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The production of milk by dairy cows far exceeds the nutritional needs of the calf and is vital for the economical use of dairy cattle. High milk yield is a unique production trait that can be effectively enhanced through traditional selection methods. The process of lactation in cows serves as an excellent model for studying the biological aspects of lactation with the aim of exploring the mechanistic base of this complex trait at the cellular level. In this study, we analyzed the milk transcriptome at the single-cell level by conducting scRNA-seq analysis on milk samples from two Holstein Friesian cows at mid-lactation (75 and 93 days) using the 10× Chromium platform. Cells were pelleted and fat was removed from milk by centrifugation. The cell suspension from each cow was loaded on separate channels, resulting in the recovery of 9313 and 14,544 cells. Library samples were loaded onto two lanes of the NovaSeq 6000 (Illumina) instrument. After filtering at the cell and gene levels, a total of 7988 and 13,973 cells remained, respectively. We were able to reconstruct different cell types (milk-producing cells, progenitor cells, macrophages, monocytes, dendritic cells, T cells, B cells, mast cells, and neutrophils) in bovine milk. Our findings provide a valuable resource for identifying regulatory elements associated with various functions of the mammary gland such as lactation, tissue renewal, native immunity, protein and fat synthesis, and hormonal response.

Background: Older adults represent a large oncologic demographic and are under-represented within oncology research despite constituting nearly two-thirds of the oncologic population in the United States. Because many social factors influence research participation, those who enroll in research do not reflect the oncology population at large, introducing bias and creating issue with external validity of studies. The same factors that influence study enrollment may also impact cancer outcomes, meaning that those who enroll in studies may already have an improved chance of cancer survival, further skewing results of these studies. This study evaluates characteristics that influence study enrollment in older adults and explore to what degree these factors may influence survival after allogeneic blood or marrow transplantation., Methods: This retrospective comparison study evaluates 63 adults aged 60 and above undergoing allogenic transplantation at one institution. Patients who elected and declined enrollment in a non-therapeutic observational study were evaluated. Demographic and clinical characteristics between groups were compared and assessed as predictors of transplant survival, including decision to enroll in the study., Results: Participants who chose to enroll in the parent study were not different with regard to gender, race/ethnicity, age, insurance type, donor age, and neighborhood income/poverty level compared to patients who were invited to participate but declined enrollment. The research participant group had higher proportion assessed as being fully active (23.8% vs. 12.7%, p = 0.034) and lower mean comorbidity scores (1.0 vs 2.47, p = 0.008). Enrollment in an observational study independently predicted transplant survival (HR = 0.316, 95% CI 0.12-0.82, p = 0.017). When controlling for relevant confounders of disease severity, comorbidities, and transplant age, enrolling in the parent study was associated with a lower hazards of death following transplant (HR = 0.302, 95% CI 0.10-0.87, p = 0.027)., Conclusions: Despite being demographically comparable, persons who enrolled in one non-therapeutic transplant study had significantly improved survivorship than those who did not participate in observational research. These findings suggest that there are unidentified factors that influence study involvement that may also impact disease survivorship, over-estimating outcomes from these studies. Results from prospective observational studies should be interpreted with the consideration that study participants have an improved chance of survival at baseline., (© 2023. The Author(s).)

