SUMMARY N-acetyl-aspartyl-glutamate (NAAG) is a peptide-based neurotransmitter that has been extensively studied in many neurological diseases. In this study, we show a specific role of NAAG in cancer. We found that NAAG is more abundant in higher grade cancers and is a source of glutamate in cancers expressing glutamate carboxypeptidase II (GCPII), the enzyme that hydrolyzes NAAG to glutamate and N-acetyl-aspartate (NAA). Knocking down GCPII expression through genetic alteration or pharmacological inhibition of GCPII results in a reduction of both glutamate concentrations and cancer growth. Moreover, targeting GCPII in combination with glutaminase inhibition accentuates these effects. These findings suggest that NAAG serves as an important reservoir to provide glutamate to cancer cells through GCPII when glutamate production from other sources is limited. Thus, GCPII is a viable target for cancer therapy, either alone or in combination with glutaminase inhibition.

Ribosomal protein synthesis in eubacteria and eukaryotic organelles initiates with an N-formylmethionyl-tRNA(i), resulting in N-terminal formylation of all nascent polypeptides. Peptide deformylase (PDF) catalyzes the subsequent removal of the N-terminal formyl group from the majority of bacterial proteins. Deformylation was for a long time thought to be a feature unique to the prokaryotes, making PDF an attractive target for designing novel antibiotics. However, recent genomic sequencing has revealed PDF-like sequences in many eukaryotes, including man. In this work, the cDNA encoding Homo sapiens PDF (HsPDF) has been cloned and a truncated form that lacks the N-terminal 58-amino-acid targeting sequence was overexpressed in Escherichia coli. The recombinant, Co(2+)-substituted protein is catalytically active in deformylating N-formylated peptides, shares many of the properties of bacterial PDF, and is strongly inhibited by specific PDF inhibitors. Expression of HsPDF fused to the enhanced green fluorescence protein in human embryonic kidney cells revealed its location in the mitochondrion. However, HsPDF is much less active than its bacterial counterpart, providing a possible explanation for the apparent lack of deformylation in the mammalian mitochondria. The lower catalytic activity is at least partially due to mutation of a highly conserved residue (Leu-91 in E. coli PDF) in mammalian PDF. PDF inhibitors had no detectable effect on two different human cell lines. These results suggest that HsPDF is likely an evolutional remnant without any functional role in protein formylation/deformylation and validates PDF as an excellent target for antibacterial drug design.

The targeting of glutamine metabolism specifically via pharmacological inhibition of glutaminase 1 (GLS1) has been translated into clinical trials as a novel therapy for several cancers. The results, though encouraging, show room for improvement in terms of tumor reduction. In this study, the glutaminase II pathway is found to be upregulated for glutamate production upon GLS1 inhibition in pancreatic tumors. Moreover, genetic suppression of glutamine transaminase K (GTK), a key enzyme of the glutaminase II pathway, leads to the complete inhibition of pancreatic tumorigenesis in vivo unveiling GTK as a new metabolic target for cancer therapy. These results suggest that current trials using GLS1 inhibition as a therapeutic approach targeting glutamine metabolism in cancer should take into account the upregulation of other metabolic pathways that can lead to glutamate production; one such pathway is the glutaminase II pathway via GTK.

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Peptide deformylase catalyzes the deformylation reaction of the amino terminal fMet residue of newly synthesized proteins in bacteria, and most likely in Plasmodium falciparum, and has therefore been identified as a potential antibacterial and antimalarial drug target. The structure of P. falciparum peptide deformylase, determined at 2.8 A resolution with ten subunits per asymmetric unit, is similar to the bacterial enzyme with the residues involved in catalysis, the position of the bound metal ion, and a catalytically important water structurally conserved between the two enzymes. However, critical differences in the substrate binding region explain the poor affinity of E. coli deformylase inhibitors and substrates toward the Plasmodium enzyme. The Plasmodium structure serves as a guide for designing novel antimalarials.

