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The effects of fungal polysaccharides on probiotic bacteria from the genus Lactobacillus
No full textRezultati nedavnih istraživanja pokazuju važan utjecaj crijevne mikrobiote na opće zdravlje i stanje ljudskog organizma. Nove vrste prebiotika, poput polisaharida iz kvasaca i nekih vrsta viših gljiva, pokazuju potencijal kao sastojci novih formulacija tzv. sinbiotika – dodataka prehrani koji kombiniraju probiotike i prebiotike u svrhu postizanja sinergističkih učinaka. Međutim, malo je poznato o osnovama mehanizma prebiotičkog djelovanja takvih spojeva, kao i o pojedinačnim preferencijama različitih vrsta probiotičkih bakterija po pitanju vrste i izvora polisaharida iz gljiva.
U ovome istraživanju različiti ekstrakti polisaharida iz gljiva, pripremljeni metodom ekstrakcije vrućom vodom ili nabavljeni iz komercijalnih izvora, ispitani su po pitanju prebiotičkih svojstava. Uzorci su testirani pomoću metoda enzimatske kvantifikacije kako bi se odredio udio glukana u njihovom sadržaju. Kako bi se ispitala prebiotička svojstva pripremljenih ekstrakata, uvedena je nova visokoprotočna metoda koja se temelji na mjerenju promjene optičke gustoće. Ova metoda omogućila je istovremeno ispitivanje utjecaja više različitih ekstrakata na različite sojeve probiotičkih bakterija. Provedeni su eksperimenti u kojima se istražio utjecaj polisaharida iz gljiva na kinetiku rasta probiotičkih sojeva iz roda Lactobacillus, kao i na patogene bakterije, što je dalo informacije o ključnim parametrima poput duljine trajanja LAG faze te o stopi rasta. Druga svojstva su također istražena, poput promjene sastava bakterijske stanice te preživljenja antibiotika.
Rezultati su pokazali da različite vrste ekstrakata iz gljiva, poglavito pročišćenih beta-glukana iz gljive Pleurotus ostreatus te kvasca Saccharomyces cerevisiae, potiču rast i proliferaciju određenih vrsta iz roda Lactobacillus te moduliraju njihovo svojstvo preživljenja antibiotika. Uz dodatak polisaharida iz gljiva, svih 7 sojeva iz roda Lactobacillus - L. casei VIIB/6, L. helveticus XA/2, L. plantarum IIA/5, L. acidophilus 145, L. rhamnosus GG, L. paracasei VIIB5, i L. paracasei spp. paracasei 1753, imali su kraće trajanje LAG faze (prosječno skraćenje od 7 do 8 sati), dok su stope rasta svih sojeva osim L. paracasei VIIB5, i L. paracasei spp. paracasei 1753 bile povećane (prosječno dvostruko povećanje). Različite magnitude učinaka primijećene su u različitim kombinacijama ekstrakata i sojeva.
Ovo istraživanje pružilo je osnovu za razjašnjenje mehanizma stimulacije rasta koju polisaharidi iz gljiva uzrokuju kod probiotičkih sojeva bakterija, kao i za brži probir njihovih optimalnih kombinacija u svrhu formuliranja inovativnih sinbiotika.Recent studies have emphasized the role of gut microbiota on the overall health and wellbeing of humans. Novel types of prebiotics, such as fungal polysaccharides found in yeast and mushrooms, show potential in the formulation of new formulas of synbiotics – dietary supplements which combine probiotics and prebiotics to induce synergistic effects. However, little is known of the underlying mechanism of prebiotic action of such compounds, or the specific preferences of different probiotic bacteria in terms of fungal polysaccharide types and sources.
In this research, different fungal polysaccharide extracts, prepared either by hot-water extraction or acquired from commercial producers, were tested for their prebiotic properties. Samples were tested for glucan content using enzymatic quantification methods. To assess the prebiotic properties of prepared extracts, a novel, high-throughput method was introduced using optical density measurement. This allowed screening of the effects of multiple extracts on different strains of probiotic bacteria, simultaneously. Experiments were conducted to evaluate the effect of fungal polysaccharides on the growth kinetics of probiotic strains from the genus Lactobacillus, as well as pathogenic bacteria, providing information on key parameters such as LAG phase duration and growth rate. Other properties, such as bacterial cell composition and antibiotic survival were also investigated.
