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Fluorescence-based assay for polyprenyl phosphate-GlcNAc-1-phosphate transferase (WecA) and identification of novel antimycobacterial WecA inhibitors.
Get PDFPolyprenyl phosphate-GlcNAc-1-phosphate transferase (WecA) is an essential enzyme for the growth of Mycobacterium tuberculosis (Mtb) and some other bacteria. Mtb WecA catalyzes the transformation from UDP-GlcNAc to decaprenyl-P-P-GlcNAc, the first membrane-anchored glycophospholipid that is responsible for the biosynthesis of mycolylarabinogalactan in Mtb. Inhibition of WecA will block the entire biosynthesis of essential cell wall components of Mtb in both replicating and non-replicating
states, making this enzyme a target for development of novel drugs. Here, we report a fluorescencebased method for the assay of WecA using a modified UDP-GlcNAc, UDP-Glucosamine-C6-FITC (1), a membrane fraction prepared from an M. smegmatis strain, and the E. coli B21WecA. Under the optimized conditions, UDP-Glucosamine-C6-FITC (1) can be converted to the corresponding decaprenyl-P-P Glucosamine-C6-FITC (3) in 61.5% yield. Decaprenyl-P-P-Glucosamine-C6-FITC is readily extracted with nbutanol
and can be quantified by ultravioletevisible (UV-vis) spectrometry. Screening of the compound libraries designed for bacterial phosphotransferases resulted in the discovery of a selective WecA inhibitor, UT-01320 (12) that kills both replicating and non-replicating Mtb at low concentration. UT-01320 (12) also kills the intracellular Mtb in macrophages. We conclude that the WecA assay reported here is amenable to medium- and high-throughput screening, thus facilitating the discovery of novel WecA
inhibitors
The impact of β-azido(or 1-piperidinyl) methylamino acids in position 2 or 3 on biological activity and conformation of dermorphin analogues
Get PDFThe synthesis of new dermorphin analogues is described. The (R)-alanine or phenylalanine residues of natural dermorphin were
substituted by the corresponding α-methyl-β-azidoalanine or α-benzyl-β-azido(1-piperidinyl)alanine residues. The potency and
selectivity of the newanalogueswere evaluated by a competitive receptor binding assay in rat brain using [3H]DAMGO (a μ ligand)
and [3H]DELT (a δ ligand). The most active analogue in this series, Tyr-(R)-Ala-(R)-α-benzyl-β-azidoAla-Gly-Tyr-Pro-Ser-NH2 and its
epimer were analysed by 1H and 13C NMR spectroscopy and restrainedmolecular dynamics simulations. The dominant conformation
of the investigated peptides depended on the absolute configuration around Cα in the α-benzyl-β-azidoAla residue in position
3. The (R) configuration led to the formation of a type I β-turn, whilst switching to the (S) configuration gave rise to an inverse β-
turn of type I′, followed by the formation of a very short β-sheet. The selectivity of Tyr-(R)-Ala-(R) and (S)-α-benzyl-β-azidoAla-Gly-
Tyr-Pro-Ser-NH2 was shown to be very similar; nevertheless, the two analogues exhibited different conformational preferences
Identification of G8969>A in mitochondrial ATP6 gene that severely compromises ATP synthase function in a patient with IgA nephropathy
Get PDFHere we elucidated the pathogenesis of a 14-year-old Chinese female who initially developed an
isolated nephropathy followed by a complex clinical presentation with brain and muscle problems,
which indicated that the disease process was possibly due to a mitochondrial dysfunction. Careful
evaluation of renal biopsy samples revealed a decreased staining of cells induced by COX and
NADH dehydrogenase activities, and a strong fragmentation of the mitochondrial network. These
anomalies were due to the presence of a mutation in the mitochondrial ATP6 gene, G8969>A. This
mutation leads to replacement of a highly conserved serine residue at position 148 of the a-subunit
of ATP synthase. Increasing the mutation load in cybrid cell lines was paralleled by the appearance of
abnormal mitochondrial morphologies, diminished respiration and enhanced production of reactive
oxygen species. An equivalent of the G8969>A mutation in yeast had dramatic consequences on ATP
synthase, with a block in proton translocation. The mutation was particularly abundant (89%) in the
kidney compared to blood and urine, which is likely the reason why this organ was affected first. Based
on these findings, we suggest that nephrologists should pay more attention to the possibility of a
mitochondrial dysfunction when evaluating patients suffering from kidney problems
TRANSAUTOPHAGY: European network for multidisciplinary research and translation of autophagy knowledge
Get PDFA collaborative consortium, named "TRANSAUTOPHAGY," has been created among European research groups, comprising more than 150 scientists from 21 countries studying diverse branches of basic and translational autophagy. The consortium was approved in the framework of the Horizon 2020 Program in November 2015 as a COST Action of the European Union (COST means: CO-operation in Science and Technology), and will be sponsored for 4 years. TRANSAUTOPHAGY will form an interdisciplinary platform for basic and translational researchers, enterprises and stakeholders of diverse disciplines (including nanotechnology, bioinformatics, physics, chemistry, biology and various medical disciplines). TRANSAUTOPHAGY will establish 5 different thematic working groups, formulated to cooperate in research projects, share ideas, and results through workshops, meetings and short term exchanges of personnel (among other initiatives). TRANSAUTOPHAGY aims to generate breakthrough multidisciplinary knowledge about autophagy regulation, and to boost translation of this knowledge into biomedical and biotechnological applications
Interplay between Structure and Charge as a Key to Allosteric Modulation of Human 20S Proteasome by the Basic Fragment of HIV-1 Tat Protein
Get PDFThe proteasome is a giant protease responsible for degradation of themajority of cytosolic
proteins. Competitive inhibitors of the proteasome are used against aggressive blood cancers.
