Changes in the urinary content of uromodulin oligomeric forms in rats during development of urolithiasis

Введение. Изучение роли олигомерных форм уромодулина в развитии уролитиаза является важной фундаментальной и прикладной задачей. Несмотря на разнообразие моделей уролитиаза на лабораторных животных в настоящее время отсутствует информация относительно динамики концентрации и фракционного состава олигомерных форм уромодулина в моче животных на различных этапах развития патологического процесса. Цель - исследование динамики содержания олигомерных форм уромодулина в моче животных на фоне развития гипероксалатного уролитиаза индуцированного экзогенным введением 1% раствора этиленгликоля в качестве безальтернативного источника питья. Методика. Проводили общеклинический и биохимический анализ образцов крови и мочи на различных этапах развития патологического процесса. До начала моделирования патологии и на фоне экзогенного введения этиленгликоля был исследован осадок мочи. Оценка содержания олигомерных форм уромодулина в моче животных проводилась методом анализа треков наночастиц и динамического рассеяния света. Результаты. Показано, что на фоне развития патологии наблюдается уменьшение концентрации олигомерных форм уромодулина в моче, на начальных этапах развития патологии за счёт увеличения фракции крупных частиц (более 200 нм, олигомерная форма 28 МДа). При дальнейшем развитии патологического процесса на завершающем этапе наблюдается радикальное уменьшение концентрации частиц в моче (более чем в 2 раза). Заключение. Полученные данные показали относительно низкую корреляцию между длительностью моделирования патологии и тяжестью проявления уролитиаза (r-Пирсона = 0,49, p-value = 0,0003). Концентрация олигомеров уромодулина в моче животных уменьшается на фоне увеличения количества кристаллов в осадке мочи, что вероятно связанно с включением уромодулина в структуру кристаллов осадка. Studying uromodulin oligomeric forms in urolithiasis development is important fundamental and applied problem. Despite the variety of in vivo models of urolithiasis, there is currently no information about concentration dynamics and fractional composition of uromodulin oligomeric forms in urine for animals at various stages of pathological process developmen. The purpose We investigate dynamics uromodulin oligomeric forms in urine of animals against the background of development hyperoxalate urolithiasis induced by exogenous administration of 1% ethylene glycol solution as a non-alternative source of drinking. Methods. For urine samples at various stages of pathogenesis, general clinical and biochemical analysis were carried out, for urine samples before the start of pathology modeling and against the background of exogenous administration of ethylene glycol, urine sediment was examined. The study of urine sediment and content uromodulin oligomeric forms was carried out on 0th, 7th, 14th, 21st and 28th days of pathology modeling. Evaluation of the content of uromodulin oligomeric forms in urine of animals was carried out by nanoparticles track analysing and dynamic light scattering. Results. It is shown that against the background of the pathology development there is a decrease in the concentration of oligomeric forms of uromodulin in the urine, at the initial stages of pathology development due to an increase in the fraction of large particles (over 200 nm, oligomeric form 28 МDa). With further development of the pathological process at the final stage, there is a radical decrease in the concentration of particles in the urine (more than 2-fold). Conclusion. The obtained data showed a relatively low correlation between the duration of pathology modeling and the severity of urolithiasis manifestation (Pearson's r = 0.49, p-value = 0.0003). Concentration of uromodulin oligomers in animals urine decreases with an increase in the amount of crystals in urine sediment, which is probably associated with inclusion of uromodulin in structure of sediment crystals.

