Crystal structures of human procathepsin B at 3.2 and 3.3 Å resolution reveal an interaction motif between a papain-like cysteine protease and its propeptide

AbstractA wild-type human procathepsin B was expressed, crystallized in two crystal forms and its crystal structure determined at 3.2 and 3.3 Å 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

Report on in-depth market research in the field of RFT

The main objective of the FUTURA project is to improve the quality and safety of life of over 55 million EU citizens who live or work near train tracks, and prepare our ambitious, close to market technical solution – Divided, axle mounted Rail Freight Brake Disc (further referred to as DRFB disc) for commercialisation, as this is a world novelty in the field of braking systems in rail freight transport (further referred to as RFT). With its innovative solution, FUTURA project will considerably influence the following 3 key elements: 1) safety and security, 2) health and environment and 3) cost-effectiveness in the RFT. Based on the market analysis, competitior analysis, face to face interviews with existing and potential customers and regulations we defined the most prospective markets and market segments for our new developed product – divided brake discs for freight

Zinc pyrithione is a potent inhibitor of PLPro and cathepsin L enzymes with ex vivo inhibition of SARS-CoV-2 entry and replication

SARS-CoV-2; Inhibition; ZincSARS-CoV-2; Inhibició; ZincSARS-CoV-2; Inhibición; ZincZinc pyrithione (1a), together with its analogues 1b–h and ruthenium pyrithione complex 2a, were synthesised and evaluated for the stability in biologically relevant media and anti-SARS-CoV-2 activity. Zinc pyrithione revealed potent in vitro inhibition of cathepsin L (IC50=1.88 ± 0.49 µM) and PLPro (IC50=0.50 ± 0.07 µM), enzymes involved in SARS-CoV-2 entry and replication, respectively, as well as antiviral entry and replication properties in an ex vivo system derived from primary human lung tissue. Zinc complexes 1b–h expressed comparable in vitro inhibition. On the contrary, ruthenium complex 2a and the ligand pyrithione a itself expressed poor inhibition in mentioned assays, indicating the importance of the selection of metal core and structure of metal complex for antiviral activity. Safe, effective, and preferably oral at-home therapeutics for COVID-19 are needed and as such zinc pyrithione, which is also commercially available, could be considered as a potential therapeutic agent against SARS-CoV-2.The authors acknowledge the financial support from the Slovenian Research Agency ARRS [Research Core Funding No. P1-0175 together with an increase in research programme funding related to the COVID-19 pandemic], and the grant from the Health Department of the Government of Catalonia [DGRIS 1_5] and the Fundació La Marató TV3 [Grants 202104FMTV3 and 202112FMTV3] to M.G. and MJ.B. MJ.B is supported by the Miguel Servet Program funded by the Spanish Health Institute Carlos III [CP17/00179]

What drives the binding of minor groove-directed ligands to DNA hairpins?

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

Specific Binding of the Pathogenic Prion Isoform: Development and Characterization of a Humanized Single-Chain Variable Antibody Fragment

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

Slomšek\u27s understanding of Christian unity: his sermon »On Christian concordance and Unity in faith«

