A new highly efficient substrate-trapping mutant of protein tyrosine phosphatase 1B (PTP1B) reveals full autoactivation of the insulin receptor precursor.

PTP1B is a protein tyrosine-phosphatase located on the cytosolic side of the endoplasmic reticulum that plays an important role in the regulation of the insulin receptor (IR). Replacement of the conserved Asp-181 by alanine is known to convert PTP1B into a substrate-trapping protein that binds to but cannot dephosphorylate its substrates. In this work, we have studied the effect of an additional mutation (Y46F) on the substrate-trapping efficiency of PTP1B-D181A. We observed that this mutation converts PTP1B-D181A into a highly efficient substrate-trapping mutant, resulting in much higher recovery of tyrosine-phosphorylated proteins coimmunoprecipitated with PTP1B. Bioluminescence resonance energy transfer (BRET) experiments were also performed to compare the dynamics of interaction of the IR with these mutants. Basal BRET, which mainly reflects the interaction of PTP1B with the IR precursor during its biosynthesis in the endoplasmic reticulum, was markedly increased with the PTP1B-D181A-Y46F mutant. In contrast, insulin-induced BRET was markedly reduced with PTP1B-D181A-Y46F. I(125) insulin binding experiments indicated that PTP1B-D181-Y46F reduced the expression of IR at the plasma membrane. Reduced expression at the cell surface was associated with higher amounts of the uncleaved IR precursor in the cell. Moreover, we observed that substantial amounts of the uncleaved IR precursor reached the Tris-phosphorylated, fully activated form in an insulin independent fashion. These results support the notion that PTP1B plays a crucial role in the control of the activity of the IR precursor during its biosynthesis. In addition, this new substrate-trapping mutant may be a valuable tool for the identification of new PTP1B substrates.

Molecular pathogenesis of sialic acid storage diseases: insight gained from four missense mutations and a putative polymorphism of human sialin.

BACKGROUND INFORMATION: Free sialic acid storage diseases are caused by mutations of a lysosomal sialic acid transporter called sialin. We showed recently that the milder clinical form, Salla disease, and a related non-Finish case, are characterized by residual transport, whereas sialin mutants found in lethal infantile cases are inactive. In the present study, we have characterized the molecular effects of a putative polymorphism (M316I) and of four pathogenic mutations associated with either infantile (G127E and R57C) or Salla-like (G409E) phenotypes, or both (G328E). The transport activity of human sialin was analysed using a novel assay that was based on a construct without the functional lysosomal sorting motif, which is expressed at the plasma membrane. RESULTS: The lysosomal localization of human sialin was not (M316I and G328E) or only partially (R57C, G127E and G409E) affected by the missense mutations. In contrast, all pathogenic mutations abolished transport, whereas the putative M316I polymorphism induced an approx. 5-fold decrease of sialic acid transport. CONCLUSIONS: The molecular effects of the R57C and G127E mutations strengthen the conclusion that the infantile phenotype is caused by loss-of-function mutations. On the other hand, the milder severity of the heterozygous G409E patient may reflect an incomplete expression of the splicing mutation present on the second allele. In the case of the G328E mutation, found in the homozygous state in a clinically heterogeneous family, the apparent severity of the transport phenotype suggests that the genetic or environmental factors underlying this clinical heterogeneity might be protective.

The glycoprotein of a fish rhabdovirus profiles the virus-specific T-cell repertoire in rainbow trout.

T-cell responses to viruses are still poorly investigated in lower vertebrates. In rainbow trout, a specific clonal expansion of T cells in response to infection with viral haemorrhagic septicaemia virus (VHSV) was recently identified. Expanded T-cell clones expressed a unique 8 aa Vbeta4-Jbeta1 junction (SSGDSYSE) in different individuals, reminiscent of a typical public response. To get further insight into the nature of this response the modifications of the T-cell repertoire following immunization with plasmid expressing the VHSV external glycoprotein (G), which is the only protein involved in protective immunity, was analysed. After G-based DNA immunization, CDR3-length spectratypes were skewed for several Vbeta-Jbeta combinations, including Vbeta4-Jbeta1. In Vbeta4-Jbeta1, biases consisted of 6 and 8 aa junctions that were detected from day 52, and were still present 3 months after DNA immunization. Sequence analysis of the Vbeta4-Jbeta1 junctions showed that the 8 aa junction (SSGDSYSE) was clearly expanded, indicating that viral G protein was probably the target of the anti-VHSV public response. Additional 6 and 8 aa Vbeta4-Jbeta1 junctions were also expanded in G-DNA-vaccinated fish, showing that significant clonotypic diversity was selected in response to the plasmid-delivered G protein. This higher clonotypic diversity may be related to the demonstrated higher efficiency of G-based DNA vaccines over whole virus immunization. The use of infectious hematopietic necrosis virus (IHNV) recombinant viruses, expressing the VHSV G protein, further substantiated the VHSV G-protein specificity of the 8 aa Vbeta4-Jbeta1 response and designated the 6 aa Vbeta4-Jbeta1 response as potentially directed to a T-cell epitope common to VHSV and IHNV.

