The nonsense-mediated mRNA decay (NMD) pathway rapidly detects and degrades mRNA containing premature termination codons (PTCs). UP-frameshift 1 (UPF1), the master regulator of the NMD process, has two alternatively-spliced isoforms; one carries 353-GNEDLVIIWLR-363 insertion in the 'regulatory loop (involved in mRNA binding)'. Such insertion can induce catalytic and/or ATPase activity, as determined experimentally; however, the kinetics and molecular level information are not fully understood. Herein, applying all-atom molecular dynamics, we probe the binding specificity of UPF1 with different GC- and AU-rich mRNA motifs and the influence of insertion to the viable control over UPF1 catalytic activity. Our results indicate two distinct conformations between 1B and RecA2 domains of UPF1: 'open (isoform_2; without insertion)' and 'closed (isoform_1; with insertion)'. These structural movements correspond to an important stacking pattern in mRNA motifs, i.e., absence of stack formation in mRNA, with UPF1 isoform_2 results in the 'open conformation'. Particularly, for UPF1 isoform_1, the increased distance between 1B and RecA2 domains has resulted in reducing the mRNA-UPF1 interactions. Lower fluctuating GC-rich mRNA motifs have better binding with UPF1, compared with AU-rich sequences. Except CCUGGGG, all other GC-rich motifs formed a 4-stack pattern with UPF1. High occupancy R363, D364, T627, and G862 residues were common binding GC-rich motifs, as were R363, N535, and T627 for the AU-rich motifs. The GC-rich motifs behave distinctly when bound to either of the isoforms; lower stability was observed with UPF1 isoform_2. The cancer-associated UPF1 variants (P533L/T and A839T) resulted in decreased protein-mRNA binding efficiency. Lack of mRNA stacking poses in the UPF1P533T system significantly decreased UPF1-mRNA binding efficiency and increased distance between 1B-RecA2. These novel findings can serve to further inform NMD-associated mechanistic and kinetic studies.

• Klíčová slova
• AU-rich, GC-rich, NMD, PTC, UPF1, alternatively spliced, degradation, isoform, mRNA, molecular dynamics, motifs, regulatory loop, stability,
• MeSH
• alternativní sestřih * MeSH
• fosforylace MeSH
• lidé MeSH
• messenger RNA genetika   metabolismus   MeSH
• nonsense mediated mRNA decay * MeSH
• protein - isoformy MeSH
• regulace genové exprese * MeSH
• RNA-helikasy genetika   metabolismus   MeSH
• trans-aktivátory genetika   metabolismus   MeSH
• vazba proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• messenger RNA MeSH
• protein - isoformy MeSH
• RNA-helikasy MeSH
• trans-aktivátory MeSH
• UPF1 protein, human MeSH Prohlížeč

The TAP1-TAP2 complex transports antigenic peptide substrates into the endoplasmic reticulum (ER). In ER, the peptides are further processed and loaded on the major histocompatibility class (MHC) I molecules by the peptide loading complex (PLC). The TAP transporters are linked with the PLC; a target for cancers and viral immune evasion. But the mechanisms whereby the cancer-derived mutations in TAP1-TAP2 or viral factors targeting the PLC, interfere peptide transport are only emerging. This study describes that transit of peptides through TAP can take place via two different channels (4 or 8 helices) depending on peptide length and sequence. Molecular dynamics and binding affinity predictions of peptide-transporters demonstrated that smaller peptides (8-10 mers; e.g. AAGIGILTV, SIINFEKL) can transport quickly through the transport tunnel compared to longer peptides (15-mer; e.g. ENPVVHFFKNIVTPR). In line with a regulated and selective peptide transport by TAPs, the immunopeptidome upon IFN-γ treatment in melanoma cells induced the shorter length (9-mer) peptide presentation over MHC-I that exhibit a relatively weak binding affinity with TAP. A conserved distance between N and C terminus residues of the studied peptides in the transport tunnel were reported. Furthermore, by adversely interacting with the TAP transport passage or affecting TAPNBD domains tilt movement, the viral proteins and cancer-derived mutations in TAP1-TAP2 may induce allosteric effects in TAP that block conformation of the tunnel (closed towards ER lumen). Interestingly, some cancer-associated mutations (e.g. TAP1R372Q and TAP2R373H) can specifically interfere with selective transport channels (i.e. for longer-peptides). These results provide a model for how viruses and cancer-associated mutations targeting TAP interfaces can affect MHC-I antigen presentation, and how the IFN-γ pathway alters MHC-I antigen presentation via the kinetics of peptide transport.