Conjugal transfer of chromosomal DNA between strains of Mycobacterium smegmatis occurs by a novel mechanism. In a transposon mutagenesis screen, three transfer-defective insertions were mapped to the lsr2 gene of the donor strain mc 2 155. Because lsr2 encodes a nonspecific DNA-binding protein, mutations of lsr2 give rise to a variety of phenotypes, including an inability to form biofilms. In this study, we show that efficient DNA transfer between strains of M. smegmatis occurs in a mixed biofilm and that the process requires expression of lsr2 in the donor but not in the recipient strain. Testing cells from different strata of standing cultures showed that transfer occurred predominantly at the biofilm air-liquid interface, as other strata containing higher cell densities produced very few transconjugants. These data suggest that the biofilm plays a role beyond mere facilitation of cell-cell contact. Surprisingly, we found that under standard assay conditions the recipient strain does not form a biofilm. Taking these results together, we conclude that for transfer to occur, the recipient strain is actively recruited into the biofilm. In support of this idea, we show that donor and recipient cells are present in almost equal numbers in biofilms that produce transconjugants. Our demonstration of genetic exchange between mycobacteria in a mixed biofilm suggests that conjugation occurs in the environment. Since biofilms are considered to be the predominant natural microhabitat for bacteria, our finding emphasizes the importance of studying biological and physical processes that occur between cells in mixed biofilms.Biofilms are dynamic communities of microorganisms that form on surfaces or at air-liquid interfaces (17,20,41). They arise following the attachment of bacteria to a surface; the bacteria then grow, differentiate, and multiply. The colonizing bacteria produce extracellular polymers, which encapsulate the cells and trap particulate matter, nutrients, and other bacteria that in turn contribute to the further development of the biofilm. Thus, as the biofilm develops it becomes increasingly heterogeneous. Microbial life is thought to exist predominantly in a biofilm, and biofilms can have either beneficial or harmful impacts on their environments (23). From a medical standpoint, biofilms can create serious problems. Bacteria within a biofilm are inherently more resistant to antibiotics, which makes their eradication difficult and is particularly problematic for patients with surgical implants resulting in chronic infections (19,33).Mycobacteria are known to form biofilms; however, relatively little is known about the mechanism of biofilm formation and development or its role in the biology of Mycobacterium species. For practical reasons, most biofilm studies have focused on the more rapidly growing and less pathogenic species, namely, Mycobacterium fortuitum, M. marinum, and M. smegmatis (16, 18, 36). In particular, genetic studies of M. smegmatis have provided insight into some of the key factors required f...

A macrocyclic, peptidomimetic inhibitor of peptide deformylase was designed by covalently cross-linking the P1' and P3' side chains. The macrocycle, which contains an N-formylhydroxylamine side chain as the metal-chelating group, was synthesized from a diene precursor via olefin metathesis using Grubbs's catalyst. The cyclic inhibitor showed potent inhibitory activity toward Escherichia coli deformylase (K(I) = 0.67 nM) and antibacterial activity against both Gram-positive and Gram-negative bacteria (MIC = 0.7-12 microg/mL).

Peptide deformylase (PDF, E.C. 3.5.1.88) catalyzes the removal of N-terminal formyl groups from nascent ribosome-synthesized polypeptides. PDF contains a catalytically essential divalent metal ion, which is tetrahedrally coordinated by three protein ligands (His, His, and Cys) and a water molecule. Previous studies revealed that the metal cofactor is a Fe2+ ion in Escherichia coli and many other bacterial PDFs. In this work, we found that PDFs from two iron-deficient bacteria, Borrelia burgdorferi and Lactobacillus plantarum, are stable and highly active under aerobic conditions. The native B. burgdorferi PDF (BbPDF) was purified 1200-fold and metal analysis revealed that it contains approximately 1.1 Zn2+ ion/polypeptide but no iron. Our studies suggest that PDF utilizes different metal ions in different organisms. These data have important implications in designing PDF inhibitors and should help address some of the unresolved issues regarding PDF structure and catalytic function.