Francesco Lipari

Crystal structure of a class I α1,2‐mannosidase involved in N ‐glycan processing and endoplasmic reticulum quality control

Mannose trimming is not only essential for N‐glycan maturation in mammalian cells but also triggers degradation of misfolded glycoproteins. The crystal structure of the class I α1,2‐mannosidase that trims Man9GlcNAc2 to Man8GlcNAc2 isomer B in the endoplasmic reticulum of Saccharomyces cerevisiae reveals a novel (αα)7‐barrel in which an N‐glycan from one molecule extends into the barrel of an adjacent molecule, interacting with the essential acidic residues and calcium ion. The observed protein–carbohydrate interactions provide the first insight into the catalytic mechanism and specificity of this eukaryotic enzyme family and may be used to design inhibitors that prevent degradation of misfolded glycoproteins in genetic diseases.

Role of the Cysteine Residues in the α1,2-Mannosidase Involved in N-Glycan Biosynthesis in Saccharomyces cerevisiae THE CONSERVED Cys340 AND Cys385 RESIDUES FORM AN ESSENTIAL DISULFIDE BOND

The Saccharomyces cerevisiae α1,2-mannosidase, which removes one specific mannose residue from Man9GlcNAc2 to form Man8GlcNAc2, is a member of a family of α1,2-mannosidases with similar amino acid sequences. The yeast α1,2-mannosidase contains five cysteine residues, three of which are conserved. Recombinant yeast α1,2-mannosidase, produced as the soluble catalytic domain, was shown to contain two disulfide bonds and one free thiol group using 2-nitro-5-thiosulfobenzoate and 5,5′-dithiobis(2-nitrobenzoate), respectively. Cys485 contains the free thiol group, as demonstrated by sequencing of labeled peptides following modification with [3H]ICH2COOH and by high performance liquid chromatography/mass spectrometry tryptic peptide mapping. A Cys340-Cys385 disulfide was demonstrated by sequencing a purified peptide containing this disulfide and by tryptic peptide mapping. Cys468 and Cys471 were not labeled with [3H]ICH2COOH and a peptide containing these two residues was identified in the tryptic peptide map, showing that Cys468 and Cys471 form the second disulfide bond. The α1,2-mannosidase loses its activity in the presence of dithiothreitol with first order kinetics, suggesting that at least one disulfide bond is essential for activity. Mutagenesis of each cysteine residue to serine showed that Cys340 and Cys385 are essential for production of recombinant enzyme, whereas Cys468, Cys471, and Cys485 are not required for production and enzyme activity. These results indicate that the sensitivity to dithiothreitol is due to reduction of the Cys340-Cys385 disulfide. Since Cys340 and Cys385 are conserved residues, it is likely that this disulfide bond is important to maintain the correct structure in the other members of the α1,2-mannosidase family.

Single-Well Monitoring of Protein-Protein Interaction and Phosphorylation-Dephosphorylation Events

We combined oxygen channeling assays with two distinct chemiluminescent beads to detect simultaneously protein phosphorylation and interaction events that are usually monitored separately. This novel method was tested in the ERK1/2 MAP kinase pathway. It was first used to directly monitor dissociation of MAP kinase ERK2 from MEK1 upon phosphorylation and to evaluate MAP kinase phosphatase (MKP) selectivity and mechanism of action. In addition, MEK1 and ERK2 were probed with an ATP competitor and an allosteric MEK1 inhibitor, which generated distinct phosphorylation-interaction patterns. Simultaneous monitoring of protein-protein interactions and substrate phosphorylation can provide significant mechanistic insight into enzyme activity and small molecule action.

Catalytic Specificity of Human Protein Tyrosine Kinases Revealed by Peptide Substrate Profiling

Out of the 90 human protein tyrosine kinases, 81 were assayed with short peptides derived from well-characterized [CDK1(Tyr15), IRS1(Tyr983), and JAK1(Tyr1023)] or generic [polyGlu:Tyr(4:1) and poly-Glu:Ala:Tyr(1:1:1)] substrates. As expected, the CDK1 peptide is a substrate for all Src family kinases. On the other hand, some of the activities are novel and lead to a better understanding of the function of certain kinases. Specifically, the CDK1 peptide is a substrate for many of the Eph family members. Interestingly, profiling of nearly all the human protein tyrosine kinases revealed a distinct pattern of selectivity towards the CDK1 and IRS1 peptides.