Biogenesis of the peptide-derived redox cofactor pyrroloquinoline quinone

Pyrroloquinoline quinone (PQQ) is a peptide-derived redox cofactor produced by prokaryotes that also plays beneficial roles in organisms from other kingdoms. We review recent developments on the pathway of PQQ biogenesis, focusing on the mechanisms of PqqE, PqqF/G, and PqqB. These advances may shed light on other, uncharacterized biosynthetic pathways.     

Peptide transport in rabbit kidney. Studies with l-carnosine

l-Carnosine was shown to be transported into rabbit renal brush-border membrane vesicles by an Na⁺ - independent mechanism. The transport was competitively inhibited by glycyl-l-proline. Various di- and tripeptides inhibited l-carnosine transport, whereas free amino acids did not. Inhibition studies showed that blocking the free amino and carboxyl groups of the peptide reduced its affinity for the transport carrier. Under the conditions in which there was no detectable hydrolysis of l-carnosine in the medium, intravesicular contents showed a 30% hydrolysis of the peptide within the vesicles. Disruption of membrane vesicles with deoxycholate resulted in a 3-fold increase in l-carnosine hydrolyzing activity over untreated intact vesicles. Based on these observations, a model for peptide transport is proposed in which transport of the intact peptide across the membrane is followed by its partial or complete hydrolysis by a membrane peptidase whose active site is on the cytoplasmic side of the membrane.     

Characterization of a chromosomal mutant that blocks hemolysin excretion in Escherichia coli

Abstract Alanine dipeptides normally penetrate into Pseudomonas aeruginosa by the way of two transport systems. In peptidase N-deficient mutants, dialanine is unable to use its low affinity transport system. Uptake competition showed that this system harboured a permease common to the transport of the amino acid alanine. This permease permits the penetration of both alanine and alanyl peptides uniquely in the presence of active peptidase N. The uptake of trialanine is independent of the presence of active peptidase N inside bacteria despite the fact that hydrolysis of this tripeptide absolutely requires this activity.     

Prolyl Oligopeptidase Enhances α-Synuclein Dimerization via Direct Protein-Protein Interaction

Prolyl oligopeptidase (PREP) accelerates the aggregation of α-synuclein (aSyn), a key protein involved in development of Parkinson disease and other synucleinopathies. PREP inhibitors reduce aSyn aggregation, but the mechanism has remained unknown. We have now used protein-fragment complementation assays (PCA) and microscale thermophoresis in parallel to show that PREP interacts directly with aSyn in both intact cells and in a cell-free system. Using split luciferase-based PCA, we first showed that PREP enhances the formation of soluble aSyn dimers in live Neuro-2A neuroblastoma cells. A PREP inhibitor, KYP-2047, reduced aSyn dimerization in PREP-expressing cells but not in cells lacking PREP expression. aSyn dimerization was also enhanced by PREP(S554A), an enzymatically inactive PREP mutant, but this was not affected by KYP-2047. PCA and microscale thermophoresis studies showed that aSyn interacts with both PREP and PREP(S554A) with low micromolar affinity. Neither the proline-rich, C-terminal domain of aSyn nor the hydrolytic activity of PREP was required for the interaction with PREP. Our results show that PREP binds directly to aSyn to enhance its dimerization and may thus serve as a nucleation point for aSyn aggregation. Native gel analysis showed that KYP-2047 shifts PREP to a compact monomeric form with reduced ability to promote aSyn nucleation. As PREP inhibition also enhances autophagic clearance of aSyn, PREP inhibitors may reduce accumulation of aSyn inclusions via a dual mechanism and are thus a novel therapeutic candidate for synucleinopathies. Our results also suggest that PREP has other cellular functions in addition to its peptidase activity.     

Biostable multi-Aib analogs of tachykinin-related peptides demonstrate potent oral aphicidal activity in the pea aphid Acyrthosiphon pisum (Hemiptera: Aphidae)

The tachykinin-related peptides (TRPs) are multifunctional neuropeptides found in a variety of arthropod species, including the pea aphid Acyrthosiphon pisum (Hemiptera: Aphidae). Two new biostable TRP analogs containing multiple, sterically hindered Aib residues were synthesized and found to exhibit significantly enhanced resistance to hydrolysis by angiotensin converting enzyme and neprilysin, membrane-bound enzymes that degrade and inactivate natural TRPs. The two biostable analogs were also found to retain significant myostimulatory activity in an isolated cockroach hindgut preparation, the bioassay used to isolate and identify the first members of the TRP family. Indeed one of the analogs (Leuma-TRP-Aib-1) matched the potency and efficacy of the natural, parent TRP peptide in this myotropic bioassay. The two biostable TRP analogs were further fed in solutions of artificial diet to the pea aphid over a period of 3 days and evaluated for antifeedant and aphicidal activity and compared with the effect of treatment with three natural, unmodified TRPs. The two biostable multi-Aib TRP analogs were observed to elicit aphicidal effects within the first 24 h. In contrast natural, unmodified TRPs, including two that are native to the pea aphid, demonstrated little or no activity. The most active analog, double-Aib analog Leuma-TRP-Aib-1 (pEA[Aib]SGFL[Aib]VR-NH₂), featured aphicidal activity calculated at an LC₅₀ of 0.0083 nmol/μl (0.0087 μg/μl) and an LT₅₀ of 1.4 days, matching or exceeding the potency of commercially available aphicides. The mechanism of this activity has yet to be established. The aphicidal activity of the biostable TRP analogs may result from disruption of digestive processes by interfering with gut motility patterns and/or with fluid cycling in the gut; processes shown to be regulated by the TRPs in other insects. These active TRP analogs and/or second generation analogs offer potential as environmentally friendly pest aphid control agents.     