Background: Developing sustainable autotrophic cell factories depends heavily on the availability of robust and well-characterized biological parts. For cyanobacteria, these still lag behind the more advanced E. coli toolkit. In the course of previous protein expression experiments with cyanobacteria, we encountered inconveniences in working with currently available RSF1010-based shuttle plasmids, particularly due to their low biosafety and low yields of recombinant proteins. We also recognized some drawbacks of the commonly used fluorescent reporters, as quantification can be affected by the intrinsic fluorescence of cyanobacteria. To overcome these drawbacks, we envisioned a new chimeric vector and an alternative reporter that could be used in cyanobacterial synthetic biology and tested them in the model cyanobacterium Synechocystis sp. PCC 6803., Methods: We designed the pMJc01 shuttle plasmid based on the broad host range RSFmob-I replicon. Standard cloning techniques were used for vector construction following the RFC10 synthetic biology standard. The behavior of pMJC01 was tested with selected regulatory elements in E. coli and Synechocystis sp. PCC 6803 for the biosynthesis of the established GFP reporter and of a new reporter protein, cystatin. Cystatin activity was assayed using papain as a cognate target., Results: With the new vector we observed a significantly higher GFP expression in E. coli and Synechocystis sp. PCC 6803 compared to the commonly used RSF1010-based pPMQAK1. Cystatin, a cysteine protease inhibitor, was successfully expressed with the new vector in both E. coli and Synechocystis sp. PCC 6803. Its expression levels allowed quantification comparable to the standardly used fluorescent reporter GFPmut3b. An important advantage of the new vector is its improved biosafety due to the absence of plasmid regions encoding conjugative transfer components. The broadhost range vector pMJc01 could find application in synthetic biology and biotechnology of cyanobacteria due to its relatively small size, stability and ease of use. In addition, cystatin could be a useful reporter in all cell systems that do not contain papain-type proteases and inhibitors, such as cyanobacteria, and provides an alternative to fluorescent reporters or complements them., Competing Interests: The authors declare that they have no competing interests., (© 2021 Juteršek and Dolinar.)

Boris Krajnc je bil prvi habilitirani učitelj ljubljanske univerze za področje biokemije - imenovanje v naziv docenta je datirano s 7. januarjem 1946. Vendar se je Krajnčeva življenjska pot zelo hitro končala, saj so ga 27. oktobra 1947 aretirali in 26. aprila 1948 na montiranem dachauskem procesu obsodili na smrt. Ustrelili naj bi ga 12. maja 1948, starega 34 let. Krajnčeva življenjska zgodba je tesno povezana z delom več slovenskih kemikov, ki so bili kot zaporniki v koncentracijskem taborišču Dachau izbrani za tehnično pomoč pri izvedbah poskusov na ljudeh ali pri delu v kliničnem laboratoriju. Na povojnih dachauskih procesih v Ljubljani je bilo skupaj obsojenih 8 kemikov, od teh jih je bilo 5 obsojenih na smrt in ustreljenih. V kasnejših presojah dachauskih procesov se je izkazalo, da so bile obtožnice skonstruirane, priznanja pa pridobljena s krutimi zasliševalskimi metodami, zato so bile vse sodbe razveljavljene. Po emigraciji encimatika Richarda Klemna ter pregonu Maksa Samca in Marte Blinc z univerze je bil Boris Krajnc tisti, ki naj bi skrbel za biokemijo na Tehniški fakulteti. Po njegovi smrti je nekaj let biokemijo predaval Krajnčev mlajši kolega Dušan Stucin z Medicinske fakultete, potem pa biokemije več let ni bilo v kemijskem kurikulumu.

The bloom-forming cyanobacterium Microcystis aeruginosa is known for its global distribution and for the production of toxic compounds. In the genome of M. aeruginosa PCC 7806, we discovered that the gene coding for MaOC1, a caspase homolog protease, is followed by a toxin-antitoxin module, flanked on each side by a direct repeat. We therefore investigated their possible interaction at the protein level. Our results suggest that this module belongs to the ParE/ParD-like superfamily of type II toxin-antitoxin systems. In solution, the antitoxin is predominantly alpha-helical and dimeric. When coexpressed with its cognate toxin and isolated from Escherichia coli , it forms a complex, as revealed by light scattering and affinity purification. The active site of the toxin is restricted to the C-terminus of the molecule. Its truncation led to normal cell growth, while the wild-type form prevented bacterial growth in liquid medium. The orthocaspase MaOC1 was able to cleave the antitoxin so that it could no longer block the toxin activity. The most likely target of the protease was the C-terminus of the antitoxin with two sections of basic amino acid residues. E. coli cells in which MaOC1 was expressed simultaneously with the toxin-antitoxin pair were unable to grow. In contrast, no effect on cell growth was found when using a proteolytically inactive MaOC1 mutant. We thus present the first case of a cysteine protease that regulates the activity of a toxin-antitoxin module, since all currently known activating proteases are of the serine type., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Klemenčič, Halužan Vasle and Dolinar.)