Results show that different types of fungal extracts, especially purified beta-glucan extracts from Pleurotus ostreatus and Saccharomyces cerevisiae, induce the growth and proliferation of certain Lactobacillus species and modulate their antibiotic survival. When supplemented with fungal polysaccharides, all 7 Lactobacillus strains - L. casei VIIB/6, L. helveticus XA/2, L. plantarum IIA/5, L. acidophilus 145, L. rhamnosus GG, L. paracasei VIIB5, and L. paracasei spp. paracasei 1753 had shorter LAG phases (average 7-8h reduction), while the growth rates of all strains, except L. paracasei VIIB5, and L. paracasei spp. paracasei 1753, were increased (average 2-fold increase). Different magnitude of effects was observed in different strain-extract combinations.
This research provides the basis for elucidation of the mechanism of growth stimulation of fungal polysaccharides on probiotic bacteria, as well as for faster screening of their optimal combinations in the aim of formulating innovative synbiotics
Aegerolysin proteins in water extracts of selected ascomycetes and basidiomycetes
No full textEgerolizini su proteini koji su dosada otkriveni u gljivama, biljkama, praživotinjama, bakterijama, čak i virusima. Prvi izolirani egerolizin je Asp-hemolizin iz plijesni Aspergillus fumigatus. Njihova točna biološka uloga još nije poznata, ali je utvrđeno da ovi proteini, molekulske mase 15-20 kDa, sami ili u kombinaciji s drugim proteinima prave pore u staničnim membranama i umjetnim lipidnim membranama (liposomima).
Cilj diplomskog rada je dokazati prisutnost egerolizinskih proteina u višim gljivama (bazidiomicetama) za koje još ne postoje podaci o prisutnosti gena za proteine iz porodice egerolizina, kao i u nižim gljivama (askomicetama) koje u svojim genomima posjeduju gene za egerolizine.
Za otkrivanje prisutnosti egerolizina iskoristili smo njihovu sposobnost da se specifično vežu na liposome koji sadrže sfingolipide ceramid fosfoetanolamin ili sfingomijelin u kombinaciji s kolesterolom.
Rezultati su pokazali da jedna gljiva posjeduje proteine egerolizine, a nema hemolitičku aktivnost niti sposobnost permeablizacije membrane.Aegerolysins are proteins discovered in fungi, plants, protozoa, bacteria, and even viruses. First isolated aegerolysin was Asp-hemolysin from mould Aspergillus fumigatus. The exact biological role of aegerolysins is not revealed yet, but it was confirmed that these 15-20kDa proteins, alone or accompanied with other proteins induce pores in natural and artificial cell membranes.
The main goal of this thesis is to prove the presence of aegerolysins in higher fungi (Basidiomycota) for which there is no available data about the presence of genes coding for aegerolysins proteins, and in the lower fungi (Ascomycota) that harbour aegerolysin-coding genes in their genomes.
To discover new aegerolysins, we used their ability to specifically bind to liposomes consisting of ceramide phosphoethanolamine or sphingomyelin in combination with cholesterol.
