The antimicrobial peptide human alpha-defensin 5 (HD5) is expressed in Paneth cells, secretory epithelial cells in the small intestine. Unlike other characterized defensins, HD5 is stored in secretory vesicles as a propeptide. The storage quantities of HD5 are approximately 90 450 microg per cm2 of mucosal surface area, which is sufficient to generate microbicidal concentrations in the intestinal lumen. HD5 peptides isolated from the intestinal lumen are proteolytically processed forms--HD5(56-94) and HD5(63-94)--that are cleaved at the Arg55-Ala56 and Arg62-Thr63 sites, respectively. We show here that a specific pattern of trypsin isozymes is expressed in Paneth cells, that trypsin colocalizes with HD5 and that this protease can efficiently cleave HD5 propeptide to forms identical to those isolated in vivo. By acting as a prodefensin convertase in human Paneth cells, trypsin is involved in the regulation of innate immunity in the small intestine.

Nicotinamide N-methyltransferase (NNMT) catalyzes the N-methylation of nicotinamide, pyridines and other analogs using S-adenosyl-L-methionine as donor. NNMT plays a significant role in the regulation of metabolic pathways and is expressed at markedly high levels in several kinds of cancers, presenting it as a potential molecular target for cancer therapy. We have determined the crystal structure of human NNMT as a ternary complex bound to both the demethylated donor S-adenosyl-L-homocysteine and the acceptor substrate nicotinamide, to 2.7Å resolution. These studies reveal the structural basis for nicotinamide binding and highlight several residues in the active site which may play roles in nicotinamide recognition and NNMT catalysis. The functional importance of these residues was probed by mutagenesis. Of three residues near the nicotinamide’s amide group, substitution of S201 and S213 had no effect on enzyme activity while replacement of D197 dramatically decreased activity. Substitutions of Y20, whose side chain hydroxyl interacts with both the nicotinamide aromatic ring and AdoHcy carboxylate, also compromised activity. Enzyme kinetics analysis revealed kcat/Km decreases of 2-3 orders of magnitude for the D197A and Y20A mutants, confirming the functional importance of these active site residues. The mutants exhibited substantially increased Km for both NCA and AdoMet and modestly decreased kcat. MD simulations revealed long-range conformational effects which provide an explanation for the large increase in Km(AdoMet) for the D197A mutant, which interacts directly only with nicotinamide in the ternary complex crystal structure.

After BNT162b2 mRNA vaccination, antibody levels to spike, receptor binding domain (RBD) and virus neutralization were examined in 149 nursing home (NH) residents and 110 health care worker controls. SARS-CoV-2-naive NH residents median post-2nd vaccine dose antibody neutralization titers are ¼ that of SARS-CoV-2-naive healthcare workers.

Thiopurine S-methyltransferase (TPMT1) modulates the cytotoxic effects of thiopurine prodrugs such as 6-mercaptopurine by methylating them in a reaction using S-adenosyl-L-methionine as the donor. Patients with TPMT variant allozymes exhibit diminished levels of protein and/or enzyme activity and are at risk for thiopurine drug-induced toxicity. We have determined two crystal structures of murine TPMT, as a binary complex with the product S-adenosyl-L-homocysteine and as a ternary complex with S-adenosyl-L-homocysteine and the substrate 6-mercaptopurine, to 1.8 Å and 2.0 Å resolution respectively. Comparison of the structures reveals that an active site loop becomes ordered upon 6-mercaptopurine binding. The positions of the two ligands are consistent with the expected S N 2 reaction mechanism. Arg147 and Arg221, the only polar amino acids near 6-mercaptopurine, are highlighted as possible participants in substrate deprotonation. To probe whether these residues are important for catalysis, point mutants were prepared in the human enzyme. Substitution of Arg152 (Arg147 in murine TPMT) with glutamic acid decreases Vmax and increases Km for 6-mercaptopurine but not Km for S-adenosyl-L-methionine. Substitution at this position with alanine or histidine, and similar substitutions of Arg226 (Arg221 in murine TPMT), result in no effect on enzyme activity. The double mutant Arg152Ala/Arg226Ala exhibits a decreased Vmax and increased Km for 6-mercaptopurine. These observations suggest that either Arg152 or Arg226 may participate in some fashion in the TPMT reaction, with one residue compensating when the other is altered, and that Arg152 may interact with substrate more directly than Arg226, consistent with observations in the murine TPMT crystal structure. with accession codes 3BGI and 3BGD, respectively. 1 Abbreviations: TPMT, thiopurine S-methyltransferase; mTPMT, murine TPMT; hTPMT, human TPMT; AdoMet, S-adenosyl-Lmethionine; AdoHcy, S-adenosyl-L-homocysteine; 6MP, 6-mercaptopurine; MAD, multiple wavelength anomalous dispersion; SAMT, salicylic acid carboxyl methyltransferase; PrmC, N 5 -glutamine methyltransferase, the product of the protein release factor methylation gene; PAPT, putrescine aminopropyltransferase; TIMP, thioinosine monophosphate; dcAdoMet, decarboxylated S-adenosyl-Lmethionine; AdoDATO, S-adenosyl-1,8-diamino-3-thiooctane. Thiopurines such as 6-mercaptopurine (6MP), 6-thioguanine, and azothioprine are cytotoxic and are immunosuppressant compounds which are used to treat childhood acute lymphoblastic leukemia, inflammatory bowel disease, and transplant rejection (1-3). They are prodrugs which are extensively metabolized to give thioguanine nucleotides that exert their cytotoxic effects by incorporation into DNA or inhibiting purine synthesis. Thiopurine S-methyltransferase (TPMT) is a cytosolic enzyme which modulates the cytotoxicity of thiopurines by taking them as substrates in an S-methylation reaction. TPMT is a classic example of pharmacogenetics, in which allelic variation in patients r...