Structural basis of SALM3 dimerization and synaptic adhesion complex formation with PTPσ

bioRxiv: doi: https://doi.org/10.1101/2020.01.09.893701Synaptic adhesion molecules play an important role in the formation, maintenance and refinement of neuronal connectivity. Recently, several leucine rich repeat (LRR) domain containing neuronal adhesion molecules have been characterized including netrin G-ligands, SLITRKs and the synaptic adhesion-like molecules (SALMs). Dysregulation of these adhesion molecules have been genetically and functionally linked to various neurological disorders. Here we investigated the molecular structure and mechanism of ligand interactions for the postsynaptic SALM3 adhesion protein with its presynaptic ligand, receptor protein tyrosine phosphatase sigma (PTP sigma). We solved the crystal structure of the dimerized LRR domain of SALM3, revealing the conserved structural features and mechanism of dimerization. Furthermore, we determined the complex structure of SALM3 with PTP sigma using small angle X-ray scattering, revealing a 2:2 complex similar to that observed for SALM5. Solution studies unraveled additional flexibility for the complex structure, but validated the uniform mode of action for SALM3 and SALM5 to promote synapse formation. The relevance of the key interface residues was further confirmed by mutational analysis with cellular binding assays and artificial synapse formation assays. Collectively, our results suggest that SALM3 dimerization is a pre-requisite for the SALM3-PTP sigma complex to exert synaptogenic activity.Peer reviewe

Effects of low frequency ultrasound on some properties of fibrinogen and its plasminolysis

<p>Abstract</p> <p>Background</p> <p>Pharmacological thrombolysis with streptokinase, urokinase or tissue activator of plasminogen (t-PA), and mechanical interventions are frequently used in the treatment of both arterial and venous thrombotic diseases. It has been previously reported that application of ultrasound as an adjunct to thrombolytic therapy offers unique potential to improve effectiveness. However, little is known about effects of the ultrasound on proteins of blood coagulation and fibrinolysis. Here, we investigated the effects of the ultrasound on fibrinogen on processes of coagulation and fibrinogenolysis in an <it>in vitro </it>system.</p> <p>Results</p> <p>Our study demonstrated that low frequency high intensity pulse ultrasound (25.1 kHz, 48.4 W/cm2, duty 50%) induced denaturation of plasminogen and t-PA and fibrinogen aggregates formation <it>in vitro</it>. The aggregates were characterized by the loss of clotting ability and a greater rate of plasminolysis than native fibrinogen. We investigated the effect of the ultrasound on individual proteins. In case of plasminogen and t-PA, ultrasound led to a decrease of the fibrinogenolysis rate, while it increased the fibrinogenolysis rate in case of fibrinogen. It has been shown that upon ultrasound treatment of mixture fibrinogen or fibrin with plasminogen, t-PA, or both, the rate of proteolytic digestion of fibrin(ogen) increases too. It has been shown that summary effect on the fibrin(ogen) proteolytic degradation under the conditions for combined ultrasound treatment is determined exclusively by effect on fibrin(ogen).</p> <p>Conclusions</p> <p>The data presented here suggest that among proteins of fibrinolytic systems, the fibrinogen is one of the most sensitive proteins to the action of ultrasound. It has been shown <it>in vitro </it>that ultrasound induced fibrinogen aggregates formation, characterized by the loss of clotting ability and a greater rate of plasminolysis than native fibrinogen in different model systems and under different mode of ultrasound treatment. Under ultrasound treatment of plasminogen and/or t-PA in the presence of fibrin(ogen) the stabilizing effect fibrin(ogen) on given proteins was shown. On the other hand, an increase in the rate of fibrin(ogen) lysis was observed due to both the change in the substrate structure and promoting of the protein-protein complexes formation.</p

Tardigrade workbench: comparing stress-related proteins, sequence-similar and functional protein clusters as well as RNA elements in tardigrades

<p>Abstract</p> <p>Background</p> <p>Tardigrades represent an animal phylum with extraordinary resistance to environmental stress.</p> <p>Results</p> <p>To gain insights into their stress-specific adaptation potential, major clusters of related and similar proteins are identified, as well as specific functional clusters delineated comparing all tardigrades and individual species (<it>Milnesium tardigradum</it>, <it>Hypsibius dujardini</it>, <it>Echiniscus testudo</it>, <it>Tulinus stephaniae</it>, <it>Richtersius coronifer</it>) and functional elements in tardigrade mRNAs are analysed. We find that 39.3% of the total sequences clustered in 58 clusters of more than 20 proteins. Among these are ten tardigrade specific as well as a number of stress-specific protein clusters. Tardigrade-specific functional adaptations include strong protein, DNA- and redox protection, maintenance and protein recycling. Specific regulatory elements regulate tardigrade mRNA stability such as lox P DICE elements whereas 14 other RNA elements of higher eukaryotes are not found. Further features of tardigrade specific adaption are rapidly identified by sequence and/or pattern search on the web-tool tardigrade analyzer <url>http://waterbear.bioapps.biozentrum.uni-wuerzburg.de</url>. The work-bench offers nucleotide pattern analysis for promotor and regulatory element detection (tardigrade specific; nrdb) as well as rapid COG search for function assignments including species-specific repositories of all analysed data.</p> <p>Conclusion</p> <p>Different protein clusters and regulatory elements implicated in tardigrade stress adaptations are analysed including unpublished tardigrade sequences.</p