• Klíčová slova
• Cancer mutations, IFN-γ, Immunopeptidome, In silico peptide screening, Kinetics, MHC-I, Molecular dynamics, Peptide, TAP1, TAP2, Transporters, Viral factors,
• Publikační typ
• časopisecké články MeSH

DNA transposons are defined as repeated DNA sequences that can move within the host genome through the action of transposases. The transposon superfamily Merlin was originally found mainly in animal genomes. Here, we describe a global distribution of the Merlin in animals, fungi, plants and protists, reporting for the first time their presence in Rhodophyceae, Metamonada, Discoba and Alveolata. We identified a great variety of potentially active Merlin families, some containing highly imperfect terminal inverted repeats and internal tandem repeats. Merlin-related sequences with no evidence of mobilization capacity were also observed and may be products of domestication. The evolutionary trees support that Merlin is likely an ancient superfamily, with early events of diversification and secondary losses, although repeated re-invasions probably occurred in some groups, which would explain its diversity and discontinuous distribution. We cannot rule out the possibility that the Merlin superfamily is the product of multiple horizontal transfers of related prokaryotic insertion sequences. Moreover, this is the first account of a DNA transposon in kinetoplastid flagellates, with conserved Merlin transposase identified in Bodo saltans and Perkinsela sp., whereas it is absent in trypanosomatids. Based on the level of conservation of the transposase and overlaps of putative open reading frames with Merlin, we propose that in protists it may serve as a raw material for gene emergence.

• MeSH
• Alveolata genetika   MeSH
• Eukaryota genetika   MeSH
• fylogeneze MeSH
• Kinetoplastida genetika   MeSH
• molekulární evoluce MeSH
• neurofibromin 2 genetika   MeSH
• polymerázová řetězová reakce MeSH
• transpozibilní elementy DNA genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• neurofibromin 2 MeSH
• transpozibilní elementy DNA MeSH

Homology modeling is a method for building protein 3D structures using protein primary sequence and utilizing prior knowledge gained from structural similarities with other proteins. The homology modeling process is done in sequential steps where sequence/structure alignment is optimized, then a backbone is built and later, side-chains are added. Once the low-homology loops are modeled, the whole 3D structure is optimized and validated. In the past three decades, a few collective and collaborative initiatives allowed for continuous progress in both homology and ab initio modeling. Critical Assessment of protein Structure Prediction (CASP) is a worldwide community experiment that has historically recorded the progress in this field. Folding@Home and Rosetta@Home are examples of crowd-sourcing initiatives where the community is sharing computational resources, whereas RosettaCommons is an example of an initiative where a community is sharing a codebase for the development of computational algorithms. Foldit is another initiative where participants compete with each other in a protein folding video game to predict 3D structure. In the past few years, contact maps deep machine learning was introduced to the 3D structure prediction process, adding more information and increasing the accuracy of models significantly. In this review, we will take the reader in a journey of exploration from the beginnings to the most recent turnabouts, which have revolutionized the field of homology modeling. Moreover, we discuss the new trends emerging in this rapidly growing field.

