This study is focused on cercarial cysteine peptidases of the trematode Diplostomum pseudospathaceum. It follows previous research which confirmed the presence of a 24kDa cysteine peptidase in cercariae biochemically and by mass spectrometry. It was postulated, that the function of this peptidase is histolytic, when cercariae penetrate the tissues. During an attempt to purify this peptidase and characterize its peptidolytic activity, it was found out that the cercarial homogenate containsmore different peptidases varying in their pI. Tests of peptidolytic activity and inhibition have shown that these peptidases are cathepsin L-like. They are active over a broad spectrum of pH with optima of activities in weakly acidicor neutral pH. Using degenerate primers based on conserved motifs of cysteine pepridases, partial sequences of three genes for cathepsin L of D. pseudospataceum (DpCL1, 2 a 3) were obtained. Then the complete sequences of DpCL2 and 3 genes and partial sequence (without 5'end) of DpCL1 were obtained by RACE PCR. To confirm function of these peptidases we tried to immunolocalize them. We assumed that they are localized in penetration glands. Preliminary results suggested that some of the cathepsins could be also localized in the gut of cercariae. For more detailed biochemical...

The blood processing mechanisms in monogeneans of the subclass Polyopisthocotylea are known from ultrastructural and histochemical analyses only. In contrast to other blood- feeding parasites, just few biochemical and molecular analyses have been done on digestive enzymes in monogeneans. Therefore, we focused on the biochemical and molecular characterization of hydrolytic enzymes (peptidases) in the hematophagous species Paradiplozoon bliccae and Eudiplozoon nipponicum. The presence of the cysteine class peptidases, mainly cathepsin L, in excretory- secretory products and soluble protein extracts of P. bliccae and E. nipponicum we found. Detection was carried out using fluorogenic substrates, specific inhibitors and the labelled probe DCG-04. On the gels / membranes after electrophoresis / blotting we detected bands of approximately size of 35 kDa in the case of both species and 24 kDa for E. nipponicum. Soluble protein extracts of worms were separated by 2D gel electrophoresis and relevant spots around 35 kDa (P. bliccae) and around 25 ˗ 35 kDa (E. nipponicum) were confirmed by mass spectrometry as cathepsins L. Using degenerate primers based on the conserved motifs of cysteine class peptidases, a partial sequence of cathepsin L gene from E. nipponicum was obtained. Furthermore, 3'RACE PCR method...

The majority of the mitochondrial proteins is synthetized on the cytosolic ribosomes in the form of the protein precursors bearing mitochondrion-targeting signal presequences. Once the protein precursor has reached the mitochondrial matrix the signal presequence is no longer necessary and is cleaved off by heterodimeric mitochondrial processing peptidase (MPP; α/β). Although the crystal structure of MPP is available, the MPP mechanism of function is still matter of discussion. An all atomic, non-restrained molecular dynamics (MD) simulation in explicit water was used to study in detail the structural features of the highly conserved glycine-rich loop (GRL) of the regulatory α-subunit of the yeast MPP. Wild-type and GRL-deleted MPP structures were studied both in the presence and absence of a substrate in the peptidase active site. Targeted MD simulations were employed to study the mechanism of substrate translocation from the GRL to the peptidase active site. We demonstrate that the natural conformational flexibility of the GRL is crucial for the substrate translocation process from outside the enzyme towards the MPP active site. We show that the α-helical conformation of the substrate is important not only during its initial interaction with MPP (i.e. substrate recognition), but also later, at...

There is a continuous search for novel enzymes to complement the abilities of today’s commercially available enzyme and find tailor-fit alternatives to suit the diverse array of bio-based industries. One application could be to increase biogas yield by finding substrate degrading proteases that can be added to the anaerobic digestion process and survive degradation themselves. A novel enzyme identified as a hypothetical dipeptidyl peptidase III, a zinc dependent metallo-protease, was found by a metaproteogenomics approach to be produced by the microorganisms of a thermophilic biogas process. The aim of this study was to express and refold a recombinant variant of the novel DPP III to its active form after production in inclusion bodies in Escherichia Coli. Assaying of refolding conditions was performed by stepwise dialysis and drip dilution. Nine attempts were performed based on findings in literature, although no other variant of DPP III has earlier been successfully refolded from inclusion bodies. The study resulted in a limited set of conditions of temperature, volumes, metal ions, salts and other additives being tested in the refolding buffers. Enzyme refolding and activation was monitored by the hydrolysis of the DPP III fluorescent substrate Arg-Arg β-naphthylamide trihydrochloride, alongside with measurements of protein concentration and SDS-PAGE. The novel DPP III was successfully purified but no definite strategy of producing correctly folded protein was found.