Transcriptome Analysis in Tardigrade Species Reveals Specific Molecular Pathways for Stress Adaptations

Tardigrades have unique stress-adaptations that allow them to survive extremes of cold, heat, radiation and vacuum. To study this, encoded protein clusters and pathways from an ongoing transcriptome study on the tardigrade Milnesium tardigradum were analyzed using bioinformatics tools and compared to expressed sequence tags (ESTs) from Hypsibius dujardini, revealing major pathways involved in resistance against extreme environmental conditions. ESTs are available on the Tardigrade Workbench along with software and databank updates. Our analysis reveals that RNA stability motifs for M. tardigradum are different from typical motifs known from higher animals. M. tardigradum and H. dujardini protein clusters and conserved domains imply metabolic storage pathways for glycogen, glycolipids and specific secondary metabolism as well as stress response pathways (including heat shock proteins, bmh2, and specific repair pathways). Redox-, DNA-, stress- and protein protection pathways complement specific repair capabilities to achieve the strong robustness of M. tardigradum. These pathways are partly conserved in other animals and their manipulation could boost stress adaptation even in human cells. However, the unique combination of resistance and repair pathways make tardigrades and M. tardigradum in particular so highly stress resistant

Липофильность BODIPY флуорофоров и их распределение в системе октанол-1–вода

The work covers synthesis and lipophilicity estimation of several BODIPY dyes. For these compounds, the distribution between 1-octanol and water layers is experimentally described and the corresponding partition coefficients LogP are calculated. The experimental LogP values are compared with popular fragment-based methods XLopP3, ALogPS, WLogP, SILICOS-IT and MLogP. Additionally, the hydrophobic and polar surface areas are found with quantum-mechanical calculations. That allowed to find a correlation between the LogP coefficient and the molecular surface topology, as well as to determine the corresponding incremental values of the methyl, acetyl, and phenyl substituents. Выполнен синтез нескольких BODIPY флуорофоров и рассмотрено их распределение в системе октанол-1–вода. Для оценки эффективности использования расчетных методов при описании липофильности BODIPY производных обсуждены такие подходы, как XLopP3, ALogPS, WLogP, SILICOS-IT и MLogP. С помощью квантово-механических расчетов найдены гидрофобная и полярная площади молекулярных поверхностей соединений. Это позволило установить корреляцию между коэффициентом LogP и топологией молекулярной поверхности, а также определить соответствующие величины инкрементов для метильного, ацетильного и фенильного заместителей

Дикетоны природного происхождения как потенциальные ковалентные лиганды белков SARS-CoV-2: исследование in silico методом докинга

Our computer-aided protein-ligand docking test using Autodock Vina software allowed to reveal the potential of few α- and β-diketones from plants and alternative living organisms as covalent ligands for few proteins of coronavirus SARS-CoV-2 – a causative agent of COVID-19. It has been established that values for energy of binding (docking score, Ebind, kcal/mol) less than –7.5 and for distances of ligands’ carbonyl groups to side chain nitrogens of arginine residues of some coronaviral enzymes within 0.4 nm have been true for β-diketones 6-gingerdione (Pubchem code CID162952), 8-gingerdione (CID14440537), tetrahydrocurcumine (CID124072) as well as α-diketone wallitaxane E (CID132967478). The in silico revealed interactions are interesting to be verified in vitro and they point out a possibility of investigation of the compounds and related natural materials as tools for struggle against coronaviral infections.Компьютерный докинг, проведенный с использованием программы Autodock Vina, позволил выявить потенциал нескольких α- и β-дикетонов растений и других природных объектов в качестве ковалентных лигандов ряда белков коронавируса SARS-CoV-2 – возбудителя COVID-19. Выявлено, что энергией связывания (docking score, Ebind, ккал/мол) менее –7,5 с колоколизацией карбонильных групп на расстоянии не более 0,4 нм от атомов азота боковой цепи остатков аргинина белков коронавируса. β-Дикетоны 6-гингердион (код структуры по базе данных Pubchem CID162952), 8-гингердион (CID14440537), тетрагидрокуркумин (CID124072), а также α-дикетон валлитаксан E (CID132967478) обладали такими свойствами. Выявленные in silico взаимодействия указывают на возможность обнаружения их в эксперименте и исследования этих веществ или содержащих их природных материалов как средств борьбы с короновирусной инфекцией

