The Rab-binding profiles of bacterial virulence factors during infection

Legionella pneumophila, the causative agent of Legionnaire's disease, uses its type IV secretion system to translocate over 300 effector proteins into host cells. These effectors subvert host cell signaling pathways to ensure bacterial proliferation. Despite their importance for pathogenesis, the roles of most of the effectors are yet to be characterized. Key to understanding the function of effectors is the identification of host proteins they bind during infection. We previously developed a novel tandem-affinity purification (TAP) approach using hexahistidine and BirA-specific biotinylation tags for isolating translocated effector complexes from infected cells whose composition were subsequently deciphered by mass spectrometry. Here we further advanced the workflow for the TAP approach and determined the infection-dependent interactomes of the effectors SidM and LidA, which were previously reported to promiscuously bind multiple Rab GTPases in vitro. In this study we defined a stringent subset of Rab GTPases targeted by SidM and LidA during infection, comprising of Rab1A, 1B, 6, and 10; in addition, LidA targets Rab14 and 18. Taken together, this study illustrates the power of this approach to profile the intracellular interactomes of bacterial effectors during infection

Inhibition of peptide aggregation by means of enzymatic phosphorylation

As is the case in numerous natural processes, enzymatic phosphorylation can be used in the laboratory to influence the conformational populations of proteins. In nature, this information is used for signal transduction or energy transfer, but has also been shown to play an important role in many diseases like tauopathies or diabetes. With the goal of determining the effect of phosphorylation on amyloid fibril formation, we designed a model peptide which combines structural characteristics of α-helical coiled-coils and β-sheets in one sequence. This peptide undergoes a conformational transition from soluble structures into insoluble amyloid fibrils over time and under physiological conditions and contains a recognition motif for PKA (cAMP- dependent protein kinase) that enables enzymatic phosphorylation. We have analyzed the pathway of amyloid formation and the influence of enzymatic phosphorylation on the different states along the conformational transition from random-coil to β-sheet-rich oligomers to protofilaments and on to insoluble amyloid fibrils, and we found a remarkable directing effect from β -sheet-rich structures to unfolded structures in the initial growth phase, in which small oligomers and protofilaments prevail if the peptide is phosphorylated

Characterisation of the Toxoplasma gondii tyrosine transporter and its phosphorylation by the calcium-dependent protein kinase 3.

Toxoplasma gondii parasites rapidly exit their host cell when exposed to calcium ionophores. Calcium-dependent protein kinase 3 (TgCDPK3) was previously identified as a key mediator in this process, as TgCDPK3 knockout (∆cdpk3) parasites fail to egress in a timely manner. Phosphoproteomic analysis comparing WT with ∆cdpk3 parasites revealed changes in the TgCDPK3-dependent phosphoproteome that included proteins important for regulating motility, but also metabolic enzymes, indicating that TgCDPK3 controls processes beyond egress. Here we have investigated a predicted direct target of TgCDPK3, ApiAT5-3, a putative transporter of the major facilitator superfamily, and show that it is rapidly phosphorylated at serine 56 after induction of calcium signalling. Conditional knockout of apiAT5-3 results in transcriptional upregulation of most ribosomal subunits, but no alternative transporters, and subsequent parasite death. Mutating the S56 to a non-phosphorylatable alanine leads to a fitness cost, suggesting that phosphorylation of this residue is beneficial, albeit not essential, for tyrosine import. Using a combination of metabolomics and heterologous expression, we confirmed a primary role in tyrosine import for ApiAT5-3. However, no significant differences in tyrosine import could be detected in phosphorylation site mutants showing that if tyrosine transport is affected by S56 phosphorylation, its regulatory role is subtle

Chemical proteomics approaches for identifying the cellular targets of natural products.

Covering: 2010 up to 2016. Deconvoluting the mode of action of natural products and drugs remains one of the biggest challenges in chemistry and biology today. Chemical proteomics is a growing area of chemical biology that seeks to design small molecule probes to understand protein function. In the context of natural products, chemical proteomics can be used to identify the protein binding partners or targets of small molecules in live cells. Here, we highlight recent examples of chemical probes based on natural products and their application for target identification. The review focuses on probes that can be covalently linked to their target proteins (either via intrinsic chemical reactivity or via the introduction of photocrosslinkers), and can be applied "in situ" - in living systems rather than cell lysates. We also focus here on strategies that employ a click reaction, the copper-catalysed azide-alkyne cycloaddition reaction (CuAAC), to allow minimal functionalisation of natural product scaffolds with an alkyne or azide tag. We also discuss 'competitive mode' approaches that screen for natural products that compete with a well-characterised chemical probe for binding to a particular set of protein targets. Fuelled by advances in mass spectrometry instrumentation and bioinformatics, many modern strategies are now embracing quantitative proteomics to help define the true interacting partners of probes, and we highlight the opportunities this rapidly evolving technology provides in chemical proteomics. Finally, some of the limitations and challenges of chemical proteomics approaches are discussed

Recurrent syncope and hypocalcaemic cardiomyopathy as manifestations of Fahr's syndrome