Richard Klemen was the first teacher of enzymology at the University of Ljubljana. His early career in Ljubljana ended in January 1942 when he moved to Vienna, Austria. During the war he conducted experiments that led him to describe the so-called Hofmann-Klemen effect in clay. Later he was a research assistant and titular associate professor in the field of biochemical technology at the Vienna Technical University and finally a lecturer at the University of Natural Resources in Vienna. His life is an interesting example of a scientist and educator whose Gottscheer German origin would probably prevent him from continuing his career in post-war Yugoslavia. At the same time, he did not achieve in Austria the positions and status that his former colleagues and students had achieved in Slovenia. Although he was almost forgotten, he remains important as the first trained enzymologist and teacher of enzymology in Slovenia. This article also presents his full bibliography.

Orthocaspases are prokaryotic caspase homologs - proteases, which cleave their substrates after positively charged residues using a conserved histidine - cysteine (HC) dyad situated in a catalytic p20 domain. However, in orthocaspases pseudo-variants have been identified, which instead of the catalytic HC residues contain tyrosine and serine, respectively. The presence and distribution of these presumably proteolytically inactive p20-containing enzymes has until now escaped attention. We have performed a detailed analysis of orthocaspases in all available prokaryotic genomes, focusing on pseudo-orthocaspases. Surprisingly we identified type I metacaspase homologs in filamentous cyanobacteria. While genes encoding pseudo-orthocaspases seem to be absent in Archaea, our results show conservation of these genes in organisms performing either anoxygenic photosynthesis (orders Rhizobiales, Rhodobacterales, and Rhodospirillales in Alphaproteobacteria) or oxygenic photosynthesis (all sequenced cyanobacteria, except Gloeobacter , Prochlorococcus , and Cyanobium ). Contrary to earlier reports, we were able to detect pseudo-orthocaspases in all sequenced strains of the unicellular cyanobacteria Synechococcus and Synechocystis. In silico comparisons of the primary as well as tertiary structures of pseudo-p20 domains with their presumably proteolytically active homologs suggest that differences in their amino acid sequences have no influence on the overall structures. Mutations therefore affect most likely only the proteolytic activity. Our data provide an insight into diversification of pseudo-orthocaspases in Prokaryotes, their taxa-specific distribution, and allow suggestions on their taxa-specific function.

Cyanobacteria are an important group of microorganisms displaying a range of morphologies that enable phenotypic differentiation between the major lineages of cyanobacteria, often to the genus level, but rarely to species or strain level. We focused on the unicellular genus Synechocystis that includes the model cyanobacterial strain PCC 6803. For 11 Synechocystis members obtained from cell culture collections, we sequenced the variable part of the 16S rRNA-encoding region and the 16S - 23S internally transcribed spacer (ITS), both standardly used in taxonomy. In combination with microscopic examination we observed that 2 out of 11 strains from cell culture collections were clearly different from typical Synechocystis members. For the rest of the samples, we demonstrated that both sequenced genomic regions are useful for discrimination between investigated species and that the ITS region alone allows for a reliable differentiation between Synechocystis strains.

Programmed cell death in multicellular organisms is a coordinated and precisely regulated process. On the other hand, in bacteria we have little clue about the network of interacting molecules that result in the death of a single cell within a population or the death of almost complete population, such as often observed in cyanobacterial blooms. With the recent discovery that orthocaspase MaOC1 of the cyanobacterium Microcystis aeruginosa is an active proteolytic enzyme, we have gained a possible hint about at least one step in the process, but the picture is far from complete. Interestingly, the genomic context of MaOC1 revealed the presence of multiple copies of genes that belong to toxin-antitoxin modules. It has been speculated that these also play a role in bacterial programmed cell death. The discovery of two components linked to cell death within the same genomic region could open new ways to deciphering the underlying mechanisms of cyanobacterial cell death.