Results showed that only one fungus has aegerolysins proteins, but doesn’t have hemolytical activity nor membrane permeability ability
Investigation of silver(I) complexes with Schiff's bases by NMR spectroscopy
Get PDFReakcijom piridoksal hidroklorida, odnosno neutralnog piridoksala s 2-amin-4-klorfenolom u molarnom omjeru 1 : 1 u otopini etanola pripravljene su Schiffove baze (C14H14ClN2O3)Cl, odnosno C14H13ClN2O3. Mehaničkim miješanjem dobivenih spojeva u ahatnom tarioniku bez dodatka otapala, ali i reakcijom kondenzacije u otopini etanola i struji dušika sa srebrovim(I) nitratom, odnosno srebrovim(I) acetatom u molarnom omjeru 1 : 2 pripravljeni su srebrovi kompleksi opće formule [Ag(C14H14ClN2O3)2]Cl∙A, odnosno [Ag(C14H13ClN2O3)2]A (A = NO3―, CH3COO―). Strukture svih izoliranih spojeva u otopini okarakterizirane su 1D tehnikama višenuklearne spektroskopije NMR (1H, 13C APT, 13C{1H}, 15N) te potvrđene 2D tehnikama NMR (1H—1H COSY, 1H—1H NOESY, 1H—13C HMQC, 1H—13C HMBC, 1H—15N HMBC). Kompleksi su u čvrstom stanju okarakterizirani spektroskopijom IR zračenja.The reaction of pyridoxal hydrocloride or neutral piridoxal with 2-amine-4-chlorophenols in a 1 : 1 molar ratio in ethanol solution Schiff base were prepared of formula (C14H14ClN2O3)Cl and C14H13ClN2O3 respectively. By mechanical stirring of the obtained bases in agate mortar and pestle, but also by condensation reaction in a solution of ethanol, nitrogen stream at room temperature, with silver(I) nitrate or silver(I) acetate at a molar ratio of 1 : 2, the silver complexes of the compounds of general formula [Ag(C14H14ClN2O3)2]Cl∙A, and [Ag(C14H13ClN2O3)2]A (A = NO3―, CH3COO―) were obtained. All synthesized compounds were characterized in solution by 1D multinuclear NMR spectroscopy (1H, 13C APT, 13C{1H}, 15N) and confirmed by 2D NMR spectroscopy techniques (1H—1H COSY, 1H—1H NOESY, 1H—13C HMQC, 1H—13C HMBC, 1H—15N HMBC). In solid state, all compounds were characterized by IR spectroscopy
Chemical modification and physico-chemical characterization of the borosilicate glass surfaces for a covalent protein immobilization
No full textVezanje proteina na površine čvrstih nosača predstavlja neizbježan korak
u proizvodnji proteinskih bio uređaja. Ono se može postići kao reverzibilno
vezanje (fizisorpcija) ili ireverzibilno (kemisorpcija) putem stvaranja
kovalentne veze između površinskih skupina i proteina. Potonji način ima
prednost zbog trajnosti površinskog sloja, no vrlo često se kovalentnim
vezanjem na površinske skupine narušava tercijarna struktura proteina,
zbog čega gubi svoju aktivnost. Za postizanje kovalentne veze proteina s
površinom najčešće se koriste molekule tzv. cross-linkeri, a njihov odabir
ovisi o vrsti kemijskih skupina na površini, te ograncima aminokiselina
koje su dostupne na vanjskoj površini proteina. Za kemijsku modifikaciju
stakla, na čijoj se površini nalaze silanolne skupine, najčešće se koriste
organosilani. To su organske molekule koje na jednom kraju nose silanske
skupine te putem njih kondenziraju sa silanolnim skupinama stakla, a na
drugom terminalnu funkcionalnu skupinu koja omogućuje stvaranje
kovalentne veze s ciljanom molekulom. U slijedećim koracima se
terminalna skupina površinski vezanih organosilana prevodi u skupinu
koja može reagirati s nekim od aminokiselinskih ostataka proteina. Doseg
reakcija na površinama ograničen je geometrijskim utjecajem površine na
difuziju, kao i drugim fizikalno-kemijskim svojstvima, kako otopljenih
reaktanata tako i površine (prvenstveno liofilnost te površinski naboj).
Stoga je za uspješnost vezanja prekursora te proteina na površinu,
potrebno optimizirati fizikalno-kemijske uvjete provođenja tih reakcija
(pH, ionska jakost, odabir pomoćnog otapala…). Također, s obzirom da
standardne tehnike najčešće nisu prikladne za kemijsku analizu sastava
površine zbog relativno malih koncentracija analita, prilikom razvoja
uspješne i reproducibilne metode za vezanje proteina, u svrhu detekcije
vezanja je nužno mjeriti gore spomenuta fizikalno-kemijska svojstva
površine, kao posljedicu promjene kemijskog sastava. Cilj je uspostaviti
skup analitičkih metoda kojima je moguće nedvosmisleno dokazati
vezanje kako proteina, tako i pojedinih prekursora na površinu
borosilikatnog stakla. Za mjerenje hidrofobnosti površine korišteno je
mjerenje kontaktnog kuta sjedeće kapi, zatim mjerenje ɀ-potencijala za
analizu površinskog naboja, a za određivanje elementarnog sastava
površine korištena je spektroskopija fotoelektrona rendgenskim zrakama
(XPS). Gore navedeni cilj je ovim istraživanjem i ostvaren, tj.