• Klíčová slova
• Artificial intelligence, Collective intelligence, Homology modeling, Machine learning, Protein 3D structure, Structural bioinformatics,
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

Fe-S clusters are ubiquitous cofactors of proteins involved in a variety of essential cellular processes. The biogenesis of Fe-S clusters in the cytosol and their insertion into proteins is accomplished through the cytosolic iron-sulphur protein assembly (CIA) machinery. The early- and middle-acting modules of the CIA pathway concerned with the assembly and trafficking of Fe-S clusters have been previously characterised in the parasitic protist Trypanosoma brucei. In this study, we applied proteomic and genetic approaches to gain insights into the network of protein-protein interactions of the late-acting CIA targeting complex in T. brucei. All components of the canonical CIA machinery are present in T. brucei including, as in humans, two distinct CIA2 homologues TbCIA2A and TbCIA2B. These two proteins are found interacting with TbCIA1, yet the interaction is mutually exclusive, as determined by mass spectrometry. Ablation of most of the components of the CIA targeting complex by RNAi led to impaired cell growth in vitro, with the exception of TbCIA2A in procyclic form (PCF) trypanosomes. Depletion of the CIA-targeting complex was accompanied by reduced levels of protein-bound cytosolic iron and decreased activity of an Fe-S dependent enzyme in PCF trypanosomes. We demonstrate that the C-terminal domain of TbMMS19 acts as a docking site for TbCIA2B and TbCIA1, forming a trimeric complex that also interacts with target Fe-S apo-proteins and the middle-acting CIA component TbNAR1.

• MeSH
• cytosol metabolismus   MeSH
• interakční proteinové domény a motivy MeSH
• konformace proteinů MeSH
• myši inbrední BALB C MeSH
• myši MeSH
• proteiny obsahující železo a síru chemie   metabolismus   MeSH
• protozoální proteiny chemie   metabolismus   MeSH
• Trypanosoma brucei brucei růst a vývoj   metabolismus   MeSH
• trypanozomiáza metabolismus   parazitologie   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• proteiny obsahující železo a síru MeSH
• protozoální proteiny MeSH

Studies have suggested a role for the mammalian (or mechanistic) target of rapamycin (mTOR) in skeletal development and homeostasis, yet there is no evidence connecting mTOR with the key signaling pathways that regulate skeletogenesis. We identified a parathyroid hormone (PTH)/PTH-related peptide (PTHrP)-salt-inducible kinase 3 (SIK3)-mTOR signaling cascade essential for skeletogenesis. While investigating a new skeletal dysplasia caused by a homozygous mutation in the catalytic domain of SIK3, we observed decreased activity of mTOR complex 1 (mTORC1) and mTORC2 due to accumulation of DEPTOR, a negative regulator of both mTOR complexes. This SIK3 syndrome shared skeletal features with Jansen metaphyseal chondrodysplasia (JMC), a disorder caused by constitutive activation of the PTH/PTHrP receptor. JMC-derived chondrocytes showed reduced SIK3 activity, elevated DEPTOR, and decreased mTORC1 and mTORC2 activity, indicating a common mechanism of disease. The data demonstrate that SIK3 is an essential positive regulator of mTOR signaling that functions by triggering DEPTOR degradation in response to PTH/PTHrP signaling during skeletogenesis.

• MeSH
• HEK293 buňky MeSH
• homozygot MeSH
• intracelulární signální peptidy a proteiny metabolismus   MeSH
• lidé MeSH
• missense mutace genetika   MeSH
• mTORC1 metabolismus   MeSH
• mTORC2 metabolismus   MeSH
• mutantní proteiny chemie   metabolismus   MeSH
• osteogeneze * MeSH
• parathormon metabolismus   MeSH
• protein podobný parathormonu metabolismus   MeSH
• proteinkinasy chemie   nedostatek   genetika   metabolismus   MeSH
• proteolýza MeSH
• růstová ploténka metabolismus   MeSH
• sekvence aminokyselin MeSH
• signální transdukce * MeSH
• TOR serin-threoninkinasy metabolismus   MeSH
• typy dědičnosti genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• DEPTOR protein, human MeSH Prohlížeč
• intracelulární signální peptidy a proteiny MeSH
• mTORC1 MeSH
• mTORC2 MeSH
• mutantní proteiny MeSH
• parathormon MeSH
• protein podobný parathormonu MeSH
• proteinkinasy MeSH
• SIK3 protein, human MeSH Prohlížeč
• TOR serin-threoninkinasy MeSH