Fish Cytolysins in All Their Complexity

The majority of the effects observed upon envenomation by scorpaenoid fish species can be reproduced by the cytolysins present in their venoms. Fish cytolysins are multifunctional proteins that elicit lethal, cytolytic, cardiovascular, inflammatory, nociceptive, and neuromuscular activities, representing a novel class of protein toxins. These large proteins (MW 150–320 kDa) are composed by two different subunits, termed α and β, with about 700 amino acid residues each, being usually active in oligomeric form. There is a high degree of similarity between the primary sequences of cytolysins from different fish species. This suggests these molecules share similar mechanisms of action, which, at least regarding the cytolytic activity, has been proved to involve pore formation. Although the remaining components of fish venoms have interesting biological activities, fish cytolysins stand out because of their multifunctional nature and their ability to reproduce the main events of envenomation on their own. Considerable knowledge about fish cytolysins has been accumulated over the years, although there remains much to be unveiled. In this review, we compiled and compared the current information on the biochemical aspects and pharmacological activities of fish cytolysins, going over their structures, activities, mechanisms of action, and perspectives for the future.

Discovery and initial characterization of YloC, a novel endoribonuclease in Bacillus subtilis

Bacillus subtilis genome is predicted to encode numerous ribonucleases, including four 3’ exoribonucleases that have been characterized to some extent. A strain containing gene knockouts of all four known 3’ exoribonucleases is viable, suggesting that one or more additional RNases remain to be discovered. A protein extract from the quadruple RNase mutant strain was fractionated and RNase activity was followed, resulting in identification of an enzyme activity catalyzed by the YloC protein. YloC is an endoribonuclease and is a member of the highly conserved “YicC family” of proteins that is widespread in bacteria. YloC is a metal-dependent enzyme that catalyzes cleavage of single-stranded RNA, preferentially at U residues, and exists in an oligomeric form, most likely a hexamer. As such, YloC shares some characteristics with the SARS-CoV Nsp15 endoribonuclease. While the in vivo function of YloC in B. subtilis is yet to be determined, YloC was found to act similarly to YicC in an Escherichia coli in vivo assay that assesses decay of the small RNA, RyhB. Thus, YloC may play a role in small RNA regulation.

Comprehensive multidimensional study of the self-assembly properties of a three residue substituted β3 oligoamide

Substituted β3 oligoamides form a unique self-assembling system where each monomer folds into a helix containing approximately three β3 amino acids per turn, yielding a geometrically well-defined cylindrical building block that, when N-acylated, is able to self-assemble head-to-tail into nanorods that can reach several 100 μm length. It was shown in previous works that self-assembly can be achieved with a three residue long oligoamide as well that lacks any intramolecular H-bonds, yet it crystallizes in a helix-like conformation. The self-assembly properties of these small oligoamides are however elusive, suggesting a more complex system than the self-assembly of the H-bond stabilized helical monomers. Here we focus on the self-assembly behaviour of a three residue oligoamide, Ac-β3[LIA] where the letters denote the side chain of the analogous α amino acid. Ac-β3[LIA] can yield highly inhomogeneous suspensions in water with a broad range of large fibrous structures that seem to be very stable, yet occasionally fibre growth is only observed upon heating. The small size of the monomer suggests a highly dynamic equilibrium yet all previous attempts failed to clearly identify low molecular weight species. Therefore a special methodology was employed in this study to characterize the suspensions at different size ranges: SANS that is optimal to measure the small oligomers and cross sectional diameter of the assemblies, DLS that is sensitive to the large populations and therefore the length of the superstructures, and NMR that is sensitive to monomeric and small oligomeric form, in conjunction with IR spectroscopy to probe the folding and AFM to image the morphology of the assemblies. Temperature ramping was used to perturb the system to probe the dynamicity of the self-assembly. It was found that the anomalous self-assembly behaviour of Ac-β3[LIA] is caused by its two stable conformations, a helix-building “horseshoe” fold and a linear conformer. The latter is exclusively found in monomeric form in solution whereas the horseshoe fold is stable in solid phase and in fibrous assemblies. Small oligomers were absent. Thus the self-assembly of Ac-β3[LIA] is arrested by the activation energy need of the conformation change; fibre growth might be triggered by conditions that allow increased conformational freedom of the monomers. This observation may be used to develop strategies for controlled switchable self-assembly.