In recent years, photosynthetic autotrophic cyanobacteria have attracted interest for biotechnological applications for sustainable production of valuable metabolites. Although biosafety issues can have a great impact on public acceptance of cyanobacterial biotechnology, biosafety of genetically modified cyanobacteria has remained largely unexplored. We set out to incorporate biocontainment systems in the model cyanobacteriumSynechocystissp. PCC 6803. Plasmid-encoded safeguards were constructed using the nonspecific nuclease NucA fromAnabaenacombined with different metal-ion inducible promoters. In this manner, conditional lethality was dependent on intracellular DNA degradation for regulated autokilling as well as preclusion of horizontal gene transfer. In cells carrying the suicide switch comprising thenucAgene fused to a variant of thecopMpromoter, efficient inducible autokilling was elicited. Parallel to nuclease-based safeguards, cyanobacterial toxin/antitoxin (TA) modules were examined in biosafety switches. Rewiring ofSynechocystisTA pairsssr1114/slr0664andslr6101/slr6100for conditional lethality using metal-ion responsive promoters resulted in reduced growth, rather than cell killing, suggesting cells could cope with elevated toxin levels. Overall, promoter properties and translation efficiency influenced the efficacy of biocontainment systems. Several metal-ion promoters were tested in the context of safeguards, and selected promoters, including anrsBvariant, were characterized by beta-galactosidase reporter assay., (© 2016. Published by The Company of Biologists Ltd.)

Caspases are a family of cysteine-dependent proteases known to be involved in the process of programmed cell death in metazoans. Recently, cyanobacteria were also found to contain caspase-like proteins, but their existence has only been identified in silico up to now. Here, we present the first experimental characterisation of a prokaryotic caspase homologue. We have expressed the putative caspase-like gene MaOC1 from the toxic bloom-forming cyanobacterium Microcystis aeruginosa PCC 7806 in Escherichia coli. Kinetic characterisation showed that MaOC1 is an endopeptidase with a preference for arginine in the P1 position and a pH optimum of 7.5. MaOC1 exhibited high catalytic rates with the kcat /KM value for Z-RR-AMC substrate of the order 10(6)  M(-1)  s(-1). In contrast to plant or metazoan caspase-like proteins, whose activity is calcium-dependent or requires dimerisation for activation, MaOC1 was activated by autocatalytic processing after residue Arg219, which separated the catalytic domain and the remaining 55 kDa subunit. The Arg219Ala mutant was resistant to autoprocessing and exhibited no proteolytic activity, confirming that processing of MaOC1 is a prerequisite for its activity. Due to their structural and functional differences to other known caspase-like proteins, we suggest to name these evolutionary primitive proteins orthocaspases., (© 2015 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.)

Vascular endothelium selectively controls the transport of plasma contents across the blood vessel wall. The principal objective of our preliminary study was to quantify the electroporation-induced increase in permeability of blood vessel wall for macromolecules, which do not normally extravasate from blood into skin interstitium in homeostatic conditions. Our study combines mathematical modeling (by employing pharmacokinetic and finite element modeling approach) with in vivo measurements (by intravital fluorescence microscopy). Extravasation of fluorescently labeled dextran molecules of two different sizes (70 kDa and 2000 kDa) following the application of electroporation pulses was investigated in order to simulate extravasation of therapeutic macromolecules with molecular weights comparable to molecular weight of particles such as antibodies and plasmid DNA. The increase in blood vessel permeability due to electroporation and corresponding transvascular transport was quantified by calculating the apparent diffusion coefficients for skin microvessel wall (D [μm2/s]) for both molecular sizes. The calculated apparent diffusion coefficients were D = 0.0086 μm2/s and D = 0.0045 μm2/s for 70 kDa and 2000 kDa dextran molecules, respectively. The results of our preliminary study have important implications in development of realistic mathematical models for prediction of extravasation and delivery of large therapeutic molecules to target tissues by means of electroporation.

A number of recently developed and approved therapeutic agents based on highly specific and potent antibodies have shown the potential of antibody therapy. As the next step, antibody-based therapeutics will be bioengineered in a way that they not only bind pathogenic targets but also address other issues, including drug targeting and delivery. For antibodies that are expected to act within brain tissue, like those that are directed against the pathogenic prion protein isoform, one of the major obstacles is the blood-brain barrier which prevents efficient transfer of the antibody, even of the engineered single-chain variants. We recently demonstrated that a specific prion-specific antibody construct which was injected into the murine tail vein can be efficiently transported into brain tissue. The novelty of the work was in that the cell penetrating peptide was used as a linker connecting both specificity-determining domains of the antibody peptide, thus eliminating the need for the standard flexible linker, composed of an arrangement of three consecutive (Gly 4Ser) repeats. This paves the road toward improved bioengineered antibody variants that target brain antigens.