Structure and catalysis of acylaminoacyl peptidase: closed and open subunits of a dimer oligopeptidase

Acylaminoacyl peptidase from Aeropyrum pernix is a homodimer that belongs to the prolyl oligopeptidase family. The monomer subunit is composed of one hydrolase and one propeller domain. Previous crystal structure determinations revealed that the propeller domain obstructed the access of substrate to the active site of both subunits. Here we investigated the structure and the kinetics of two mutant enzymes in which the aspartic acid of the catalytic triad was changed to alanine or asparagine. Using different substrates, we have determined the pH dependence of specificity rate constants, the rate-limiting step of catalysis, and the binding of substrates and inhibitors. The catalysis considerably depended both on the kind of mutation and on the nature of the substrate. The results were interpreted in terms of alterations in the position of the catalytic histidine side chain as demonstrated with crystal structure determination of the native and two mutant structures (D524N and D524A). Unexpectedly, in the homodimeric structures, only one subunit displayed the closed form of the enzyme. The other subunit exhibited an open gate to the catalytic site, thus revealing the structural basis that controls the oligopeptidase activity. The open form of the native enzyme displayed the catalytic triad in a distorted, inactive state. The mutations affected the closed, active form of the enzyme, disrupting its catalytic triad. We concluded that the two forms are at equilibrium and the substrates bind by the conformational selection mechanism.     

FtsH-dependent processing of RNase colicins D and E3 means that only the cytotoxic domains are imported into the cytoplasm

It has long been suggested that the import of nuclease colicins requires protein processing; however it had never been formally demonstrated. Here we show that two RNase colicins, E3 and D, which appropriate two different translocation machineries to cross the outer membrane (BtuB/Tol and FepA/TonB, respectively), undergo a processing step inside the cell that is essential to their killing action. We have detected the presence of the C-terminal catalytic domains of these colicins in the cytoplasm of target bacteria. The same processed forms were identified in both colicin-sensitive cells and in cells immune to colicin because of the expression of the cognate immunity protein. We demonstrate that the inner membrane protease FtsH is necessary for the processing of colicins D and E3 during their import. We also show that the signal peptidase LepB interacts directly with the central domain of colicin D in vitro and that it is a specific but not a catalytic requirement for in vivo processing of colicin D. The interaction of colicin D with LepB may ensure a stable association with the inner membrane that in turn allows the colicin recognition by FtsH. We have also shown that the outer membrane protease OmpT is responsible for alternative and distinct endoproteolytic cleavages of colicins D and E3 in vitro, presumably reflecting its known role in the bacterial defense against antimicrobial peptides. Even though the OmpT-catalyzed in vitro cleavage also liberates the catalytic domain from colicins D and E3, it is not involved in the processing of nuclease colicins during their import into the cytoplasm.     

Detection of matrix metallopeptidase-9-like proteins in Trypanosoma cruzi

In this study, the cell-associated and extracellular peptidases of Trypanosoma cruzi grown in modified Roitman’s complex (MRC) medium were analyzed by measuring peptidase activity in gelatin-containing zymograms. Our results showed that the cell-associated peptidases as well as peptidases extracellularly released by T. cruzi displayed two distinct proteolytic classes: cysteine and metallopeptidase activities. The major cysteine peptidase, cruzipain, synthesized by T. cruzi cells was detected in cellular parasite content, as a 50 kDa reactive polypeptide, after probing with anti-cruzipain antibody. In addition, metallo-type peptidases belonging to the matrix metallopeptidase-9 (MMP-9) family were revealed, after Western blotting, as a 97 kDa protein band in cellular extract and an 85 kDa polypeptide in both cellular and secreted parasite extracts. The MMP-9-like activity present in cells and spent culture medium was immunoprecipitated by an anti-MMP-9 polyclonal antibody. The surface location of MMP-9-like proteins in T. cruzi was also evidenced by means of flow cytometry analysis. Furthermore, doxycycline that has direct MMP-9 inhibiting properties in vitro, inhibited MMP-9-like activities in gel zymography, immunoprecipitation and flow cytometry analyses. This is the first report of the presence of MMP-9-like molecules in T. cruzi. The presence of a matrix extracellular-degrading enzyme may play a role in the T. cruzi-host cell interaction, making this enzyme a potential target for future drug development against this pathogenic trypanosomatid.