Functional assessment of the V390F mutation in the CCTδ subunit of chaperonin containing tailless complex polypeptide 1

The chaperonin containing tailless complex polypeptide 1 (CCT) is a multi-subunit molecular chaperone. It is found in the cytoplasm of all eukaryotic cells, where the oligomeric form plays an essential role in the folding of predominantly the cytoskeletal proteins actin and tubulin. Both the CCT oligomer and monomeric subunits also display functions that extend beyond folding, which are often associated with microtubules and actin filaments. Here, we assess the functional significance of the CCTδ V390F mutation, reported in several cancer cell lines. Upon transfection into B16F1 mouse melanoma cells, GFP-CCTδV390F incorporates into the CCT oligomer more readily than GFP-CCTδ. Furthermore, unlike GFP-CCTδ, GFP-CCTδV390F does not interact with the dynactin complex component, p150Glued. As CCTδ has previously been implicated in altered migration in wound healing assays, we assessed the behaviour of GFP-CCTδV390F and other mutants of CCTδ, previously used to assess functional interactions with p150Glued, in chemotaxis assays. We developed the assay system to incorporate a layer of the inert hydrogel GrowDex® to provide a 3D matrix for chemotaxis assessment and found subtle differences in the migration of B16F1 cells, depending on the presence of the hydrogel.

Neurodegenerative disorders and the current state, pathophysiology and management of Parkinson’s disease

: In last few decades major knowledge has been gained about pathophysiological aspects and molecular pathways behind Parkinson’s disease (PD). Based on neurotoxicological studies and postmortem investigations, now there is a general concept that how environmental toxicants (neurotoxins, pesticides insecticides) and genetic factors (genetic mutations in PD-associated proteins) cause depletion of dopamine from substantia nigra pars compacta region of midbrain and modulate cellular processes leading to pathogenesis of PD. α-Synuclein, a neuronal protein accumulation in oligomeric form, called protofibrils is associated with cellular dysfunction and neuronal death thus possibly contributing to PD propagation. With advances made in identifying loci that contribute for PD, molecular pathways involved in disease pathogenesis are now clear and introducing therapeutic strategy at right time may delay the progression. Biomarkers for PD has helped to monitor PD progression, so personalized therapeutic strategies can be facilitated. In order to further improve PD diagnostic and prognostic accuracy, biomarkersfurther large independent validation is required.

Resonance assignment of coiled-coil 3 (CC3) domain of human STIM1

The protein stromal interaction molecule 1 (STIM1) plays a pivotal role in mediating store-operated calcium entry (SOCE) into cells, which is essential for adaptive immunity. It acts as a calcium sensor in the endoplasmic reticulum (ER) and extends into the cytosol, where it changes from an inactive (tight) to an active (extended) oligomeric form upon calcium store depletion. NMR studies of this protein are challenging due to its membrane-spanning and aggregation properties. Therefore follow the divide-and-conquer approach, focusing on individual domains first is in order. The cytosolic part is predicted to have a large content of coiled-coil (CC) structure. We report the 1H, 13C, 15N chemical shift assignments of the CC3 domain. This domain is crucial for the stabilisation of the tight quiescent form of STIM1 as well as for activating the ORAI calcium channel by direct contact, in the extended active form.

Lower Oligomeric Form of Surfactant Protein D in Murine Acute Lung Injury Induces M1 Subtype Macrophages Through Calreticulin/p38 MAPK Signaling Pathway

