1461 research outputs found
Sort by
The binding of the APT1 domains to phosphoinositides is regulated by metal ions in vitro
Get PDFChorein is a protein of the Vps13 family, and defects in this protein cause the rare neurodegenerative disorder chorea-acanthocytosis (ChAc). Chorein is involved in the actin cytoskeleton organization, calcium ion flux, neuronal cell excitability, exocytosis and autophagy. The function of this protein is poorly understood, and obtaining this knowledge is a key to finding a cure for ChAc. Chorein, as well as the Vps13 protein from yeast, contains the APT1 domain. Our previous research has shown that the APT1 domain from yeast Vps13 (yAPT1v) binds phosphatidylinositol 3-phosphate (PI3P) in vitro. In this study, we showed that although the APT1 domain from chorein (hAPT1) binds to PI3P it could not functionally replace yAPT1v. The hAPT1 domain binds, in addition to PI3P, to phosphatidylinositol 5-phosphate (PI5P). The binding of hAPT1 to PI3P, unlike the binding of yAPT1v to PI3P, is regulated by the bivalent ions, calcium and magnesium. Regulation of PI3P binding via calcium is also observed for the APT1 domain of yeast autophagy protein Atg2. The substitution I2771R, found in chorein of patient suffering from ChAc, reduces the binding of the hAPT1 domain to PI3P and PI5P. These results suggest that the ability of APT1 domains to bind phosphoinositides is regulated differently in yeast and human protein and that this regulation is important for chorein function
Lipophagy and lipolysis status in lipid storage and lipid metabolism diseases
Get PDFThis review discusses how lipophagy and cytosolic lipolysis degrade cellular lipids, as well as how these pathway ys communicate, how they affect lipid metabolism and energy homeostasis in cells and how their dysfunction affects the pathogenesis of lipid storage and lipid metabolism diseases. Answers to these questions will likely uncover novel strategies for the treatment of aforementioned human diseases, but, above all, will avoid destructive effects of high concentrations of lipids—referred to as lipotoxicity—resulting in cellular dysfunction and cell death
Novel perspectives of target-binding by the evolutionarily conserved PP4 phosphatase
Get PDFProtein phosphatase 4 (PP4) is an evolutionarily conserved and essential Ser/Thr phosphatase that regulates cell division, development and DNA repair in eukaryotes. The major form of PP4, present from yeast to human, is the PP4c-R2-R3 heterotrimeric complex. The R3 subunit is responsible for substrate-recognition via its EVH1 domain. In typical EVH1 domains, conserved phenylalanine, tyrosine and tryptophan residues form the specific recognition site for their target's proline-rich sequences. Here, we identify novel binding partners of the EVH1 domain of the Drosophila R3 subunit, Falafel, and demonstrate that instead of binding to proline-rich sequences this EVH1 variant specifically recognizes atypical ligands, namely the FxxP and MxPP short linear consensus motifs. This interaction is dependent on an exclusively conserved leucine that replaces the phenylalanine invariant of all canonical EVH1 domains. We propose that the EVH1 domain of PP4 represents a new class of the EVH1 family that can accommodate low proline content sequences, such as the FxxP motif. Finally, our data implicate the conserved Smk-1 domain of Falafel in target-binding. These findings greatly enhance our understanding of the substrate-recognition mechanisms and function of PP4
Retinal Degeneration Caused by Rod-Specific Dhdds Ablation Occurs without Concomitant Inhibition of ProteinN-Glycosylation
Get PDFDehydrodolichyl diphosphate synthase (DHDDS) catalyzes the committed step indolichol synthesis. Recessive mutations inDHDDScause retinitis pigmentosa(RP59), resulting in blindness. We hypothesized that rod photoreceptor-specificablation ofDhddswould cause retinal degeneration due to diminished dolichol-dependent proteinN-glycosylation.Dhddsflx/flxmice were crossed with rod-spe-cific Cre recombinase-expressing (Rho-iCre75) mice to generate rod-specificDhddsknockout mice (Dhddsflx/flxiCre+).In vivomorphological and electrophys-iological evaluation ofDhddsflx/flxiCre+retinas revealed mild retinal dysfunctionat postnatal (PN) 4 weeks, compared with age-matched controls; however, rapidphotoreceptor degeneration ensued, resulting in almost complete loss of rodsand cones by PN 6 weeks. Retina dolichol levels were markedly decreased byPN 4 weeks inDhddsflx/flxiCre+mice, relative to controls; despite this,N-glycosyl-ation of retinal proteins, including opsin (the dominant rod-specific glycoprotein),persisted inDhddsflx/flxiCre+mice. These findings challenge the conventionalmechanistic view of RP59 as a congenital disorder of glycosylation
