Plastidic type I signal peptidase 1 is a redox‐dependent thylakoidal processing peptidase

Thylakoids are the photosynthetic membranes in chloroplasts and cyanobacteria. The aqueous phase inside the thylakoid known as the thylakoid lumen plays an essential role in the photosynthetic electron transport. The presence and significance of thiol‐disulfide exchange in this compartment have been recognized but remain poorly understood. All proteins found free in the thylakoid lumen and some proteins associated to the thylakoid membrane require an N‐terminal targeting signal, which is removed in the lumen by a membrane‐bound serine protease called thylakoidal processing peptidase (TPP). TPP is homologous to Escherichia coli type I signal peptidase (SPI) called LepB. Genetic data indicate that plastidic SPI 1 (Plsp1) is the main TPP in Arabidopsis thaliana (Arabidopsis) although biochemical evidence had been lacking. Here we demonstrate catalytic activity of bacterially produced Arabidopsis Plsp1. Recombinant Plsp1 showed processing activity against various TPP substrates at a level comparable to that of LepB. Plsp1 and LepB were also similar in the pH optima, sensitivity to arylomycin variants and a preference for the residue at ?3 to the cleavage site within a substrate. Plsp1 orthologs found in angiosperms contain two unique Cys residues located in the lumen. Results of processing assays suggested that these residues were redox active and formation of a disulfide bond between them was necessary for the activity of recombinant Arabidopsis Plsp1. Furthermore, Plsp1 in Arabidopsis and pea thylakoids migrated faster under non‐reducing conditions than under reducing conditions on SDS‐PAGE. These results underpin the notion that Plsp1 is a redox‐dependent signal peptidase in the thylakoid lumen.     

Leader peptidase

The Escherichia coli leader peptidase has been vital for unravelling problems in membrane assembly and protein export. The role of this essential peptidase is to remove amino-terminal leader peptides from exported proteins after they have crossed the plasma membrane. Strikingly, almost all periplasmic proteins, many outer membrane proteins, and a few inner membrane proteins are made with cleavable leader peptides that are removed by this peptidase. This enzyme of 323 amino acid residues spans the membrane twice, with its large carboxyl-terminal domain protruding into the periplasm. Recent discoveries show that its membrane orientation is controlled by positively charged residues that border (on the cytosolic side) the transmembrane segments. Cleavable pre-proteins must have small residues at -1 and a small or aliphatic residue at -3 (with respect to the cleavage site). Leader peptidase does not require a histidine or cysteine amino acid for catalysis. Interestingly, serine 90 and aspartic acid 153 are essential for catalysis and are also conserved in a mitochondrial leader peptidase, which is 30.7% homologous with the bacterial enzyme over a 101-residue stretch.     

Dipeptidyl peptidase IV, a kidney brush-border serine peptidase.

Dipeptidyl peptidase IV, an enzyme that releases dipeptides from substrates with N-terminal sequences of the forms X-Pro-Y or X-Ala-Y, was purified 300-fold from pig kidney cortex. The kidney is the main source of the enzyme, where it is one of the major microvillus-membrane proteins. Several other tissues contained demonstrable activity against the usual assay substrate glycylproline 2-naphthylamide. In the small intestine this activity was greatly enriched in the microvillus fraction. In all tissues examined, the activity was extremely sensitive to inhibition by di-isopropyl phosphorofluoridate (Dip-F), but relatively resistant to inhibition by phenylmethylsulphonyl fluoride. It is a serine proteinase which may be covalently labelled with [32P]Dip-F, and is the only enzyme of this class in the microvillus membrane. The apparent subunit mol.wt. estimated by sodium dodecyl-sulphate/polyacrylamide-gel electrophoresis and by titration with [32P]Dip-F was 130 000. Gel-filtration and sedimentation-equilibrium methods gave values in the region of 280 000, which is consistent with a dimeric structure, a conclusion supported by electron micrographs of the purified enzyme. Among other well-characterized serine proteinases, this enzyme is unique in its membrane location and its large subunit size. Investigation of the mode of attack of the peptidase on oligopeptides revealed that it could hydrolyse certain N-blocked peptides, e.g. Z-Gly-Pro-Leu-Gly-Pro. In this respect it is acting as an endopeptidase and as such may merit reclassification and renaming as microvillus-membrane serine peptidase.     

