110M and G119E mutants. The G119E mutation impaired detection of secreted hepatitis B surface antigen (HBsAg), suggesting immune escape. The R169P mutant protein is defective in HBsAg secretion as well and has a dominant negative effect when it is coexpressed with wild-type envelope proteins. Although the S domain is present in all three envelope proteins, the I110M, G119E, and R169P mutations impair virion secretion through the small envelope protein. Conversely, coexpression of just the small envelope protein of the M133T mutant could rescue virion secretion. The M133T mutation could also overcome the secretion defect caused by the G145R immune-escape mutation or mutation at N146, the site of N-linked glycosylation. In fact, the M133T mutation creates a novel N-linked glycosylation site ((131)NST(133)). Destroying this site by N131Q/T mutation or preventing glycosylation by tunicamycin treatment of transfected cells abrogated the effect of the M133T mutation. Our findings demonstrate that N-linked glycosylation of HBV envelope proteins is critical for virion secretion and that the secretion defect caused by mutations in the S protein can be rescued by an extra glycosylation site.     

Functional analysis of the Notch ligand Jagged1 missense mutant proteins underlying Alagille syndrome

Heterozygous mutations in the JAG1 gene, encoding Notch ligand Jagged1, cause Alagille syndrome (ALGS). As most of the mutations are nonsense or frameshift mutations producing inactive truncated proteins, haplo-insufficiency is considered the major pathogenic mechanism of ALGS. However, the molecular mechanisms by which the missense mutations cause ALGS remain unclear. Here we analyzed the functional properties of four ALGS missense mutant proteins, P163L, R184H, G386R and C714Y, using transfected mammalian cells. P163L and R184H showed Notch-binding activities similar to that of the wild-type when assessed by immunoprecipitation. However, their trans-activation and cis-inhibition activities were almost completely impaired. These mutant proteins localized mainly to the endoplasmic reticulum (ER), suggesting that the mutations induced improper protein folding. Furthermore, the mutant proteins bound more strongly to the ER chaperone proteins calnexin and calreticulin than the wild-type did. C714Y also localized to the ER, but possessed significant trans-activation activity and lacked enhanced binding to the chaperones, indicating a less severe phenotype. The properties of G386R were the same as those of the wild-type. Dominant-negative effects were not detected for any mutant protein. These results indicate that accumulation in the ER and binding to the chaperones correlate with the impaired signal-transduction activities of the missense mutant proteins, which may contribute to the pathogenic mechanism of ALGS. Our findings, which suggest the requirement for cell-surface localization of Jagged1 for cis-inhibition activities, also provide important information for understanding the molecular basis of Notch-signaling pathways.     

Proinsulin lacking the A7-B7 disulfide bond, Ins2Akita, tends to aggregate due to the exposed hydrophobic surface

A single mutation (C96Y) in the Ins2 gene, which disrupts the A7-B7 disulfide bond, causes the diabetic phenotype in Akita mice. We biochemically analyzed the conformation of wild-type and Akita mutant recombinant proinsulins. Gel filtration chromatography and dynamic light scattering revealed that the apparent size of the mutant proinsulin molecules was significantly larger than that of wild-type proinsulin, even in the absence of intermolecular disulfide bonds. Titration with a hydrophobic probe, 1-anilinonaphthalene-8-sulfonate, demonstrated that the mutant proinsulin was more hydrophobic than the wild type. In addition, circular dichroism studies revealed that the conformation of the mutant proinsulin was less stable than the wild type, which is consistent with the observation that hydrophobic residues are exposed on the surface of the proinsulin molecules. Studies with antiserum against the C-peptide of proinsulin indicated that the mutant proinsulin had an immunoreactivity that was at least one-tenth weaker than wild-type proinsulin, suggesting that the C-peptide of mutant proinsulin is buried inside the aggregate of the proinsulin molecule. These findings indicate that increased hydrophobicity of mutant proinsulin facilitates aggregate formation, providing a clue to the dominant negative effect in the Akita mouse.