Mapping the substrate-binding subsite specificity of a Porphyromonas gingivalis Tpr peptidase

Calcium-dependent peptidases of the calpain family are widespread in eukaryotes but uncommon in prokaryotes. A few bacterial calpain homologs have been discovered but none of them have been characterized in detail. Here we present an in-depth substrate specificity analysis of the bacterial calpain-like peptidase Tpr from Porphyromonas gingivalis. Using the positional scanning hybrid combinatorial substrate library method, we found that the specificity of Tpr peptidase differs substantially from the papain family of cysteine proteases, showing a strong preference for proline residues at positions P2 and P3. Such a degree of specificity indicates that this P. gingivalis cell-surface peptidase has a more sophisticated role than indiscriminate protein degradation to generate peptide nutrients, and may fulfil virulence-related functions such as immune evasion

Distribution of polycyclic aromatic hydrocarbons in coke plant wastewater

The subject of examinations presented in this paper is the distribution of polycyclic aromatic hydrocarbons (PAHs) between solid and liquid phases in samples of raw wastewater and wastewater after treatment. The content of 16 PAHs according to the US EPA was determined in the samples of coke plant wastewater from the Zdzieszowice Coke Plant, Poland. The samples contained raw wastewater, wastewater after physico-chemical treatment as well as after biological treatment. The ΣPHA16 content varied between 255.050 μg L−1 and 311.907 μg L−1 in raw wastewater and between 0.940 and 4.465 μg L−1 in wastewater after full treatment. Investigation of the distribution of PAHs showed that 71–84% of these compounds is adsorbed on the surface of suspended solids and 16–29% is dissolved in water. Distribution of individual PAHs and ΣPHA16 between solid phase and liquid phase was described with the use of statistically significant, linear equations. The calculated values of the partitioning coefficient Kp changed from 0.99 to 7.90 for naphthalene in samples containing mineral-organic suspension and acenaphthylene in samples with biological activated sludge, respectively.</jats:p

Reporter system for Human Coronavirus NL63

Wraz z rozwojem technik biologii molekularnej oraz hodowli komórkowej dostępne stają się coraz doskonalsze narzędzia do wykrywania, identyfikacji oraz ilościowego oznaczania zakażeń wirusowych. Do takich narzędzi należą systemy reporterowe wykorzystujące do detekcji reporter pozwalający na łatwą identyfikację fragmentów kwasów nukleinowych, białek lub procesów biochemicznych charakterystycznych dla danego patogenu. Zastosowanie systemów reporterowych ma na celu uproszczenie, skrócenie a tym samym obniżenie kosztów wykrywania patogenów.Ludzki koronawirus NL63 został odkryty stosunkowo niedawno i jest odpowiedzialny za duży odsetek przeziębień. Szczególnie ciężkie objawy przy zakażeniu występują u dzieci, osób starszych oraz osób z obniżoną odpornością, jednak objawy chorobowe stwierdza się również u dorosłych. Niektóre z zakażeń mają na tyle poważny przebieg, że wymagają hospitalizacji pacjenta, jak również zgłaszano przypadki śmiertelne związane z zakażeniem wirusem. Mimo intensywnych badań nad tym patogenem, mechanizm zakażenia nie został do końca zbadany. W niniejszej pracy przedstawiono badania mające na celu opracowanie systemu reporterowego dla infekcji ludzkim koronawirusem NL63. Podstawą działania proponowanego systemu reporterowego jest zastosowanie konstruktu genetycznego zawierającego gen reporterowy, który koduje mRNA ulegające translacji jedynie w przypadku obecności w komórce RNA-zależnej polimerazy RNA, kodowanej w genomie ludzkiego koronawirusa NL63.Along with development of molecular biology methods and cell culture techniques new tools for detection, identification and quantitative analysis of viral infections become available. These include reporter systems, which utilize pathogen specific markers such as genetic sequence or biochemical processes. Presence of such a marker in combination with effector molecule emitting an easy to quantify signal allows simplified detection of the pathogen and thus reduce cost of such an operation.Human coronavirus NL63 is associated with a significant number of common cold cases. Children and immunodeficient persons are most susceptible to HCoV-NL63 infections. In majority of cases infection with this virus is relatively mild, though severe cases requiring hospitalization are also common and fatal outcome has been reported. Despite its high prevalence and undoubtful association with clinical illness the mechanisms of infection have not yet been fully understood.In this work an attempt was made to create the reporter system for HCoV-NL63 infection. The system is based on susceptible cell line stably transfected with reporter genetic construct which can be translated only in presence of viral RNAdependent RNA polymerase

Structural dynamics influences the antibacterial activity of a cell-penetrating peptide (KFF)3K

Abstract Given the widespread demand for novel antibacterial agents, we modified a cell-penetrating peptide (KFF)3K to transform it into an antibacterial peptide. Namely, we inserted a hydrocarbon staple into the (KFF)3K sequence to induce and stabilize its membrane-active secondary structure. The staples were introduced at two positions, (KFF)3K[5–9] and (KFF)3K[2–6], to retain the initial amphipathic character of the unstapled peptide. The stapled analogues are protease resistant contrary to (KFF)3K; 90% of the stapled (KFF)3K[5–9] peptide remained undigested after incubation in chymotrypsin solution. The stapled peptides showed antibacterial activity (with minimal inhibitory concentrations in the range of 2–16 µM) against various Gram-positive and Gram-negative strains, contrary to unmodified (KFF)3K, which had no antibacterial effect against any strain at concentrations up to 32 µM. Also, both stapled peptides adopted an α-helical structure in the buffer and micellar environment, contrary to a mostly undefined structure of the unstapled (KFF)3K in the buffer. We found that the antibacterial activity of (KFF)3K analogues is related to their disruptive effect on cell membranes and we showed that by stapling this cell-penetrating peptide, we can induce its antibacterial character

Structural characterization of human coronavirus NL63 N protein

ABSTRACT Coronaviruses are responsible for upper and lower respiratory tract infections in humans. It is estimated that 1 to 10% of the population suffers annually from cold-like symptoms related to infection with human coronavirus NL63 (HCoV-NL63), an alphacoronavirus. The nucleocapsid (N) protein, the major structural component of the capsid, facilitates RNA packing, links the capsid to the envelope, and is also involved in multiple other processes, including viral replication and evasion of the immune system. Although the role of N protein in viral replication is relatively well described, no structural data are currently available regarding the N proteins of alphacoronaviruses. Moreover, our understanding of the mechanisms of RNA binding and nucleocapsid formation remains incomplete. In this study, we solved the crystal structures of the N- and C-terminal domains (NTD, residues 10 to 140, and CTD, residues 221 to 340, respectively) of the N protein of HCoV-NL63, both at a 1.5-Å resolution. Based on our structure of NTD solved here, we proposed and experimentally evaluated a model of RNA binding. The structure of the CTD reveals the mode of N protein dimerization. Overall, this study expands our understanding of the initial steps of N protein-nucleic acid interaction and may facilitate future efforts to control the associated infections. IMPORTANCE Coronaviruses are responsible for the common cold and other respiratory tract infections in humans. According to multiple studies, 1 to 10% of the population is infected each year with HCoV-NL63. Viruses are relatively simple organisms composed of a few proteins and the nucleic acids that carry the information determining their composition. The nucleocapsid (N) protein studied in this work protects the nucleic acid from the environmental factors during virus transmission. This study investigated the structural arrangement of N protein, explaining the first steps of its interaction with nucleic acid at the initial stages of virus structure assembly. The results expand our understanding of coronavirus physiology and may facilitate future efforts to control the associated infections. </jats:p