Dipeptidyl peptidase 4 – An important digestive peptidase in Tenebrio molitor larvae

Dipeptidyl peptidase 4 (DPP 4) is a proline specific serine peptidase that plays an important role in different regulatory processes in mammals. In this report, we isolated and characterized a unique secreted digestive DPP 4 from the anterior midgut of a stored product pest, Tenebrio molitor larvae (TmDPP 4), with a biological function different than that of the well-studied mammalian DPP 4. The sequence of the purified enzyme was confirmed by mass-spectrometry, and was identical to the translated RNA sequence found in a gut EST database. The purified peptidase was characterized according to its localization in the midgut, and substrate specificity and inhibitor sensitivity were compared with those of human recombinant DPP 4 (rhDPP 4). The T. molitor enzyme was localized mainly in the anterior midgut of the larvae, and 81% of the activity was found in the fraction of soluble gut contents, while human DPP 4 is a membrane enzyme. TmDPP 4 was stable in the pH range 5.0–9.0, with an optimum activity at pH 7.9, similar to human DPP 4. Only specific inhibitors of serine peptidases, diisopropyl fluorophosphate and phenylmethylsulfonyl fluoride, suppressed TmDPP 4 activity, and the specific dipeptidyl peptidase inhibitor vildagliptin was most potent. The highest rate of TmDPP 4 hydrolysis was found for the synthetic substrate Arg-Pro-pNA, while Ala-Pro-pNA was a better substrate for rhDPP 4. Related to its function in the insect midgut, TmDPP 4 efficiently hydrolyzed the wheat storage proteins gliadins, which are major dietary proteins of T. molitor.     

Evolution of peptidase diversity

A wide variety of peptidases associate with vital biological pathways, but the origin and evolution of their tremendous diversity are poorly defined. Application of the MEROPS classification to a comprehensive set of genomes yields a simple pattern of peptidase distribution and provides insight into the organization of proteolysis in all forms of life. Unexpectedly, a near ubiquitous core set of peptidases is shown to contain more types than those unique to higher multicellular organisms. From this core group, an array of eukaryote-specific peptidases evolved to yield well known intracellular and extracellular processes. The paucity of peptidase families unique to higher metazoa suggests gains in proteolytic network complexity required a limited number of biochemical inventions. These findings provide a framework for deeper investigation into the evolutionary forces that shaped each peptidase family and a roadmap to develop a timeline for their expansion as an interconnected system.     

Membrane topology of Escherichia coli prolipoprotein signal peptidase (signal peptidase II)

The lsp gene of Escherichia coli encodes the inner membrane enzyme, signal peptidase II (SPase II). SPase II is comprised of 164 amino acid residues and contains four hydrophobic domains. A series of lsp-phoA and lsp-lacZ gene fusions have been constructed in vitro to determine the topology of SPase II. The fusion junction for each of these gene fusions was determined by DNA sequencing. The lengths of the SPase II fragment in the fusions varied from 12 to 159 amino acid residues. Strains containing SPase II-PhoA fusions to the two predicted periplasmic loops exhibited higher levels of alkaline phosphatase activity than fusions to the predicted cytoplasmic domains. In contrast, SPase II-LacZ fusions at the cytoplasmic and the periplasmic domains of SPase II showed high and low levels of beta-galactosidase activity, respectively, a result opposite to those shown by SPase II-PhoA fusions located at precisely the same amino acid of SPase II. Taken together, these results strongly support the predicted model for SPase II topology, i.e. this enzyme spans the cytoplasmic membrane four times with both the amino and the carboxyl termini facing the cytoplasm.     

Selective fluorescence probes for dipeptidyl peptidase activity-fibroblast activation protein and dipeptidyl peptidase IV

Development of suitable tools to assess enzyme activity directly from their complex cellular environment has a dramatic impact on understanding the functional roles of proteins as well as on the discovery of new drugs. In this study, a novel fluorescence-based chemosensor strategy for the direct readout of dipeptidase activities within intact living cells is described. Selective activity-based probes were designed to sense two important type II transmembrane serine proteases, fibroblast activation protein (FAP) and dipeptidyl peptidase IV (DPP-IV). These serine proteases have been implicated in diverse cellular activities, including blood coagulation, digestion, immune responses, wound healing, tumor growth, tumor invasion, and metastasis. Here, we validated that Ac-GPGP-2SBPO and GPGP-2SBPO probes are excellent reporters of both proteolytic activities. Furthermore, the novel probes can differentiate between FAP and DPP-IV proteolytic activities in cellular assay. Potentially, this assay platform is immediately useful for novel drug discovery.     

MEROPS: the peptidase database

Important additions have been made to the MEROPS database (http://www.bi.bbsrc.ac.uk/Merops/Merops.htm). These include sequence alignments and cladograms for many of the families of peptidases, and these have proved very helpful in the difficult task of distinguishing the sequences of peptidases that are simply species variants of already known enzymes from those that represent novel enzymes.     

The reaction specificities of the thylakoidal processing peptidase and Escherichia coli leader peptidase are identical.

Proteins which are transported across the bacterial plasma membrane, endoplasmic reticulum and thylakoid membrane are usually synthesized as larger precursors containing amino-terminal targeting signals. Removal of the signals is carried out by specific, membrane-bound processing peptidases. In this report we show that the reaction specificities of these three peptidases are essentially identical. Precursors of two higher plant thylakoid lumen proteins are efficiently processed by purified Escherichia coli leader peptidase. Processing of one precursor, that of the 23 kd photosystem II protein, by both the thylakoidal and E. coli enzymes generates the correct mature amino terminus. Similarly, leader (signal) peptides of both eukaryotic and prokaryotic origin are cleaved by partially purified thylakoidal processing peptidase. No evidence of incorrect processing was obtained. Both leader peptidase and thylakoidal peptidase are inhibited by a synthetic leader peptide.     