Liu, Limei. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 137-156). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Streptococcus mutans is a recognized principal etiologic agent in coronal caries. Although S. mutans has the ability to bind collagen and degrade FALGPA, a synthetic peptide mimicking collagen substrate, its role in dental root caries has not yet been fully elucidated. Degradation of collagen fibrils in dentin was attributed to S. mutans, but a collagenase enzyme has not yet been isolated from this organism. Considering the increased incidence of dental root decay among the elderly, an understanding of the role of the pathogenic factors is necessary to the development of preventive measures. The present study has focused on the cloning and analysis of S. mutans collagenase enzyme. Toward this goal, a putative collagenase gene was identified in S. mutans UA159 by genomic analysis and a primer set was designed and used to amplify the corresponding gene in S. mutans GS-5 used as a model organism. The PCR product was cloned into the vector pCR 2.1 TOPO-TA, and the gene sequenced and analyzed. Alignment of the S. mutans GS-5 and UA159 putative collagenase genes showed 99% homology. The gene was next cloned in frame into the inducible expression vector pET100/D TOPO. Induction and expression of recombinant protein in E. coli were confirmed by SDS-PAGE and Western immunoblotting, while biochemical analysis indicated that it was a calcium- dependent metalloproteinase. Enzyme analysis of the recombinant enzyme showed both gelatinolytic and collagenolytic activity. Further analysis of the GS5 gene using databases such as ExPASy, Pfam, and SMART indicated that it was highly homologous to the U32 peptidase family, which includes the PrtC collagenase of Porphyromonas gingivalis, a bacterium causing periodontitis. The present study was the first to unequivocally demonstrate the existence of a collagenase gene in S. mutans, and to identify it as a member of the U32 peptidase family. The obtaining of the S. mutans collagenase gene should help in further investigation of the role of this enzyme in dental root decay and its potential use as a dental root caries vaccine.

Peptidases are enzymes that hydrolyse peptide bonds. This potentially dangerous activity is regulated by post translational modification and peptidase inhibitors. The best characterized of the peptidase inhibitors are the serpins whilst the aspartic peptidase inhibitors are the least characterized. Aspartic peptidase inhibitors are rare with only nine known sources. However, they are of great interest because they play an important part in several human diseases such as metastasis of breast cancer cells, Candida albicans infections and HIV. The aims of this research project were to investigate the structure of Squash Aspartic peptidase inhibitor (SQAPI), using nuclear magnetic resonance spectroscopy (NMR). This required large amounts of relatively pure and isotopically labeled protein, which was achieved by heterologously expressing His-tagged rSQAPI fusion protein in Escherichia coli using a rich to minimal media transfer method. The fusion protein was purified with a nickel column and the N-terminal extension containing the His6-tag was removed by cleavage of the fusion protein with enterokinase followed by nickel column purification. Preliminary 1 dimensional NMR spectra indicated that SQAPI was folded in solution at pH 3. This was confirmed from the results of a preliminary 15N-edited HSQC. These results combined justified the production of a 15N 13C labeled SQAPI sample for the collection of further NMR spectra. From the spectra produced with double labeled protein the backbone and the side-chain atoms of SQAPI were assigned. The chemical shifts are currently 88.89% complete and have been submitted to the biological magnetic resonance bank (BMRB). A preliminary estimate of the secondary structure of SQAPI has been calculated from the HNHA spectrum suggesting that the SQAPI structure has some similarity to the previously proposed model of the inhibitor’s structure. Furthermore, the region corresponding to the putative binding loop on the model of SQAPI was found to be mobile and deuterium exchange experiments indicate that the SQAPI structure is more globular than open.

<p>The protein degradation process is of vital importance for the cell to maintain cellular functions. An important enzyme in this process is the multimeric tripeptidyl-peptidase II (TPP II). It removes tripeptides from a free N-terminus of the substrates. TPP II has broad substrate specificity and wide-spread distribution, suggesting that the TPP II gene is a house-keeping gene. However, the levels of both mRNA and TPP II protein varies during different conditions and the TPP II gene promoter was therefore identified and characterized. It is a 215 bp fragment just upstream of the coding sequence. This fragment lacks a TATA-box but contains an initiator, two inverted CCAAT-boxes and an E-box. The CCAAT-boxes and the E-box were found to bind the nuclear factor Y (NF-Y) and upstream stimulatory factor-1 (USF-1) respectively. The CCAAT-boxes appear to be most important for the transcriptional activation. Furthermore, several silencer element were identified further upstream of the 215 bp promoter and the octamer binding factor Oct-1 was found to bind one of these fragments. If Oct-1 is responsible for the inhibition of the transcription of the TPP II gene remains to be investigated. In addition, the substrate specificity was investigated. For this purpose an expression system using <i>Pichia pastoris</i> was developed. The purified recombinant TPP II was found to have the same enzymatic properties as the native enzyme. In order to identify the amino acids involved in the binding of the N-terminus of the substrate, wild-type murine TPP II and four mutants E305Q, E305K, E331Q and E331K were purified. Steady-state kinetic analysis clearly demonstrated that both Glu-305 and Glu-331 are important for this binding as the K_M^app is more than 10² higher for the mutants than wild-type. Finally, the pH-dependence for cleavage of two chromogenic substrates was compared for TPP II from different species.</p>