Tuberous sclerosis complex (TSC) is a rare autosomal dominant disorder causing benign tumors in the brain and other vital organs. The genes implicated in disease development are TSC1 and TSC2. Here, we have performed mutational analysis followed by a genotype-phenotype correlation study based on the clinical characteristics of the affected individuals. Twenty unrelated probands or families from Greece have been analyzed, of whom 13 had definite TSC, whereas another 7 had a possible TSC diagnosis. Using direct sequencing, we have identified pathogenic mutations in 13 patients/families (6 in TSC1 and 7 in TSC2), 5 of which were novel. The mutation identification rate for patients with definite TSC was 85%, but only 29% for the ones with a possible TSC diagnosis. Multiplex ligation-dependent probe amplification (MLPA) did not reveal any genomic rearrangements in TSC1 and TSC2 in the samples with no mutations identified. In general, TSC2 disease was more severe than TSC1, with more subependymal giant cell astrocytomas and angiomyolipomas, higher incidence of pharmacoresistant epileptic seizures, and more severe neuropsychiatric disorders. To our knowledge, this is the first comprehensive TSC1 and TSC2 mutational analysis carried out in TSC patients in Greece.

• MeSH
• delece genu MeSH
• dítě MeSH
• dospělí MeSH
• exony MeSH
• genetické asociační studie MeSH
• hamartin genetika   MeSH
• lidé MeSH
• missense mutace MeSH
• mutační analýza DNA MeSH
• rodokmen MeSH
• terciární struktura proteinů MeSH
• tuberin genetika   MeSH
• tuberózní skleróza genetika   patologie   MeSH
• Check Tag
• dítě MeSH
• dospělí MeSH
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Řecko MeSH
• Názvy látek
• hamartin MeSH
• tuberin MeSH

Photorhabdus asymbiotica is one of the three recognized species of the Photorhabdus genus, which consists of gram-negative bioluminescent bacteria belonging to the family Morganellaceae. These bacteria live in a symbiotic relationship with nematodes from the genus Heterorhabditis, together forming a complex that is highly pathogenic for insects. Unlike other Photorhabdus species, which are strictly entomopathogenic, P. asymbiotica is unique in its ability to act as an emerging human pathogen. Analysis of the P. asymbiotica genome identified a novel fucose-binding lectin designated PHL with a strong sequence similarity to the recently described P. luminescens lectin PLL. Recombinant PHL exhibited high affinity for fucosylated carbohydrates and the unusual disaccharide 3,6-O-Me2-Glcβ1-4(2,3-O-Me2)Rhaα-O-(p-C6H4)-OCH2CH2NH2 from Mycobacterium leprae. Based on its crystal structure, PHL forms a seven-bladed β-propeller assembling into a homo-dimer with an inter-subunit disulfide bridge. Investigating complexes with different ligands revealed the existence of two sets of binding sites per monomer-the first type prefers l-fucose and its derivatives, whereas the second type can bind d-galactose. Based on the sequence analysis, PHL could contain up to twelve binding sites per monomer. PHL was shown to interact with all types of red blood cells and insect haemocytes. Interestingly, PHL inhibited the production of reactive oxygen species induced by zymosan A in human blood and antimicrobial activity both in human blood, serum and insect haemolymph. Concurrently, PHL increased the constitutive level of oxidants in the blood and induced melanisation in haemolymph. Our results suggest that PHL might play a crucial role in the interaction of P. asymbiotica with both human and insect hosts.

• MeSH
• bakteriální proteiny genetika   imunologie   MeSH
• interakce hostitele a patogenu imunologie   MeSH
• konformace proteinů MeSH
• krystalografie rentgenová MeSH
• lektiny chemie   genetika   imunologie   MeSH
• lidé MeSH
• molekulární sekvence - údaje MeSH
• Photorhabdus genetika   imunologie   MeSH
• povrchová plasmonová rezonance MeSH
• sekvence nukleotidů MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• bakteriální proteiny MeSH
• fucose-binding lectin MeSH Prohlížeč
• lektiny MeSH