Characterization of a peptidase fromDiplococcus pneumoniae

Some properties of a purified peptidase fromDiplococcus pneumoniae have been studied. The enzyme has a broad pH optimum between 6 and 8 and a K_m (onl-leucylglycylglycine) of 2.8mm. It is activated by low levels of Hg⁺⁺ and is inhibited by Mn⁺⁺, Co⁺⁺, β-mercaptoethanol and EDTA. Substrate specificity studies show that the enzyme is an exopeptidase of the aminopeptidase type, most active on tripeptide substrates bearing bulky substituents at the NH₂ ⁻ terminal end.     

Nuclear shuttling of the peptidase nardilysin

The metalloendopeptidase nardilysin contains a putative N-terminal nuclear localization signal. The functionality of this sequence was tested with nardilysin-GFP fusion constructs. Expression in NIH3T3 cells showed approximately 90-95% of nardilysin-GFP as cytoplasmic. However, 3-6% of transfected cells showed both cytosolic and nuclear staining, while 2-4% showed predominantly nuclear staining. A nuclear localization signal mutant and an N-terminally truncated nardilysin-GFP with the nuclear localization signal deleted were completely cytoplasmic. Although endogenous nardilysin was barely detectable in the nucleus, after treatment with leptomycin B, nuclear nardilysin rose to approximately 15% and to over 25% after addition of spermine. The ability of a methionine 49 to act as the sole initiator methionine, as previously proposed, was tested by inserting a c-myc epitope between leucine28 and glycine29. Expression in HEK293 cells showed the presence of the c-myc tag, demonstrating that the enzyme can be translated from the first methionine and contains the nuclear localization signal.     

Tripeptidyl-peptidase II: a multi-purpose peptidase

Tripeptidyl-peptidase II is a high-molecular weight peptidase with a widespread distribution in eukaryotic cells. The enzyme sequentially removes tripeptides from a free N-terminus of longer peptides and also displays a low endopeptidase activity. A role for tripeptidyl-peptidase II in the formation of peptides for antigen presentation has recently become evident, and the enzyme also appears to be important for the degradation of some specific substrates, e.g. the neuropeptide cholecystokinin. However, it is likely that the main biological function of tripeptidyl-peptidase II is to participate in a general intracellular protein turnover. This peptidase may act on oligopeptides generated by the proteasome, or other endopeptidases, and the tripeptides formed would subsequently be good substrates for other exopeptidases. The fact that tripeptidyl-peptidase II activity is increased in sepsis-induced muscle wasting, a situation of enhanced protein turnover, corroborates this biological role.     

Inhibitors of dipeptidyl peptidase 8 and dipeptidyl peptidase 9. Part 2: isoindoline containing inhibitors

To obtain selective and potent inhibitors of dipeptidyl peptidases 8 and 9, we synthesized a series of substituted isoindolines as modified analogs of allo-Ile-isoindoline, the reference DPP8/9 inhibitor. The influence of phenyl substituents and different P2 residues on the inhibitors’ affinity toward other DPPs and more specifically, their potential to discriminate between DPP8 and DPP9 will be discussed. Within this series compound 8j was shown to be a potent and selective inhibitor of DPP8/9 with low activity toward DPP II.     

Vigilin interacts with signal peptide peptidase

ABSTRACT: BACKGROUND: Signal peptide peptidase (SPP), a member of the presenilin-like intra-membrane cleaving aspartyl protease family, migrates on Blue Native (BN) gels as 100 kDa, 200 kDa and 450 kDa species. SPP has recently been implicated in other non-proteolytic functions such as retro-translocation of MHC Class I molecules and binding of misfolded proteins in the endoplasmic reticulum (ER). These high molecular weight SPP complexes might contain additional proteins that regulate the proteolytic activity of SPP or support its non-catalytic functions. RESULTS: In this study, an unbiased iTRAQ-labeling mass spectrometry approach was used to identify SPP-interacting proteins. We found that vigilin, a ubiquitous multi-KH domain containing cytoplasmic protein involved in RNA binding and protein translation control, selectively enriched with SPP. Vigilin interacted with SPP and both proteins co-localized in restricted intracellular domains near the ER, biochemically co-fractionated and were part of the same 450 kDa complex on BN gels. However, vigilin does not alter the protease activity of SPP, suggesting that the SPP-vigilin interaction might be involved in the non-proteolytic functions of SPP. CONCLUSIONS: We have identified and validated vigilin as a novel interacting partner of SPP that could play an important role in the non-proteolytic functions of SPP. This data adds further weight to the idea that intramembrane-cleaving aspartyl proteases, such as presenilin and SPPs, could have other functions besides the proteolysis of short membrane stubs.