In our report we would like to present a case of a 60-year-old patient with epileptic seizures, affective disturbances, only mild neurocognitive disorders and cardiomyopathy. A female patient was taken to the internal ward with a tentative diagnosis of recurrent syncope. Laboratory results disclosed severe hypocalcaemia, hypoparathyroidism, and hypothyroidism. An echocardiogram revealed left ventricle systolic dysfunction. Computed tomography revealed massive intracranial calcifications typical for Fahr's syndrome. Our patient demonstrated only mild neurological and psychiatric symptoms, but developed hypocalcaemic heart failure. It is possible that some cases of Fahr's syndrome remain undiscovered, particularly patients taken to internal wards with mild neurological or psychiatric signs

ER-export and ARFRP1/AP-1-dependent delivery of SARS-CoV-2 Envelope to lysosomes controls late stages of viral replication

The β-coronavirus Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) is the causative agent of the global Covid-19 pandemic. Coronaviral Envelope (E) proteins are pentameric viroporins that play essential roles in assembly, release and pathogenesis. We developed a non-disruptive tagging strategy for SARS-CoV-2 E and find that at steady-state, it localises to the Golgi and to lysosomes. We identify sequences in E, conserved across Coronaviridae, responsible for Endoplasmic Reticulum (ER)-to-Golgi export, and relate this activity to interaction with COP-II via SEC24. Using proximity biotinylation, we identify an ADP Ribosylation Factor-1/Adaptor Protein-1 (ARFRP1/AP-1) dependent pathway allowing Golgi-to-lysosome trafficking of E. We identify sequences in E that bind AP-1, are conserved across β-coronaviruses and allow E to be trafficked from Golgi to lysosomes. We show that E acts to deacidify lysosomes and by developing a trans-complementation assay for SARS-CoV-2 structural proteins, we show that lysosomal delivery of E and its viroporin activity are necessary for efficient viral replication and release

Pruritus in dialysis patients — ethiopathogenesis, clinical aspects and treatment

Świąd mocznicowy jest powszechnym powikłaniem u chorych poddawanych dializie. Dolegliwość obniża jakość życia chorych, może również pogarszać rokowanie związane z przeżyciem. Mechanizmy patofizjologiczne świądu u chorych dializowanych z powodu schyłkowej niewydolności nerek pozostają nieznane. Rozważa się rogowacenie i nadmierną suchość skóry, zaburzenia immunologiczne, zaburzenia systemu endogennych opioidów, neuropeptydów, równowagi elektrolitów, jonów dwuwartościowych, wtórną nadczynność przytarczyc, niedokrwistość z niedoboru żelaza oraz zmiany pH skóry. W związku z różnorodnością możliwych przyczyn brak również metod leczenia przyczynowego. W pracy dokonano przeglądu dyskutowanych czynników patogenetycznych świądu mocznicowego u chorych z przewlekłą niewydolnością nerek oraz podejmowanych prób terapeutycznych.Uraemic pruritus is a common complication in dialysis patients which reduces the quality of life and increases the mortality. The ethiopathogenesis of uremic pruritus has not been well explained. Several explanations for ureamic pruritus have been proposed like keratosis, xerosis, dysfunction of immune system, dysfunction of endogenous opioid system, nueropeptide disturbances, water-electrolyte imbalance, dysfunction of divalent cations metabolism, iron-deficiency anaemia and skin surface pH changes. Because of complex pathomechanism there is no known causal treatment of uraemic pruritus. The paper synthesizes the current data regarding ethiopathogenesis and treatment of uraemic pruritus

Validation of N-myristoyltransferase as an antimalarial drug target using an integrated chemical biology approach

Malaria is an infectious disease caused by parasites of the genus Plasmodium, which leads to approximately one million deaths per annum worldwide. Chemical validation of new antimalarial targets is urgently required in view of rising resistance to current drugs. One such putative target is the enzyme N-myristoyltransferase, which catalyses the attachment of the fatty acid myristate to protein substrates (N-myristoylation). Here, we report an integrated chemical biology approach to explore protein myristoylation in the major human parasite P. falciparum, combining chemical proteomic tools for identification of the myristoylated and glycosylphosphatidylinositol-anchored proteome with selective small-molecule N-myristoyltransferase inhibitors. We demonstrate that N-myristoyltransferase is an essential and chemically tractable target in malaria parasites both in vitro and in vivo, and show that selective inhibition of N-myristoylation leads to catastrophic and irreversible failure to assemble the inner membrane complex, a critical subcellular organelle in the parasite life cycle. Our studies provide the basis for the development of new antimalarials targeting N-myristoyltransferase

Izolacija, biokemijske značajke i identifikacija termotolerantnih i celulolitičkih bakterija Paenibacillus lactis i Bacillus licheniformis