Unsymmetrically-Substituted 5,12-dihydrodibenzo[b,f][1,4]diazocine-6,11-dione Scaffold—A Useful Tool for Bioactive Molecules Design
Get PDFUnsymmetrically N-substituted and N,N’-disubstituted 5,12-dihydrodibenzo [b,f][1,4]diazocine-6,11-diones were synthesized in the new protocol. The desired modifications of the dibenzodiazocine scaffold were introduced at the stages of proper selection of building blocks as well as post-cyclization modifications with alkylation or acylation agents, expanding the structural diversity and possible applications of synthesized molecules. The extension of developed method resulted in the synthesis of novel: tricyclic 5,10-dihydrobenzo[b]thieno[3,4-f][1,4]diazocine-4,11-dione scaffold and fused pentacyclic framework possessing two benzodiazocine rings within its structure. Additionally, the unprecedented rearrangement of 5,12-dihydrodibenzo[b,f][1,4]diazocine-6,11-diones to 2-(2-aminophenyl)isoindoline-1,3-diones was observed under the basic conditions in the presence of sodium hydride for secondary dilactams. The structures of nine synthesized products have been established by single-crystal X-ray diffraction analysis. Detailed crystallographic analysis of the investigated tri- and pentacyclic systems has shed more light on their structural features. One cell line derived from non-cancerous cells (EUFA30—human fibroblasts) and three tumor cells (U87—human primary glioblastoma, HeLa—cervix adenocarcinoma, BICR18—laryngeal squamous cell carcinoma) were used to determine the cytotoxic effect of the newly synthesized compounds. Although these compounds showed a relatively weak cytotoxic effect, the framework obtained for 5,12-dihydrodibenzo[b,f][1,4]diazocine-6,11-dione could serve as a convenient privilege structure for the design and development of novel bioactive molecules suitable for drug design, development and optimization programs
A novel mobilizing tool based on the conjugative transfer system of the IncM plasmid pCTX-M3
Get PDFConjugative plasmids are the main players in horizontal gene transfer in Gram-negative bacteria. DNA transfer tools constructed on the basis of such plasmids enable gene manipulation even in strains of clinical or environmental origin, which are often difficult to work with. The conjugation system of the IncM plasmid pCTX-M3 isolated from a clinical strain of Citrobacter freundii has been shown to enable efficient mobilization of oriT pCTX-M3-bearing plasmids into a broad range of hosts comprising Alpha-, Beta-, and Gammaproteobacteria We constructed a helper plasmid pMOBS mediating such mobilization with an efficiency up to 1000-fold higher than that achieved with native pCTX-M3. We also constructed E. coli donor strains with chromosome-integrated conjugative transfer genes: S14 and S15, devoid of one putative regulator (orf35), and S25 and S26, devoid of two putative regulators (orf35 and orf36) of the pCTX-M3 tra genes. Strains S14 and S15, and S25 and S26 are, respectively, up to 100 and 1000 times more efficient in mobilization than pCTX-M3. Moreover, they also enable plasmid mobilization to the Gram-positive bacteria Bacillus subtilis and Lactococcus lactis Additionally, the constructed E. coli strains carried no antibiotic resistance genes that are present in pCTX-M3 to facilitate manipulations with antibiotic-resistant recipient strains, such as those of clinical origin. To demonstrate possible application of the constructed tool, an antibacterial conjugation-based system was designed. Strain S26 was used for introduction of a mobilizable plasmid coding for a toxin, resulting in the elimination of over 90% of recipient E. coli cells.IMPORTANCE The conjugation of donor and recipient bacterial cells resulting in conjugative transfer of mobilizable plasmids is the preferred method enabling the introduction of DNA into strains for which other transfer methods are difficult to establish (e.g., clinical strains). We have constructed E. coli strains carrying the conjugation system of the IncM plasmid pCTX-M3 integrated into the chromosome. To increase the mobilization efficiency up to 1000-fold, two putative regulators of this system, orf35 and/or orf36, were disabled. The constructed strains broaden the repertoire of tools for the introduction of DNA into the Gram-negative Alpha-, Beta-, and Gammaproteobacteria, as well as into Gram-positive bacteria such as Bacillus subtilis and Lactococcus lactis The antibacterial procedure based on conjugation with the use of the orf35- and orf36-deficient strain lowered the recipient cell number by over 90% owing to the mobilizable plasmid-encoded toxin
Low Content of Cyclosporine A and Its Metabolites in the Colostrum of Post-Transplant Mothers
Get PDFBreeding Colony Dynamics of Southern Elephant Seals at Patelnia Point, King George Island, Antarctica