V magistrskem delu z naslovom Razumevanje edinosti kristjanov blaženega Antona Martina Slomška avtor predstavi njegovo podobo z vidika prizadevanja za zedinjenje kristjanov, in sicer na podlagi njegovih del, organizacijskega dela in zlasti pridige »Od krščanske sloge in edinosti v veri«. Namen dela je pokazati, da je Slomškovo delo za edinost ukoreninjeno v teološkem izročilu, iz katerega lahko črpamo tudi danes. Naloga je razdeljena na tri dele. V 1. poglavju avtor predstavi življenje blaženega škofa Antona Martina Slomška in tako osvetlitvi njegovo svetniško osebnost, ki jo lahko motrimo predvsem v ljubezni do svete Cerkve. Naloga se nato osredotoči na Slomškove zasluge zanjo. V tem odseku avtor izpostavi pomembnost ustanovitve Bratovščine (Apostolstva) svetega Cirila in Metoda, ki velja za temeljni kamen vsega nadaljnjega dela za edinost na Slovenskem in širše. V 2. poglavju sledi analiza Slomškove pridige »Od krščanske sloge in edinosti v veri«, ki je bila objavljena leta 1850 v knjigi Apostolska hrana bogoljubnim dušam dana. Pri tem avtor izpostavi Slomškove pridonos na področju pridigarstva in opiše značilnosti dotične literarne vrste. Na koncu tega dela je povzetek pridige in njen prepis. V 3. poglavju avtor predstavi tri ključne teološke poudarke, ki jih škof Slomšek dojema kot temeljne značilnosti edinosti. Prvo je prizadevanje za edinost oz. edinost kot temeljna značilnost katoliškega verovanja. Drugo je zavezanost ali zvestoba svetemu izročilu, ki je pogoj edinosti v nauku. Tretja je razumevanje Cerkve kot Kristusovega mističnega telesa. V vsakem podpoglavju sledi predstavitev najpomembnejših odlomkov iz obravnavane pridige, v katerih se zrcali Slomškovo razumevanje posameznega teološkega vidika, ki je nato umeščen v širše ozadje. Na ta način avtor pokaže na kontinuiteto poudarka v celotni zgodovini Cerkve, začenši s svetopisemskim razumevanjem, ki ga osvetli učenje cerkvenih očetov. Na koncu vsakega poglavja je vsebina povzeta in nadgrajena s teološkimi razlagami cerkvenega učiteljstva. Zadnji del naloge je sinteza Slomškovega razumevanja krščanske edinosti.In his master\u27s thesis entitled Slomšek\u27s understanding of Christian Unity and his sermon »On Christian Concordance and Unity in Faith«, the author presents the blessed\u27s image from the point of view of his efforts to unify Christians, namely on the basis of his works, organizational activity and especially the sermon »On Christian Concordance and Unity in Faith«. The purpose of the work is to show that Slomšek\u27s work for unity is rooted in a deep theological tradition, from which we can still draw today. The thesis is divided into three parts. In the 1st chapter, the author presents the life of Blessed Bishop Anton M. Slomšek, shedding light on his saintly personality, which can be primarily observed in his love for the Holy Catholic Church. The thesis then focuses on Slomšek\u27s merits. In this section, the author highlights the importance of founding the Fraternity (Apostolate) of Saints Cyril and Methodius, which is considered the cornerstone of all further work for unity (and later ecumenism) in Slovenia and beyond. In the 2nd chapter, follows an analysis of Slomšek\u27s sermon »On Christian Concordance and Unity in Faith«, which was published in the book Apostolic Food for Pious Souls in 1850. Here the author points out Slomšek\u27s contributions in the field of homiletics and describes the characteristics of the respective literary genre. At the end of this part is a summary of the sermon and its transcript. In the 3rd chapter, the author presents three key theological emphases, which Bishop Slomšek perceives as the foundations of unity. The first is the commitment for unity or unity as a fundamental characteristic of the Catholic faith. The second is commitment or fidelity to the Sacred Tradition, which is a condition for doctrinal unity. The third is the understanding of the Church as Christ\u27s Mystical Body. Each part is followed by a presentation of most important passages from the above considered sermon, which reflect Slomšek\u27s understanding of a particular theological aspect. The latter is then placed in a wider background. In this way, the author shows the continuity of a emphases throughout the history of the Church, beginning with the biblical understanding and than illuminated by the teaching of Church Fathers. At the end of each part, the content is summarized and enriched by theological explanations of the Church\u27s Magisterium. The last part is a synthesis of Slomšek\u27s understanding of Christian unity

Obiski Rosalind Franklin v Sloveniji - Visits of Rosalind Franklin to Slovenia

The world-renowned structural chemist and biochemist Rosalind Franklin (1920-1958) maintained contacts with Slovenian and Croatian chemists and visited both countries on several occasions. On the basis of biographies and archived correspondence, this article summarises her visits to Slovenia and other parts of the former Yugoslavia. Interestingly, I discovered that the most famous mountaineering photograph of Rosalind Franklin was not taken on her family vacation in Norway in the late 1930s as generally believed, but on the Vršič Pass in Julian Alps, as the vegetation and rock structure, especially the so-called Heathen Maiden in the northwest face of Mount Prisojnik, show. It is likely that the photo was taken by her Slovenian colleague Dušan Hadži, probably in May 1952, so the photo was obviously wrongly labelled in the Franklin family archives. In addition, her mountaineering trip to Mt. Triglav and her stay at Lake Bled could not have happened in 1952 as mentioned in her biography, but rather in 1954 or 1955

A chimeric vector for dual use in cyanobacteria and Escherichia coli, tested with cystatin, a nonfluorescent reporter protein

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