Research background. Cellulose is an ingredient of waste materials that can be converted to other valuable substances. This is possible provided that the polymer molecule is degraded to smaller particles and used as a carbon source by microorganisms. Because of the frequently applied methods of pretreatment of lignocellulosic materials, the cellulases derived from thermophilic microorganisms are particularly desirable. Experimental approach. We were looking for cellulolytic microorganisms able to grow at 50 °C and we described their morphological features and biochemical characteristics based on carboxymethyl cellulase (CMCase) activity and the API® ZYM system. The growth curves during incubation at 50 °C were examined using the BioLector® microbioreactor. Results and conclusions. Forty bacterial strains were isolated from fermenting hay, geothermal karst spring, hot spring and geothermal pond at 50 °C. The vast majority of the bacteria were Gram-positive and rod-shaped with the maximum growth temperature of at least 50 °C. We also demonstrated a large diversity of biochemical characteristics among the microorganisms. The CMCase activity was confirmed in 27 strains. Hydrolysis capacities were significant in bacterial strains: BBLN1, BSO6, BSO10, BSO13 and BSO14, and reached 2.74, 1.62, 1.30, 1.38 and 8.02 respectively. Rapid and stable growth was observed, among others, for BBLN1, BSO10, BSO13 and BSO14. The strains fulfilled the selection conditions and were identified based on the 16S rDNA sequences. BBLN1, BSO10, BSO13 were classified as Bacillus licheniformis, whereas BSO14 as Paenibacillus lactis. Novelty and scientific contribution. We described cellulolytic activity and biochemical characteristics of many bacteria isolated from hot environments. We are also the first to report the cellulolytic activity of thermotolerant P. lactis. Described strains can be a source of new thermostable cellulases, which are extremely desirable in various branches of circular bioeconomy.Pozadina istraživanja. Celuloza je sastojak otpadnih tvari koji se može preraditi u korisne spojeve. To je moguće ako se molekule polimera razgrade na manje čestice koje mikroorganizmi mogu iskoristiti kao izvor ugljika. Celulaze proizvedene s pomoću termofilnih mikroorganizama su naročito zanimljive zbog njihove učestale primjene u prethodnoj obradi lignoceluloznog materijala. Eksperimentalni pristup. Istražili smo celulolitičke mikroorganizme koji rastu na 50 °C te opisali njihove morfološke i biokemijske značajke na osnovi aktivnosti karboksimetil celulaze i ostalih enzima ispitanih sustavom API® ZYM. Krivulje rasta tijekom inkubacije pri 50 °C ispitane su uzgojem bakterija u mikrobioreaktoru BioLector®. Rezultati i zaključci. Četrdeset sojeva bakterija izolirano je iz fermentirane slame, geotermičkog izvora u kršu, termalnog vrela i geotermalnog jezera s temperaturom od 50 °C. Većina bakterija bile su Gram-pozitivne i štapićastog oblika, a najviša temperatura pri kojoj su rasle je iznosila najmanje 50 °C. Također smo potvrdili veliku raznolikost biokemijskih značajki među ispitanim mikroorganizmima. Aktivnost karboksimetil celulaze potvrđena je u 27 sojeva. Sojevi bakterija sa značajnim hidrolitičkim kapacitetom bili su: BBLN1 s 2,74; BSO6 s 1,62; BSO10 s 1,30; BSO13 s 1.38 i BSO14 s kapacitetom od 8,02. Sojevi BBLN1, BSO10, BSO13 i BSO14 rasli su brzo i stabilno. Ispunjavali su odabrane preduvjete pa su identificirani 16S rDNA sekvenciranjem. Sojevi BBLN1, BSO10 i BSO13 klasificirani su kao sojevi bakterije Bacillus licheniformis, dok je soj BSO14 potvrđen kao soj Paenibacillus lactis. Novina i znanstveni doprinos. Opisali smo celulolitičku aktivnost i biokemijska svojstva velikog broja bakterija izoliranih iz lokacija gdje vladaju velike vrućine. Prvi smo izvijestili o celulolitičkoj aktivnosti termotolerantne bakterije P. lactis. Opisani bi sojevi mogli biti izvor novih termički stabilnih celulaza, što je izuzetno poželjno u različitim granama cirkularne bioekonomije

<i>Aronia melanocarpa Elliot</i>Reduces the Activity of Angiotensin I-Converting Enzyme—<i>In Vitro</i>and<i>Ex Vivo</i>Studies

Purpose. The aim of the study was to analyze the effects of two-month supplementation with chokeberry preparation on the activity of angiotensin I-converting enzyme (ACE) in patients with metabolic syndrome (MS). During thein vitrostage of the study, we determined the concentration of chokeberry extract, which inhibited the activity of ACE by 50% (IC50).Methods. The participants (n=70) were divided into three groups: I—patients with MS who received chokeberry extract supplements, II—healthy controls, and III—patients with MS treated with ACE inhibitors.Results. After one and two months of the experiment, a decrease in ACE activity corresponded to 25% and 30%, respectively. We documented significant positive correlations between the ACE activity and the systolic (r=0.459,P=0.048) and diastolic blood pressure, (r=0.603,P=0.005) and CRP. The IC50of chokeberry extract and captopril amounted to155.4±12.1 μg/mL and0.52±0.18 μg/mL, respectively.Conclusions. Ourin vitrostudy revealed that chokeberry extract is a relatively weak ACE inhibitor. However, the results of clinical observations suggest that the favorable hypotensive action of chokeberry polyphenols may be an outcome of both ACE inhibition and other pleotropic effects, for example, antioxidative effect.</jats:p