Diabetes reversal by inhibition of the low-molecular-weight tyrosine phosphatase.

Obesity-associated insulin resistance plays a central role in type 2 diabetes. As such, tyrosine phosphatases that dephosphorylate the insulin receptor (IR) are potential therapeutic targets. The low-molecular-weight protein tyrosine phosphatase (LMPTP) is a proposed IR phosphatase, yet its role in insulin signaling in vivo has not been defined. Here we show that global and liver-specific LMPTP deletion protects mice from high-fat diet-induced diabetes without affecting body weight. To examine the role of the catalytic activity of LMPTP, we developed a small-molecule inhibitor with a novel uncompetitive mechanism, a unique binding site at the opening of the catalytic pocket, and an exquisite selectivity over other phosphatases. This inhibitor is orally bioavailable, and it increases liver IR phosphorylation in vivo and reverses high-fat diet-induced diabetes. Our findings suggest that LMPTP is a key promoter of insulin resistance and that LMPTP inhibitors would be beneficial for treating type 2 diabetes

Molecular Signatures of Hemagglutinin Stem-Directed Heterosubtypic Human Neutralizing Antibodies against Influenza A Viruses

Recent studies have shown high usage of the IGHV1-69 germline immunoglobulin gene for influenza hemagglutinin stem-directed broadly-neutralizing antibodies (HV1-69-sBnAbs). Here we show that a major structural solution for these HV1-69-sBnAbs is achieved through a critical triad comprising two CDR-H2 loop anchor residues (a hydrophobic residue at position 53 (Ile or Met) and Phe54), and CDR-H3-Tyr at positions 98±1; together with distinctive V-segment CDR amino acid substitutions that occur in positions sparse in AID/polymerase-η recognition motifs. A semi-synthetic IGHV1-69 phage-display library screen designed to investigate AID/polη restrictions resulted in the isolation of HV1-69-sBnAbs that featured a distinctive Ile52Ser mutation in the CDR-H2 loop, a universal CDR-H3 Tyr at position 98 or 99, and required as little as two additional substitutions for heterosubtypic neutralizing activity. The functional importance of the Ile52Ser mutation was confirmed by mutagenesis and by BCR studies. Structural modeling suggests that substitution of a small amino acid at position 52 (or 52a) facilitates the insertion of CDR-H2 Phe54 and CDR-H3-Tyr into adjacent pockets on the stem. These results support the concept that activation and expansion of a defined subset of IGHV1-69-encoded B cells to produce potent HV1-69-sBnAbs does not necessarily require a heavily diversified V-segment acquired through recycling/reentry into the germinal center; rather, the incorporation of distinctive amino acid substitutions by Phase 2 long-patch error-prone repair of AID-induced mutations or by random non-AID SHM events may be sufficient. We propose that these routes of B cell maturation should be further investigated and exploited as a pathway for HV1-69-sBnAb elicitation by vaccination

Diabetes reversal by inhibition of the low-molecular-weight tyrosine phosphatase.

Obesity-associated insulin resistance plays a central role in type 2 diabetes. As such, tyrosine phosphatases that dephosphorylate the insulin receptor (IR) are potential therapeutic targets. The low-molecular-weight protein tyrosine phosphatase (LMPTP) is a proposed IR phosphatase, yet its role in insulin signaling in vivo has not been defined. Here we show that global and liver-specific LMPTP deletion protects mice from high-fat diet-induced diabetes without affecting body weight. To examine the role of the catalytic activity of LMPTP, we developed a small-molecule inhibitor with a novel uncompetitive mechanism, a unique binding site at the opening of the catalytic pocket, and an exquisite selectivity over other phosphatases. This inhibitor is orally bioavailable, and it increases liver IR phosphorylation in vivo and reverses high-fat diet-induced diabetes. Our findings suggest that LMPTP is a key promoter of insulin resistance and that LMPTP inhibitors would be beneficial for treating type 2 diabetes

Validating the structural role of Ser52 in HV1-69-sBnAbs.

<p><b>A</b>) F10 V-segment germline variants were analyzed for H5VN04 binding in the phage-Ab (5 scFv/phage) format by MSD ELISA <i>(left)</i> and for their ability to activate B-cell when expressed as B-cell receptors in the presence of H5VN04 <i>(right)</i>. <b>B</b>) HV1-69-sBnAb variants of S52I in F10 and A66, G52aP in CR6331, G17 and D8 were analyzed for H5VN04 reactivity by ELISA. <b>C</b>) Kinetic analysis by Biacore of F10 and A66 CDR-H2 variants against purified H5VN04. Residues colored in blue are non-germline amino acids. <b>D</b>) Circular dichroism measurement of F10 and the non-H5 reactive variant characterized by a germline configured CDR-H2 shows a highly similar CD profile for both constructs.</p

Characterization of HV1-69-sBnAbs VH domain.

<p><b>A</b>) Alignment of 38 published HV1-69-sBnAbs is shown with highlights referring to hydrophobic residues at position 53 (light plum), the conserved Phe54 (dark plum), the occurrence of CDR-H3-Tyr (pink) residues. Other highlights refer to panel <b>B</b>), which describes the result of a Fisher's exact test with Bonferroni adjustment that compared V-segment amino acid substitutions diversity and frequency of the 37 51p1 allele related HV1-69-sBnAbs with that of a reference <i>IGHV1-69</i> 51p1 allele related Ab dataset. 13 amino acid substitutions were determined to uniquely associate with the HV1-69-sBnAb dataset (P<0.05).</p

Understanding the structural role of the distinctive CDR-H2 amino acid substitutions in HV1-69-sBnAbs.

<p>A) VDW contact analysis (black lines) shows that Ser52 of F10 and CR9114 (orange), and Ile52 of CR6261(gray) make only intramolecular contacts; i.e., do not form contacts with their respective H5VN04s. Antibodies are shown in color; HA is in light gray. At far right, steric consequences of the germline Ile52 and the Ile52Ser substitutions are shown when the Abs are overlaid on their framework residues (RMSD ∼0.5 Å). Comparing structures of the HV1-69-sBnAbs, centered on Ile52 of CR6261 (green), with F10 (yellow) and CR9114 (cyan), the Ile52Ser mutation in F10 and CR9114 enables the 2 strands to come closer together, as indicated by the yellow and cyan arrows. Distances in red indicate hypothetical steric clashes (<3 Å) that would be created if Ile52 were present in CR9114 and F10. B) Comparison between the unbound (PDB 4FQH, left) and H5VN04-bound structures (PDB 4FQI, right) of CR9114, colored according to the magnitude of structural change after superposition on the main-chain of the VH domain (from blue = 0 Å, through white = 1 Å, to red = 1.8 Å). CDRs and side-chains of the major contact residues are shown, as depicted in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004103#ppat-1004103-g001" target="_blank">Figure 1A</a>. Distances between the Cα and Cβ atoms of Phe54 and the Cα atom of CDR-H3 Tyr98 (shown as dashed lines) are indicated. Large rotations of the side chains of CDR-H3 Tyr98, CDR-H2 Phe54 and CDR-H2 Ile53 are also evident, as previously noted <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004103#ppat.1004103-Dreyfus1" target="_blank">[7]</a>.</p