Surfactant protein D (SP-D) plays an important role in innate and adaptive immune responses. In this study, we found that the expression of total and de-oligomerized SP-D was significantly elevated in mice with lipopolysaccharide (LPS)-induced acute lung injury (ALI). To investigate the role of the lower oligomeric form of SP-D in the pathogenesis of ALI, we treated bone marrow-derived macrophages (BMDMs) with ALI-derived bronchoalveolar lavage (BAL) and found that SP-D in ALI BAL predominantly bound to calreticulin (CALR) on macrophages, subsequently increasing the phosphorylation of p38 mitogen-activated protein kinase (MAPK) and expression of interleukin (IL)-6, tumor necrosis factor (TNF)-alpha, IL-10, and CD80. However, anti-SP-D (aSP-D) and anti-calreticulin (aCALR) pretreatment reversed the SP-D binding and activation of macrophages induced by ALI BAL or de-oligomerized recombinant murine SP-D (rSP-D). Lack of signal transducer and activator of transcription (STAT)6 in STAT6-/- macrophages resulted in resistance to suppression by aCALR. Further studies in an ALI mouse model showed that blockade of pulmonary SP-D by intratracheal (i.t.), but not intraperitoneal (i.p.), administration of aSP-D attenuated the severity of ALI, accompanied by lower neutrophil infiltrates and expression of IL-1beta and IL-6. Furthermore, i.t. administration of de-oligomerized rSP-D exacerbated the severity of ALI in association with more pro-inflammatory CD45+Siglec-F(-) M1 subtype macrophages and production of IL-6, TNF-alpha, IL-1beta, and IL-18. The results indicated that SP-D in the lungs of murine ALI was de-oligomerized and participated in the pathogenesis of ALI by predominantly binding to CALR on macrophages and subsequently activating the pro-inflammatory downstream signaling pathway. Targeting de-oligomerized SP-D is a promising therapeutic strategy for the treatment of ALI and acute respiratory distress syndrome (ARDS).

The Role of Soluble Molecules CD25, CD38, and CD95 in the Development of Immunosuppression in Cytomegalovirus Infection

Introduction: Cytomegalovirus (CMV) infection is a common beta-herpesvirus infection widely spread in the human population. The proportion of infected population increases with age and approaches 100 % in elderly people. The infection is usually latent but is capable of reactivation when immunosuppression develops. The mechanisms of reactivation are not fully understood. The objective of our study was to evaluate the role of soluble molecules CD25, CD38, CD95 in the development of immunosuppression in CMV infection. Materials and methods: We used 18 serum samples from cases of CMV disease in the stage of reactivation, all confirmed by clinical and laboratory data. The patients received treatment in Nizhny Novgorod Infectious Disease Hospital No. 2. The serum content of the total and oligomeric soluble molecules CD25, CD38, and CD95 was identified by ELISA using monoclonal and polyclonal antibodies against human peripheral blood mononuclear cell proteins. The results were recorded spectrophotometrically and evaluated by converting optical density units to conventional units (U/mL). Results: We established an increase in the serum content of total and oligomeric fractions of soluble molecules CD25, CD38, and CD95 in the cases of CMV disease. While the serum content of the total and oligomeric fractions of molecules CD25 and CD38 increased equally, the oligomeric fraction of molecules СD95 demonstrated a more pronounced increase compared to the total fraction of these molecules. Our findings suggest the immune response suppression mechanism associated with initiation of apoptosis of effector T lymphocytes involving oligomeric form of molecules CD95. Conclusion: Changes in the content, structural and functional state of soluble differentiating molecules CD25, CD38, and CD95 indicate their involvement in immunosuppression mechanisms in patients with CMV infection.

The 2.4 Å cryo-EM structure of a heptameric light-harvesting 2 complex reveals two carotenoid energy transfer pathways

We report the 2.4 Ångström resolution structure of the light-harvesting 2 (LH2) complex from Marichromatium (Mch.) purpuratum determined by cryogenic electron microscopy. The structure contains a heptameric ring that is unique among all known LH2 structures, explaining the unusual spectroscopic properties of this bacterial antenna complex. We identify two sets of distinct carotenoids in the structure and describe a network of energy transfer pathways from the carotenoids to bacteriochlorophyll a molecules. The geometry imposed by the heptameric ring controls the resonant coupling of the long-wavelength energy absorption band. Together, these details reveal key aspects of the assembly and oligomeric form of purple bacterial LH2 complexes that were previously inaccessible by any technique.