Dynamics of the interaction between the insulin receptor and protein tyrosine-phosphatase 1B in living cells.

The dynamics of the interaction of the insulin receptor with a substrate-trapping mutant of protein-tyrosine phosphatase 1B (PTP1B) were monitored in living human embryonic kidney cells using bioluminescence resonance energy transfer (BRET). Insulin dose-dependently stimulates this interaction, which could be followed in real time for more than 30 minutes. The effect of insulin on the BRET signal could be detected at early time-points (30 seconds), suggesting that in intact cells the tyrosine-kinase activity of the insulin receptor is tightly controlled by PTP1B. Interestingly, the basal (insulin-independent) interaction of the insulin receptor with PTP1B was much weaker with a soluble form of the tyrosine-phosphatase than with the endoplasmic reticulum (ER)-targeted form. Inhibition of insulin-receptor processing using tunicamycin suggests that the basal interaction occurs during insulin-receptor biosynthesis in the ER. Therefore, localization of PTP1B in this compartment might be important for the regulation of insulin receptors during their biosynthesis.

Primary structure and complementarity-determining region (CDR) 3 spectratyping of rainbow trout TCRbeta transcripts identify ten Vbeta families with Vbeta6 displaying unusual CDR2 and differently spliced forms.

VDJ rearrangement at the teleost TCRbeta locus leads to a highly diverse repertoire of junctions for each VbetaJbeta combination. From a rainbow trout 5' RACE library of TCRbeta transcripts, 47 clones encompassing a full Vbeta-Dbeta-Jbeta-Cbeta sequence were selected and analyzed. A similarity analysis of the sequences evidenced 10 Vbeta families, of which 6 were not previously described. Immunoscope and sequence analysis of the Vbeta-Dbeta-Jbeta junctions of the new families confirmed that they create a polyclonal and diverse repertoire. Multiple alignments showed that rainbow trout Vbetas possess most of the conserved residues typical of Vbeta segments. However, this study revealed a high complementarity-determining region 2 (CDR2) and CDR1 length diversity among rainbow trout Vbeta families, suggesting that the spatial orientation of the TCR could fluctuate in the TCR/peptide/MHC complex, depending on the Vbeta expressed. Among the new Vbeta families, Vbeta6 displayed the strongest deviance from typical hypervariable CDR1 and CDR2 loops, with an unusually short CDR2. Moreover, the Vbeta6 sequence is overall divergent from typical Vbeta sequence, raising the question of its functional relevance. Immunoscope experiments identified a Vbeta6-Jbeta3 junction, which was amplified during the response against viral hemorrhagic septicemia virus, a fish rhabdovirus. Vbeta6 seems therefore to be expressed functionally in a selected TCR. However, the shorter Vbeta6 transcripts produced through an alternative splicing lack the C', C", D, and E strands of the Vbeta domain and are probably nonfunctional.

Kinetic regulation of the mitochondrial glycerol-3-phosphate dehydrogenase by the external NADH dehydrogenase in Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, the two most important systems for conveying excess cytosolic NADH to the mitochondrial respiratory chain are external NADH dehydrogenase (Nde1p/Nde2p) and the glycerol-3-phosphate dehydrogenase shuttle. In the latter system, NADH is oxidized to NAD+ and dihydroxyacetone phosphate is reduced to glycerol 3-phosphate by the cytosolic Gpd1p; glycerol 3-phosphate gives two electrons to the respiratory chain via mitochondrial glycerol-3-phosphate dehydrogenase (Gut2p)-regenerating dihydroxyacetone phosphate. Both Nde1p/Nde2p and Gut2p are located in the inner mitochondrial membrane with catalytic sites facing the intermembranal space. In this study, we showed kinetic interactions between these two enzymes. First, deletion of either one of the external dehydrogenases caused an increase in the efficiency of the remaining enzyme. Second, the activation of NADH dehydrogenase inhibited the Gut2p in such a manner that, at a saturating concentration of NADH, glycerol 3-phosphate is not used as respiratory substrate. This effect was not a consequence of a direct action of NADH on Gut2p activity because both NADH dehydrogenase and its substrate were needed for Gut2p inhibition. This kinetic regulation of the activity of an enzyme as a function of the rate of another having a similar physiological function may be allowed by their association into the same supramolecular complex in the inner membrane. The physiological consequences of this regulation are discussed.