The protein degradation process is of vital importance for the cell to maintain cellular functions. An important enzyme in this process is the multimeric tripeptidyl-peptidase II (TPP II). It removes tripeptides from a free N-terminus of the substrates. TPP II has broad substrate specificity and wide-spread distribution, suggesting that the TPP II gene is a house-keeping gene. However, the levels of both mRNA and TPP II protein varies during different conditions and the TPP II gene promoter was therefore identified and characterized. It is a 215 bp fragment just upstream of the coding sequence. This fragment lacks a TATA-box but contains an initiator, two inverted CCAAT-boxes and an E-box. The CCAAT-boxes and the E-box were found to bind the nuclear factor Y (NF-Y) and upstream stimulatory factor-1 (USF-1) respectively. The CCAAT-boxes appear to be most important for the transcriptional activation. Furthermore, several silencer element were identified further upstream of the 215 bp promoter and the octamer binding factor Oct-1 was found to bind one of these fragments. If Oct-1 is responsible for the inhibition of the transcription of the TPP II gene remains to be investigated. In addition, the substrate specificity was investigated. For this purpose an expression system using Pichia pastoris was developed. The purified recombinant TPP II was found to have the same enzymatic properties as the native enzyme. In order to identify the amino acids involved in the binding of the N-terminus of the substrate, wild-type murine TPP II and four mutants E305Q, E305K, E331Q and E331K were purified. Steady-state kinetic analysis clearly demonstrated that both Glu-305 and Glu-331 are important for this binding as the KMapp is more than 102 higher for the mutants than wild-type. Finally, the pH-dependence for cleavage of two chromogenic substrates was compared for TPP II from different species.

Introduction. The outermost layer of the epidermis, the stratum corneum (SC), plays a fundamental role in our defense against microorganisms, chemicals, and dehydration. The SC is composed of tightly packed keratinized skin cells, corneocytes. For a functioning skin it is essential that corneocytes are constantly shed (desquamated). Kallikrein-related peptidase (KLK) 5 and KLK7 may be important in the desquamation process through degradation of desmosomal proteins. Severe hereditary diseases, where inhibition of KLK5 and/or KLK7 is missing, points to the importance of regulation of protease activity. KLKs may be regulated in various ways: tissue expression, activation of proforms, specific inhibitors, and physico-chemical properties like pH. Besides their involvement in desquamation, KLKs may also be important in immune defense and inflammation by processing of mediators and via activation of proteinase-activated receptors (PARs). Aims. 1. To identify and characterize previously unknown proteases in the SC. 2. To further characterize KLK5 and KLK7 with special focus on activation mechanisms. 3. To identify new inhibitors of KLKs in human SC. 4. To further characterize KLKs regarding effects of various inhibitors and substrates. 5. To study possible functions of KLKs in inflammation, in particular via activation of PAR-2. Methods. Plantar SC was used as a source for purification of proteins. Recombinant proteins were produced in different expression systems (insect cells, yeast cells, and bacteria). Different activity assays and kinetic studies were performed. Tissue expression was studied by immunohistochemistry, immunoblot and PCR. PAR-2 activation was studied by measurement of intracellular [Ca2+] and immunofluorescense in KNRK-PAR2 cells. Results. Active KLK14 was purified from extracts of plantar SC. KLK14 showed a superior catalytic efficiency as compared to KLK5 when measuring trypsin-like activity. This indicated that KLK14, despite being present in low amounts in skin, may have great relevance for skin physiology. Among enzymes tested only KLK5 showed autocatalytic activity and is so far the only enzyme found in SC that can activate proKLK7. KLK5 could also activate proKLK14. This together with studies of pH dependence on activation placed KLK5 as a possible key activating enzyme in a proposed proteolytic cascade in the SC. In plantar SC extracts we have also identified the novel Kazal-type serine protease inhibitor 9 (SPINK9). Our results indicate that SPINK9 is preferentially expressed in palmo-plantar skin and specific for KLK5. Differences found regarding substrate specificity and inhibition profile can be useful in evaluating the contribution of individual KLKs to the proteolytic activity in crude SC extracts. One interesting finding was that KLK8, present at high protein levels in the epidermis, could not be inhibited by any protease inhibitor found in the extracts. PAR-2 activation studies showed that KLK5 and 14 but neither KLK7 nor 8 can activate PAR-2. Immunohistochemistry preferentially detected KLK14 in intraepidermal parts of the sweat ducts and in dermal sweat glands but we could also show coexpression of KLK14 and PAR-2 in the SC and stratum granulosum of the epidermis in inflammatory skin disorders. To summarize, KLK involvement in desquamation may be dependent on a proteolytic activation cascade regulated by an intrinsic pH gradient and specific inhibitors present in SC. Another possible function of KLKs is as mediators of inflammation through activation of PAR-2.