Multi‐scale ensemble properties of the Escherichia coli RNA degradosome

Abstract: In organisms from all domains of life, multi‐enzyme assemblies play central roles in defining transcript lifetimes and facilitating RNA‐mediated regulation of gene expression. An assembly dedicated to such roles, known as the RNA degradosome, is found amongst bacteria from highly diverse lineages. About a fifth of the assembly mass of the degradosome of Escherichia coli and related species is predicted to be intrinsically disordered – a property that has been sustained for over a billion years of bacterial molecular history and stands in marked contrast to the high degree of sequence variation of that same region. Here, we characterize the conformational dynamics of the degradosome using a hybrid structural biology approach that combines solution scattering with ad hoc ensemble modelling, cryo‐electron microscopy, and other biophysical methods. The E. coli degradosome can form punctate bodies in vivo that may facilitate its functional activities, and based on our results, we propose an electrostatic switch model to account for the propensity of the degradosome to undergo programmable puncta formation

Синтез новых тиазоло[3,2-а]пиримидинов и in silico анализ их биоактивности

An effective method of synthesis thiazolo[3,2-a]pyrimidine derivatives was developed and the compounds with n-pentyl or β-acetoxycyclopropyl as well as fluorescent benzo[f]coumarin substituents were obtained with yields 60 % and more. Using computational (in silico) approaches we demonstrated the ability of the obtained compounds to permeate lipid bilayer as well as their affinity to some protein kinases (compounds 4 and 6 bind with a protein kinase AKT1 with PDB code 3о96; Autodock Vina-computed energy of binding (Ebind) values were -10.9 and -10.6 kcal/mol, respectively), acethylcholine esterase and some human cytochromes P450 (for P450 3A4, pdb 5vcd, Ebind -12.3 kcal/mol).Разработан эффективный метод получения и синтезированы новые тиазоло[3,2-a]пиримидины, содержащие н-пентановый или β-гидроксициклопропановый, а также флуоресцирующий бензо[f]кумариновый заместители с выходами более 60 %. Компьютерными методами (in silico) показана способность полученных соединений проникать через липидный бислой и оценено их сродство к ряду протеинкиназ (соединения 4 и 6 связываются с протеинкиназой AKT1 с кодом PDB 3о96; величины, рассчитываемые программой Autodock Vina энергии связывания (Ebind), составили: -10,9 и -10,6 ккал/моль), ацетилхолинэстеразе и цитохромам P450 человека (для цитохрома P450 3A4, pdb 5vcd, Ebind -12,3 ккал/моль)

In silico оценка новых аффинных взаимодействий метилкумарина с цитохромами P450

4-methyl-7-methoxycoumarin (CumOMe) has been synthesized and in silico calculations demonstrated localization of methoxy group within 0.4 nm from Fe ion of hem groups for some structures of human CYP19 &amp; CYP46 as well as CYP152 S. paucimobilis, CYP158 St. coelicolor, HMUO C. diphtheriae, XPLA R. rhodochrous, CYP199A4 Rh. palustris, CYP101A1 Ps. putida and CYP51 M. tuberculosis.Синтезирован 4-метил-7-метоксикумарин (CumOMe) и in silico показано, что локализация метоксильного фрагмената на расстоянии не более 0,4 нм от железа гема возможны для отдельных структур CYP19A1 и CYP46 человека, а также CYP152 S. paucimobilis, CYP158 St. coelicolor, HMUO C. diphtheriae, XPLA R. rhodochrous, CYP199A4 Rh. palustris, CYP101A1 Ps. Putida и CYP51 M. tuberculosis

Analysis of the natively unstructured RNA/protein-recognition core in the Escherichia coli RNA degradosome and its interactions with regulatory RNA/Hfq complexes.

The RNA degradosome is a multi-enzyme assembly that plays a central role in the RNA metabolism of Escherichia coli and numerous other bacterial species including pathogens. At the core of the assembly is the endoribonuclease RNase E, one of the largest E. coli proteins and also one that bears the greatest region predicted to be natively unstructured. This extensive unstructured region, situated in the C-terminal half of RNase E, is punctuated with conserved short linear motifs that recruit partner proteins, direct RNA interactions, and enable association with the cytoplasmic membrane. We have structurally characterized a subassembly of the degradosome-comprising a 248-residue segment of the natively unstructured part of RNase E, the DEAD-box helicase RhlB and the glycolytic enzyme enolase, and provide evidence that it serves as a flexible recognition centre that can co-recruit small regulatory RNA and the RNA chaperone Hfq. Our results support a model in which the degradosome captures substrates and regulatory RNAs through the recognition centre, facilitates pairing to cognate transcripts and presents the target to the ribonuclease active sites of the greater assembly for cooperative degradation or processing