Get PDFDuring the 2019 breeding season (October-December), a battery-powered DIJ Inspire 2 drone was used to investigate a breeding southern elephant seal colony located at Patelnia Point (ASPA 128, King George Island, South Shetland Islands, maritime Antarctica). Twelve unmanned aerial vehicle (UAV) missions conducted 50–70 m above ground level (AGL) were completed to monitor the breeding ground with a maximum of 0.348 km2. The missions were planned in Pix4D Capture software. A drone, with the support of ground cameras and observations, was used to derive population counts, map harems, and track the phenology of the southern elephant seals. Based on data obtained from the UAV missions, orthophotomaps were created in PIX4D Mapper and then analyzed in QGIS. Calculated values of body size parameters such as body length and orthogonal body surface area were used to determine the age and sex of individuals. Analysis of the ranges of the harems on particular days, supported by an analysis of land conditions that generate physical barriers to the movement of animals, allowed zones in which the transformations of groups of harems took place to be determined. The hypothesized hermeticity of the designated zones was supported by statistical tests. The use of drones allows for comprehensive population analyses of the breeding colonies of elephant seals such as censuses of pups and adult individuals, determination of the sex ratio, and spatial analysis of the distribution of breeding formations. In addition, it allows for a more accurate result than ground counting
The Palladium(II) Complex of A β4-16 as Suitable Model for Structural Studies of Biorelevant Copper(II) Complexes of N-Truncated Beta-Amyloids
Get PDFThe Aβ4-42 peptide is a major beta-amyloid species in the human brain, forming toxic aggregates related to Alzheimer's Disease. It also strongly chelates Cu(II) at the N-terminal Phe-Arg-His ATCUN motif, as demonstrated in Aβ4-16 and Aβ4-9 model peptides. The resulting complex resists ROS generation and exchange processes and may help protect synapses from copper-related oxidative damage. Structural characterization of Cu(II)Aβ4-x complexes by NMR would help elucidate their biological function, but is precluded by Cu(II) paramagneticism. Instead we used an isostructural diamagnetic Pd(II)-Aβ4-16 complex as a model. To avoid a kinetic trapping of Pd(II) in an inappropriate transient structure, we designed an appropriate pH-dependent synthetic procedure for ATCUN Pd(II)Aβ4-16, controlled by CD, fluorescence and ESI-MS. Its assignments and structure at pH 6.5 were obtained by TOCSY, NOESY, ROESY, 1H-13C HSQC and 1H-15N HSQC NMR experiments, for natural abundance 13C and 15N isotopes, aided by corresponding experiments for Pd(II)-Phe-Arg-His. The square-planar Pd(II)-ATCUN coordination was confirmed, with the rest of the peptide mostly unstructured. The diffusion rates of Aβ4-16, Pd(II)-Aβ4-16 and their mixture determined using PGSE-NMR experiment suggested that the Pd(II) complex forms a supramolecular assembly with the apopeptide. These results confirm that Pd(II) substitution enables NMR studies of structural aspects of Cu(II)-Aβ complexes
Polymerase iota - an odd sibling among Y family polymerases
Get PDFIt has been two decades since the discovery of the most mutagenic human DNA polymerase, polymerase iota (Polι). Since then, the biochemical activity of this translesion synthesis (TLS) enzyme has been extensively explored, mostly through in vitro experiments, with some insight into its cellular activity. Polι is one of four members of the Y-family of polymerases, which are the best characterized DNA damage-tolerant polymerases involved in TLS. Polι shares some common Y-family features, including low catalytic efficiency and processivity, high infidelity, the ability to bypass some DNA lesions, and a deficiency in 3'→5' exonucleolytic proofreading. However, Polι exhibits numerous properties unique among the Y-family enzymes. Polι has an unusual catalytic pocket structure and prefers Hoogsteen over Watson-Crick pairing, and its replication fidelity strongly depends on the template; further, it prefers Mn2+ ions rather than Mg2+ as catalytic activators. In addition to its polymerase activity, Polι possesses also 5'-deoxyribose phosphate (dRP) lyase activity, and its ability to participate in base excision repair has been shown. As a highly error-prone polymerase, its regulation is crucial and mostly involves posttranslational modifications and protein-protein interactions. The upregulation and downregulation of Polι are correlated with different types of cancer and suggestions regarding the possible function of this polymerase have emerged from studies of various cancer lines. Nonetheless, after twenty years of research, the biological function of Polι certainly remains unresolved