However, broadening the use of proteasome-targeting drugs requires new mechanistic
approaches to the enzyme’s inhibition. In our previous studies we described Tat1 peptide, an
allosteric inhibitor of the proteasome derived from a fragment of the basic domain of HIV-Tat1
protein. Here, we attempted to dissect the structural determinants of the proteasome inhibition
by Tat1. Single- and multiple- alanine walking scans were performed. Tat1 analogs with stabilized
beta-turn conformation at positions 4–5 and 8–9, pointed out by the molecular dynamics
modeling and the alanine scan, were synthesized. Structure of Tat1 analogs were analyzed
by circular dichroism, Fourier transform infrared and nuclear magnetic resonance spectroscopy
studies, supplemented by molecular dynamics simulations. Biological activity tests and
structural studies revealed that high flexibility and exposed positive charge are hallmarks of
Tat1 peptide. Interestingly, stabilization of a beta-turn at the 8–9 position was necessary to
significantly improve the inhibitory potency
Newly identified protein Imi1 affects mitochondrial integrity and glutathione homeostasis in Saccharomyces cerevisiae
Get PDFGlutathione homeostasis is crucial for cell functioning. We describe a novel Imi1 protein of Saccharomyces cerevisiae affecting mitochondrial integrity and involved in controlling glutathione level. Imi1 is cytoplasmic and, except for its N-terminal Flo11 domain, has a distinct solenoid structure. A lack of Imi1 leads to mitochondrial lesions comprising aberrant morphology of cristae and multifarious mtDNA rearrangements and impaired respiration. The mitochondrial malfunctioning is coupled to significantly decrease of the level of intracellular reduced glutathione without affecting oxidized glutathione, which decreases the reduced/oxidized glutathione ratio. These defects are accompanied by decreased cadmium sensitivity and increased phytochelatin-2 level
TFIIS-Dependent Non-coding Transcription Regulates Developmental Genome Rearrangements.
Get PDFBecause of their nuclear dimorphism, ciliates provide a unique opportunity to study the role of non-coding RNAs (ncRNAs) in the communication between germline and somatic lineages. In these unicellular eukaryotes, a new somatic nucleus develops at each sexual cycle from a copy of the zygotic (germline) nucleus, while the old somatic nucleus degenerates. In the ciliate Paramecium tetraurelia, the genome is massively rearranged during this process through the reproducible elimination of repeated sequences and the precise excision of over 45,000 short, single-copy Internal Eliminated Sequences (IESs). Different types of ncRNAs resulting from genome-wide transcription were shown to be involved in the epigenetic regulation of genome rearrangements. To understand how ncRNAs are produced from the entire genome, we have focused on a homolog of the TFIIS elongation factor, which regulates RNA polymerase II transcriptional pausing. Six TFIIS-paralogs, representing four distinct families, can be found in P. tetraurelia genome. Using RNA interference, we showed that TFIIS4, which encodes a development-specific TFIIS protein, is essential for the formation of a functional somatic genome. Molecular analyses and high-throughput DNA sequencing upon TFIIS4 RNAi demonstrated that TFIIS4 is involved in all kinds of genome rearrangements, including excision of ~48% of IESs. Localization of a GFP-TFIIS4 fusion revealed that TFIIS4 appears specifically in the new somatic nucleus at an early developmental stage, before IES excision. RT-PCR experiments showed that TFIIS4 is necessary for the synthesis of IES-containing non-coding transcripts. We propose that these IES+ transcripts originate from the developing somatic nucleus and serve as pairing substrates for germline-specific short RNAs that target elimination of their homologous sequences. Our study, therefore, connects the onset of zygotic non coding transcription to the control of genome plasticity in Paramecium, and establishes for the first time a specific role of TFIIS in non-coding transcription in eukaryotes
The steady-state level and stability of TLS polymerase eta are cell cycle dependent in the yeast S. cerevisiae.