Delivery of therapeutic proteins into tissues and across the blood-brain barrier (BBB) is limited by the size and biochemical properties of the proteins. Efficient delivery across BBB is generally restricted to small, highly lipophilic molecules. However, in the last decades, several peptides that can pass cell membranes have been identified. It has been shown that these peptides are also capable of delivering large hydrophilic cargoes into cells and are therefore a powerful biological tool for transporting drugs across cell membranes and even into the brain. We designed and prepared a single-chain antibody fragment (scFvs), specific for the pathological form of the prion protein (PrP(Sc)), where a cell-penetrating peptide (CPP) was used as a linker between the two variable domains of the scFv. The intravenously administered recombinant scFv-CPP was successfully targeted to and delivered into mouse brain cells. Our single-chain antibody fragments are of special interest in view of possible therapeutic reagents design not only for prion diseases but also for other neurodegenerative diseases.

Murine monoclonal antibody V5B2 which specifically recognizes the pathogenic form of the prion protein represents a potentially valuable tool in diagnostics or therapy of prion diseases. As murine antibodies elicit immune response in human, only modified forms can be used for therapeutic applications. We humanized a single-chain V5B2 antibody using variable domain resurfacing approach guided by computer modelling. Design based on sequence alignments and computer modelling resulted in a humanized version bearing 13 mutations compared to initial murine scFv. The humanized scFv was expressed in a dedicated bacterial system and purified by metal-affinity chromatography. Unaltered binding affinity to the original antigen was demonstrated by ELISA and maintained binding specificity was proved by Western blotting and immunohistochemistry. Since monoclonal antibodies against prion protein can antagonize prion propagation, humanized scFv specific for the pathogenic form of the prion protein might become a potential therapeutic reagent.

Prion diseases are incurable neurodegenerative diseases that affect both humans and animals. The infectious agent is a pathogenic form of the prion protein that accumulates in brain as amyloids. Currently, there is neither cure nor reliable preclinical diagnostics on the market available. The growing number of reports shows that passive immunisation is one of the most promising strategies for prion disease therapy, where antibodies against prions may prevent and even cure the infection. Since antibodies are large molecules and, thus, might not be suitable for the therapy, different antibody fragments are a good alternative. Therefore, we have designed and prepared single-chain antibody fragments (scFvs) derived from the PrP(Sc)-specific murine monoclonal antibody V5B2. Using a new expression vector pMD204, we produced scFvs in two opposing chain orientations in the periplasm of Escherichia coli. Both recombinant antibody fragments retained the specificity of the parent antibody and one of these exhibited binding properties comparable to the corresponding murine Fab fragments with the affinity in nM range. Our monovalent antibody fragments are of special interest in view of possible therapeutic reagents for prion diseases as well as for development of a new generation of diagnostics.

In genetic engineering, gene expression is often modulated by replacements in promoter regions. Any deliberate intervention into the regulatory elements requires a subsequent evaluation based on analysis of reporter proteins. We have developed a new and rapid approach for characterization of promoter activity in which promoter strengths are determined by antibiotic resistance level. Values are expressed in comparison with those obtained from the reference promoter using the kanamycin resistance (aminoglycoside 3'-phosphotransferase) gene as a reporter. The new assay vector pSB1K0prom enables straightforward cloning of promoters or their subparts; therefore, mutations in different elements of the promoter region are easily introduced and analyzed. A series of promoters can be examined in parallel because no protein analysis is required other than determination of bacterial growth rates in the presence of increasing kanamycin concentrations. An internet application called PromCal for evaluation of experimental data has also been developed and is freely accessible at http://web.fkkt.uni-lj.si/biokemija/nskrlj/tools/PromCal.php.

