Byung Ha Oh

PGRP-LC and PGRP-LE have essential yet distinct functions in the drosophila immune response to monomeric DAP-type peptidoglycan.

Drosophila rely entirely on an innate immune response to combat microbial infection. Diaminopimelic acid–containing peptidoglycan, produced by Gram-negative bacteria, is recognized by two receptors, PGRP-LC and PGRP-LE, and activates a homolog of transcription factor NF-κB through the Imd signaling pathway. Here we show that full-length PGRP-LE acted as an intracellular receptor for monomeric peptidoglycan, whereas a version of PGRP-LE containing only the PGRP domain functioned extracellularly, like the mammalian CD14 molecule, to enhance PGRP-LC-mediated peptidoglycan recognition on the cell surface. Interaction with the imd signaling protein was not required for PGRP-LC signaling. Instead, PGRP-LC and PGRP-LE signaled through a receptor-interacting protein homotypic interaction motif–like motif. These data demonstrate that like mammals, drosophila use both extracellular and intracellular receptors, which have conserved signaling mechanisms, for innate immune recognition.

Structural Basis for Preferential Recognition of Diaminopimelic Acid-type Peptidoglycan by a Subset of Peptidoglycan Recognition Proteins

Drosophila peptidoglycan recognition protein (PGRP)-LCx and -LCa are receptors that preferentially recognize meso-diaminopimelic acid (DAP)-type peptidoglycan (PGN) present in Gram-negative bacteria over lysine-type PGN of Gram-positive bacteria and initiate the IMD signaling pathway, whereas PGRP-LE plays a synergistic role in this process of innate immune defense. How these receptors can distinguish the two types of PGN remains unclear. Here the structure of the PGRP domain of Drosophila PGRP-LE in complex with tracheal cytotoxin (TCT), the monomeric DAP-type PGN, reveals a buried ionic interaction between the unique carboxyl group of DAP and a previously unrecognized arginine residue. This arginine is conserved in the known DAP-type PGN-interacting PGRPs and contributes significantly to the affinity of the protein for the ligand. Unexpectedly, TCT induces infinite head-to-tail dimerization of PGRP-LE, in which the disaccharide moiety, but not the peptide stem, of TCT is positioned at the dimer interface. A sequence comparison suggests that TCT induces heterodimerization of the ectodomains of PGRP-LCx and -LCa in a closely analogous manner to prime the IMD signaling pathway, except that the heterodimer formation is nonperpetuating.

The TRAPP Complex: Insights into its Architecture and Function

Vesicle‐mediated transport is a process carried out by virtually every cell and is required for the proper targeting and secretion of proteins. As such, there are numerous players involved to ensure that the proteins are properly localized. Overall, transport requires vesicle budding, recognition of the vesicle by the target membrane and fusion of the vesicle with the target membrane resulting in delivery of its contents. The initial interaction between the vesicle and the target membrane has been referred to as tethering. Because this is the first contact between the two membranes, tethering is critical to ensuring that specificity is achieved. It is therefore not surprising that there are numerous ‘tethering factors’ involved ranging from multisubunit complexes, coiled‐coil proteins and Rab guanosine triphosphatases. Of the multisubunit tethering complexes, one of the best studied at the molecular level is the evolutionarily conserved TRAPP complex. There are two forms of this complex: TRAPP I and TRAPP II. In yeast, these complexes function in a number of processes including endoplasmic reticulum‐to‐Golgi transport (TRAPP I) and an ill‐defined step at the trans Golgi (TRAPP II). Because the complex was first reported in 1998 (1), there has been a decade of studies that have clarified some aspects of its function but have also raised further questions. In this review, we will discuss recent advances in our understanding of yeast and mammalian TRAPP at the structural and functional levels and its role in disease while trying to resolve some apparent discrepancies and highlighting areas for future study.

Synthesis of acarbose transfer products by Bacillus stearothermophilus maltogenic amylase with simmondsin.

Simmondsin, a material related to food intake inhibition from jojoba (Simmondsia chinensis), was transglycosylated by Bacillus stearothermophilus maltogenic amylase (BSMA) reaction with acarbose to synthesize an antiobese compound with hypoglycemic activity. Ten percent each of acarbose and simmondsin were mixed and incubated with BSMA at 55°C. Glycosylation products of simmondsin were observed by thin layer chromatography (TLC) and high performance ion chromatography (HPIC). The major transfer product was purified by using Biogel P-2 column. The structure was determined by matrix-assisted laser desorption ionization with time of flight (MALDI-TOF):mass spectrometry (MS) and 13 C-NMR. The major transglycosylation product was pseudotrisaccharide (PTS)-simmondsin, in which PTS was attached by an a-(1i6) glycosidic linkage to simmondsin. The administration of transglycosylated simmondsin with acarbose (200 mg:kg per day for 6 days) significantly reduced the food intake by 74%, comparable to 62% of simmondsin versus control in ob:ob mice. The transfer product (10 mg:kg) significantly suppressed the postprandial blood glucose response to starch (2 g:kg) by 68%, comparable to 60% of acarbose in Zucker fa:fa rats. The results indicated that the transfer products would be effective agents in lowering both food intake and blood glucose.

Small exterior hydrophobic cluster contributes to conformational stability and steroid binding in ketosteroid isomerase from Pseudomonas putida biotype B

A structural motif called the small exterior hydrophobic cluster (SEHC) has been proposed to explain the stabilizing effect mediated by solvent‐exposed hydrophobic residues; however, little is known about its biological roles. Unusually, in Δ5‐3‐ketosteroid isomerase from Pseudomonas putida biotype B (KSI‐PI) Trp92 is exposed to solvent on the protein surface, forming a SEHC with the side‐chains of Leu125 and Val127. In order to identify the role of the SEHC in KSI‐PI, mutants of those amino acids associated with the SEHC were prepared. The W92A, L125A/V127A, and W92A/L125A/V127A mutations largely decreased the conformational stability, while the L125F/V127F mutation slightly increased the stability, indicating that hydrophobic packing by the SEHC is important in maintaining stability. The crystal structure of W92A revealed that the decreased stability caused by the removal of the bulky side‐chain of Trp92 could be attributed to the destabilization of the surface hydrophobic layer consisting of a solvent‐exposed β‐sheet. Consistent with the structural data, the binding affinities for three different steroids showed that the surface hydrophobic layer stabilized by SEHC is required for KSI‐PI to efficiently recognize hydrophobic steroids. Unfolding kinetics based on analysis of the ΦU value also indicated that the SEHC in the native state was resistant to the unfolding process, despite its solvent‐exposed site. Taken together, our results demonstrate that the SEHC plays a key role in the structural integrity that is needed for KSI‐PI to stabilize the hydrophobic surface conformation and thereby contributes both to the overall conformational stability and to the binding of hydrophobic steroids in water solution.