Get PDFPolymerase eta (Pol eta) is a ubiquitous translesion DNA polymerase that is capable of bypassing UV-induced pyrimidine dimers in an error-free manner. However, this specialized polymerase is error prone when synthesizing through an undamaged DNA template. In S. cerevisiae, both depletion and overproduction of Pol eta result in mutator phenotypes. Therefore, regulation of the cellular abundance of this enzyme is of particular interest. However, based on the investigation of variously tagged forms of Pol eta, mutually contradictory conclusions have been reached regarding the stability of this polymerase in yeast. Here, we optimized a protocol for the detection of untagged yeast Pol eta and established that the half-life of the native enzyme is 80±14 min in asynchronously growing cultures. Experiments with synchronized cells indicated that the cellular abundance of this translesion polymerase changes throughout the cell cycle. Accordingly, we show that the stability of Pol eta, but not its mRNA level, is cell cycle stage dependent. The half-life of the polymerase is more than 4-fold shorter in G1-arrested cells than in those at G2/M. Our results, in concert with previous data for Rev1, indicate that cell cycle regulation is a general property of Y family TLS polymerases in S. cerevisiae
Yeast as a system for modeling mitochondrial disease mechanisms and discovering therapies
Get PDFMitochondrial diseases are severe and largely untreatable. Owing to
the many essential processes carried out by mitochondria and the
complex cellular systems that support these processes, these
diseases are diverse, pleiotropic, and challenging to study. Much of
our current understanding of mitochondrial function and dysfunction
comes from studies in the baker’s yeast Saccharomyces cerevisiae.
Because of its good fermenting capacity, S. cerevisiae can survive
mutations that inactivate oxidative phosphorylation, has the ability to
tolerate the complete loss of mitochondrial DNA (a property referred
to as ‘petite-positivity’), and is amenable to mitochondrial and nuclear
genome manipulation. These attributes make it an excellent model
system for studying and resolving the molecular basis of numerous
mitochondrial diseases. Here, we review the invaluable insights this
model organism has yielded about diseases caused by mitochondrial
dysfunction, which ranges from primary defects in oxidative
phosphorylation to metabolic disorders, as well as dysfunctions in
maintaining the genome or in the dynamics of mitochondria. Owing to
the high level of functional conservation between yeast and human
mitochondrial genes, several yeast species have been instrumental in
revealing the molecular mechanisms of pathogenic human
mitochondrial gene mutations. Importantly, such insights have
pointed to potential therapeutic targets, as have genetic and
chemical screens using yeast
Characterization of Polish Phytophthora lacustris Isolates Obtained from Water Environments
Get PDFP. lacustris sp. nov. (formerly known as Phytophthora taxon Salixsoil) was first isolated in 1972 in the UK and then in many other European countries, including Poland. The aim of this work was a morphological, physiological, and genetic characterization of the P. lacustris isolates by means of the daily growth rate, mycelium morphology, and generative and vegetative structures, depending on the temperature of incubation and growth media. In addition, the ability to colonize willow shoots and leaves was estimated.
Out of 114 isolates of P. lacustris obtained from water habitats located near plant nurseries in central and southeastern Poland in 2007-10 that were identified on the basis of molecular tests which showed high diversity in colony growth patterns and daily growth rates, 10 groups were separated by means of Duncan’s test. Representatives of these 10 groups together with three reference isolates – P. lacustris P245 as the holotype, P. gonapodyides CBS 117380 as a specimen most closely related phenotypically to P. lacustris, and P. cactorum as a positive control of forest trees’ pathogen – were researched.
Great heterogeneity in the growth rates and morphology of mycelium, as well as in the structure of zoosporangia and hyphal swellings, were observed. Moreover, the isolates differed in their ability to colonize the willow leaves and shoots in the in vitro tests. Some correlation may be found between the daily growth rates and colonizing abilities, and between the daily growth rate and the dimension of the sporangia. Also, a large genotypic variation between the isolates based on the fingerprint patterns generated by molecular techniques (RAPD and ISSR) was obtained