We have developed an Escherichia coli expression vector that is particularly useful for construction and production of fusion proteins. Based on the synthetic biology pSB1C3 platform, the resulting vector offers a combination of useful features: the strong T7 promoter combined with lac operator, OmpA signal sequence, a selection of cloning sites located at convenient positions and a 3'-terminal His-10 tag. Each of these regions is flanked by a restriction site that allows for easy vector modification, including removal of the signal sequence without perturbation of the reading frame. All the elements were assembled by stepwise addition of three cassettes for which the design was made de novo. To prove the efficiency of the new vector, named pMD204, we successfully produced a cysteine proteinase inhibitor variant in the periplasm and in the cytoplasm of E. coli, in both cases as a soluble and active protein.

Cathepsin B (EC 3.4.22.1) and other cysteine proteases are synthesized as zymogens, which are processed to their mature forms autocatalytically or by other proteases. Autocatalytic processing was suggested to be a bimolecular process, whereas initiation of the processing has not yet been clarified. Procathepsin B was shown by zymography to hydrolyze the synthetic substrate 7-N-benzyloxycarbonyl-L-arginyl-L-arginylamide-4-methylcoumarin (Z-Arg-Arg-NH-MEC), suggesting that procathepsin B is catalytically active. The activity-based probe DCG-04, which is an E-64-type inhibitor, was found to label both mature cathepsin B and its zymogen, confirming the zymography data. Mutation analyses in the linker region between the propeptide and the mature part revealed that autocatalytic processing of procathepsin B is largely unaffected by mutations in this region, including mutations to prolines. On the basis of these results, a model for autocatalytic activation of cysteine cathepsins is proposed, involving propeptide dissociation from the active-site cleft as the first step during zymogen activation. This unimolecular conformational change is followed by a bimolecular proteolytic removal of the propeptide, which can be accomplished in one or more steps. Such activation, which can be also facilitated by glycosaminoglycans or by binding to negatively charged surfaces, may have important physiological consequences because cathepsin zymogens were often found secreted in various pathological states.

Understanding the molecular basis of ligand-DNA-binding events, and its application to the rational design of novel drugs, requires knowledge of the structural features and forces that drive the corresponding recognition processes. Existing structural evidence on DNA complexation with classical minor groove-directed ligands and the corresponding studies of binding energetics have suggested that this type of binding can be described as a rigid-body association. In contrast, we show here that the binding-coupled conformational changes may be crucial for the interpretation of DNA (hairpin) association with a classical minor groove binder (netropsin). We found that, although the hairpin form is the only accessible state of ligand-free DNA, its association with the ligand may lead to its transition into a duplex conformation. It appears that formation of the fully ligated duplex from the ligand-free hairpin, occurring via two pathways, is enthalpically driven and accompanied by a significant contribution of the hydrophobic effect. Our thermodynamic and structure-based analysis, together with corresponding theoretical studies, shows that none of the predicted binding steps can be considered as a rigid-body association. In this light we anticipate our thermodynamic approach to be the basis of more sophisticated nucleic acid recognition mechanisms, which take into account the dynamic nature of both the nucleic acid and the ligand molecule.

The genome of Trypanosoma cruzi, the Protozoan parasite causing the American Trypanosomiasis, Chagas disease, contains two genes, TcMCA3 and TcMCA5, with homology to those encoding metacaspases, distantly related to the caspases involved in programmed cell death (PCD) in higher eukaryotes. TcMCA3 is present in the CL Brener clone at 16 copies per haploid genome, arrayed in two tandems located in chromosomes of 0.54 and 0.98 Mbp. TcMCA5, on the other hand, is present as a single copy gene. The proteins encoded were expressed in Escherichia coli BL21 [DE3] cells, and used to generate antibodies, which allowed demonstrating that TcMCA3 is expressed in the four major developmental stages of the parasite, whereas TcMCA5 is expressed only in the epimastigote form. Moreover, recombinant TcMCA3, but not TcMCA5, was recognized by most sera from chronic Chagasic patients, showing that the protein is expressed during natural infections. All attempts to show processing and enzyme activity in the recombinant proteins have been unsuccessful so far; however, indirect evidence suggests that the metacaspases might be involved in PCD of the parasite. (1) Immunofluorescence experiments showed that both proteins change their subcellular localization during fresh human serum (FHS)-induced PCD migrating into the nucleus. (2) Epimastigotes over-expressing TcMCA5 were more sensitive to FHS-induced PCD than the controls. (3) PCD was parallelled by an increase in peptidase activity against Z-YVAD-AFC, a typical caspase substrate, and the apoptotic nuclei cells were labeled in vivo with the pan-caspase fluorescent inhibitor SR-VAD-FMK. Further experiments will be required to complete the characterization of these proteins and elucidate their role in the parasite.