GpsB regulatory protein and StkP protein kinase have been proposed as molecular switches that balance septal and peripheral (side-wall like) peptidoglycan (PG) synthesis in Streptococcus pneumoniae (pneumococcus); yet, mechanisms of this switching remain unknown. We report that ΔdivIVA mutations are not epistatic to ΔgpsB division-protein mutations in progenitor D39 and related genetic backgrounds; nor is GpsB required for StkP localization or FDAA labeling at septal division rings. However, we confirm that reduction of GpsB amount leads to decreased protein phosphorylation by StkP and report that the essentiality of ΔgpsB mutations is suppressed by inactivation of PhpP protein phosphatase, which concomitantly restores protein phosphorylation levels. ΔgpsB mutations are also suppressed by other classes of mutations, including one that eliminates protein phosphorylation and may alter division. Moreover, ΔgpsB mutations are synthetically lethal with Δpbp1a, but not Δpbp2a or Δpbp1b mutations, suggesting GpsB activation of PBP2a activity. Consistent with this result, co-IP experiments showed that GpsB complexes with EzrA, StkP, PBP2a, PBP2b and MreC in pneumococcal cells. Furthermore, depletion of GpsB prevents PBP2x migration to septal centers. These results support a model in which GpsB negatively regulates peripheral PG synthesis by PBP2b and positively regulates septal ring closure through its interactions with StkP-PBP2x.

• MeSH
• aminoacyltransferasy genetika   metabolismus   MeSH
• bakteriální proteiny genetika   metabolismus   MeSH
• buněčná stěna metabolismus   MeSH
• buněčné dělení genetika   fyziologie   MeSH
• faktory virulence genetika   metabolismus   MeSH
• fosforylace MeSH
• membránové proteiny genetika   metabolismus   MeSH
• mutace genetika   MeSH
• peptidoglykan biosyntéza   MeSH
• proteiny vázající penicilin genetika   metabolismus   MeSH
• Streptococcus pneumoniae genetika   metabolismus   MeSH
• zastoupení bazí genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• aminoacyltransferasy MeSH
• bakteriální proteiny MeSH
• faktory virulence MeSH
• GpsB protein, Streptococcus pneumoniae MeSH Prohlížeč
• membránové proteiny MeSH
• penicillin-binding protein 2b, Streptococcus MeSH Prohlížeč
• peptidoglykan MeSH
• proteiny vázající penicilin MeSH

The short rib polydactyly syndromes (SRPS) are a group of recessively inherited, perinatal-lethal skeletal disorders primarily characterized by short ribs, shortened long bones, varying types of polydactyly and concomitant visceral abnormalities. Mutations in several genes affecting cilia function cause SRPS, revealing a role for cilia function in skeletal development. To identify additional SRPS genes and discover novel ciliary molecules required for normal skeletogenesis, we performed exome sequencing in a cohort of patients and identified homozygosity for a missense mutation, p.E80K, in Intestinal Cell Kinase, ICK, in one SRPS family. The p.E80K mutation abolished serine/threonine kinase activity, resulting in altered ICK subcellular and ciliary localization, increased cilia length, aberrant cartilage growth plate structure, defective Hedgehog and altered ERK signalling. These data identify ICK as an SRPS-associated gene and reveal that abnormalities in signalling pathways contribute to defective skeletogenesis.

• MeSH
• cilie genetika   patologie   MeSH
• exom genetika   MeSH
• kojenec MeSH
• kostra abnormality   růst a vývoj   MeSH
• lidé MeSH
• MAP kinasový signální systém MeSH
• mnohočetné abnormality genetika   patofyziologie   MeSH
• protein-serin-threoninkinasy genetika   MeSH
• proteiny hedgehog genetika   MeSH
• rodokmen MeSH
• sekvenční analýza DNA MeSH
• signální transdukce MeSH
• syndrom krátkého žebra a polydaktylie genetika   patologie   MeSH
• těhotenství MeSH
• Check Tag
• kojenec MeSH
• lidé MeSH
• těhotenství MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• CILK1 protein, human MeSH Prohlížeč
• protein-serin-threoninkinasy MeSH
• proteiny hedgehog MeSH