Cathepsin S is unique among mammalian cysteine cathepsins in being active and stable at neutral pH. We show that autocatalytic activation of procathepsin S at low pH is a bimolecular process that is considerably accelerated (approximately 20-fold) by glycosaminoglycans and polysaccharides such as dextran sulfate, chondroitin sulfates A and E, and dermatan sulfate through electrostatic interaction with the proenzyme. Procathepsin S is also shown to undergo autoactivation at neutral pH in the presence of dextran sulfate with t1/2 of approximately 20 min at pH 7.5. This novel property of procathepsin S may have implications in pathological conditions associated with the appearance of active cathepsins outside lysosomes.

Human procathepsin H was expressed in the form of inclusion bodies in Escherichia coli. Following refolding and autocatalytic activation, a recombinant cathepsin H form lacking the mini chain was produced. Removal of the mini chain completely abolished aminopeptidase activity of the enzyme and largely increased its endopeptidase activity (approximately 40-fold). Similarly to cathepsin S, Bz-FVR-AMC (k(cat)/K(m) value of 1070 mM(-1) s(-1)) was found to be the preferred substrate of recombinant cathepsin H. However, substrate inhibition was observed at a higher substrate (Z-FR-AMC, Bz-FVR-AMC) concentration. Endopeptidase activity of recombinant cathepsin H was seen also with the protein substrate insulin beta-chain with the major cleavage site between Glu13-Ala14. Recombinant human cathepsin H was inhibited by chicken cystatin, stefin A, and stefin B with the K(i) values in the range of 0.05-0.1 nM, which is slightly tighter than the inhibition of purified cathepsin H by the same inhibitors. These results thus indicate that the cathepsin H mini chain is essential for the aminopeptidase activity of the enzyme but has only a minor effect on the inhibition by cystatins.

Thyroglobulin contains 11 repeats of a motif called thyroglobulin type-1 domain that show sequence similarity to some proteins exhibiting inhibitory activity against cysteine proteinases. Here we report that thyroglobulin decreases the activity of cathepsins B, H, L, and papain. To examine the possible involvement of particular type-1 domains in that decrease of activity, some individual thyroglobulin type-1 domains were expressed in E. coli. These recombinant domains proved to be substrates for cathepsins B, H, L, and papain instead of inhibitors. The cleavage points with cathepsins B and L on the second and the fourth domains were determined. The possible reasons for degradation are discussed.

Human procathepsin L has been expressed in E. coli in the form of inclusion bodies. The recombinant protein was isolated, refolded and processed at pH 5.5 by the addition of dextran sulfate which increased the overall yield of cathepsin L almost 10-fold. After the auto-activation of the 38 kDa procathepsin L at least three processing sites were determined by N-terminal amino acid sequencing. After replacing the Ala205 residue by glutamic acid, cathepsin B-like specificity was introduced into cathepsin L. This mutation resulted in a 15-fold increased activity toward the substrate Z-Arg-Arg-AMC and in a 29-fold decreased activity toward Z-Phe-Arg-AMC. Residue 205 is thereby confirmed experimentally to be critical for the specificity of cathepsins B and L.

A wild-type human procathepsin B was expressed, crystallized in two crystal forms and its crystal structure determined at 3.2 and 3.3 Angstroms resolution. The structure reveals that the propeptide folds on the cathepsin B surface, shielding the enzyme active site from exposure to solvent. The structure of the enzymatically active domains is virtually identical to that of the native enzyme [Musil et al. (1991) EMBO J. 10, 2321-2330]: the main difference is that the occluding loop residues are lifted above the body of the mature enzyme, supporting the propeptide structure.

Human procathepsin S was produced in the form of insoluble inclusion bodies in Escherichia coli using an inducible T7-based expression system. After cell disruption, the dissolved inclusion body proteins were S-sulphonated with 2-nitro-5-thiosulphobenzoate and purified by gel filtration. Recombinant procathepsin S was renatured at pH 7.6 by a two-step dilution which significantly increased the yield of production compared to single-step dilution. The proenzyme was autocatalytically processed to active cathepsin S at pH 4.5 in the presence of an excess of cysteine and catalytic amounts of dextran sulphate. Most of the loss of procathepsin S occurred during folding, probably because of aggregation. Concentrations lower than 20 microgram/ml of procathepsin S were necessary to minimise such aggregation. The recombinant cathepsin S was catalytically active on fluorogenic substrates and had kinetic properties similar to those of recombinant enzyme produced in yeast. The expression, renaturation, and activation procedures used enable the production of up to 2 mg of catalytically active recombinant human cathepsin S/l fermentation broth.

From human embrional lung fibroblasts mRNA was obtained and converted to cDNA. The procathepsin L coding region was amplified by PCR, inserted into pALTER and, after checking the nucleotide sequence, transferred into pET81F1+. Procathepsin L was expressed by induction of recombinant E. coli strain BL21[DE3](pLysS) with IPTG and was found to be deposited into inclusion bodies. These were isolated and solubilized in guanidinium hydrochloride. The soluble proteins were sulphonated and procathepsin L was obtained after gel filtration. Purified proenzyme was refolded by dialysis and autoactivated into a form of the expected size and enzymatic activity against a fluorogenic substrate.

A cDNA clone encoding human procathepsin B was expressed at a high level in Escherichia coli using a T7 polymerase expression system, resulting in the formation of insoluble cytoplasmic protein aggregates (inclusion bodies). The recombinant product was solubilized and renatured by refolding and reoxidation. The proenzyme was subsequently processed with pepsin to produce an enzymically active enzyme. By systematic variation of the parameters influencing the folding, formation of disulphide bonds, and processing of procathepsin B to the catalytically active mature form, a simple renaturation procedure was designed, allowing the production of about 3 mg purified active cathepsin B/l E. coli culture broth. The enzyme obtained in this way consists of a single chain and, as a consequence of pepsin treatment, possesses a three-amino-acid extension at its N-terminus. The enzyme has similar kinetic and immunological properties to native human cathepsin B.

We have isolated L- and H-kininogens from chicken egg white and plasma using affinity chromatography on CM-papain Sepharose, Con-A Sepharose, gel filtration and ion exchange chromatography, in pure form. In chicken plasma mostly HK was present, whereas in chicken egg white both LK and HK were identified. The determined Mr of LK and HK were 69000 and about 96000, respectively. They occurred in multiple forms with pI 4.3-5.2. The inhibitory activity of chicken HK was tested against cysteine proteinases cathepsins B and L, isolated from chicken liver, and papain. The obtained Ki values demonstrated that chicken H-kininogen is a strong inhibitor of chicken cathepsin L and papain, but much weaker inhibitor of chicken cathepsin B.

The (K15R M52E) aprotinin is a recombinant molecule with a broader inhibition spectrum against serine proteinases and a higher affinity towards certain proteinases as compared to native aprotinin. This aprotinin variant was produced in E. coli, isolated and purified to homogeneity. The inhibitor was further tested for its effectiveness to reduce human immunodeficiency virus type 1 (HIV-1) replication. Virus growth was followed by an ELISA which detects the amount of virus core protein p24. At a concentration of 50 microM, the recombinant (K15R M52E) aprotinin clearly reduced HIV-1 replication in H9 cells.

A synthetic master gene coding for rat trypstatin was designed, assembled of eight oligonucleotides, ligated into the cloning vector pUC8 and cloned in E. coli. In addition to the expected product DNA sequencing revealed the presence of several gene variants. The gene coding for (-1M F44G) rat trypstatin was mutated to a wild type form (-1M) rat trypstatin by cassette mutagenesis. For the first expression experiments the E. coli pEx31 system was used. After SDS-PAGE analysis fusion proteins were detected consisting of the N-terminal part of MS2-polymerase, a linker region and of the appropriate rat trypstatin variant. These fusion proteins were synthesized in amounts as high as 25% of total E. coli proteins.