uspostavljena je metodologija za kemijsku modifikaciju površine
borosilikatnog stakla za vezanje proteina i njenu analizu.The binding of proteins on solid surfaces represents an unavoidable step
in protein bio-devices production. It can be achieved as reversible
(physisorption) or irreversible (chemisorption) binding via covalent bonds
between surface groups of the solids and proteins. The latter has the
advantage due to the longevity of the surface layer, but often tertiary
structure of the protein is disrupted because of which proteins lose their
activity. For achieving of the covalent binding of the protein to the surface
groups, cross-linking molecules are most often used. Their selection is
based on the type of the chemical groups on the solid surface and amino
acid residues on the outer surface of the protein. For chemical
modification of glass, with silanol groups on the surface, organosilanes are
most often used. These are molecules featured with silane groups on the
one end which allow condensation with silanol groups at the glass surface,
and a terminal functional group on the other end which allows creation of
a covalent bond with the target molecule. In the next steps the terminal
group of surface-bound organosilanes is converted to a chemical group
that can react with some of the amino acid residues of the protein. The
reach of the surface reactions is limited by the geometrical influence of
the surface on diffusion, as well as other physico-chemical properties of
solvated reactants and the surface (primarily liophilicity and surface
charge). Therefore, in order to achieve the bonding of the precursors and
binding of proteins on the surface, optimization of the physico-chemical
conditions for the involved reactions (pH, ionic strength, auxiliary solvent
etc.) is required. Since the standard analytical techniques are most often
not suitable for the chemical analysis of the surface, due to relatively low
concentrations of the analyte, during the development of successful and
reproducible methods for protein binding, changes in physico-chemical
properties that accompany variation in chemical composition have to be
measured for the detection of binding. The aim is to establish an
assemblage of analytical methods which can be used as unequivocal proof
of protein binding, as well as individual precursors on the surface of
borosilicate glass. For surface hydrophobicity measurements, sessile drop
contact angle measurements were used, zeta-potential measurements
were utilized for the analysis of surface charge, while the X-ray
photoelectron spectroscopy was employed for the analysis of elemental
composition. This allowed successful assessment of the methods and
protocols necessary for chemical modification of the borosilicate glass
surface for protein binding, and accomplishment all of the thesis goals
In vitro SUMOylation of proteins involved in mental illness
No full textPosttranslacijske modifikacije implicirane su u velikom broju bolesti, uključujući kronične mentalne poremećaje. SUMOilacija jest vrsta posttranslacijske modifikacije koja uključuje reverzibilno, kovalentno vezanje SUMO (small ubiquitin-related modifiers) proteina za lizin supstrata. Uloge ove modifikacije su brojne, kao i njezini supstrati, a uključuju razvoj mozga, formaciju sinapsi, kao i maturaciju neurona. Disrupted in Schizophrenia 1 (DISC1) protein također spada u mete SUMOilacije, a kodiran je DISC1 genom, rizičnim faktorom za razvoj bolesti poput shizofrenije, bipolarnog poremećaja te depresije. No, točan mehanizam regulacije ovog proteina još je uvijek nepoznat, dijelom radi manjka informacija o njegovoj strukturi. Usprkos tome, nedavno je otkriveno kako se DISC1 sastoji od četiri prepoznatljive regije nazvane “D”, “I”, “S” i “C”. U ovome smo radu odlučili potvrditi SUMOilaciju DISC1 proteina in vitro, uzevši u obzir njegovu novootkrivenu strukturu. Nadalje, istražili smo dvije moguće mete SUMOilacije, proteine TRIOBP-1 (TRIO F-actin-binding protein 1) i dysbindin 1A, također implicirane u patologiji mentalnih bolesti. Za eksprimiranje novootkrivenih DISC1 regija koje u sebi sadrže potvrđeno mjesto SUMOilacije na lizinu 643 (K643) koristili smo in vitro metodu rekombinantne SUMOilacije. Također, predvidjeli smo moguća mjesta SUMOilacije u proteinima TRIOBP-1 i dysbindin 1A koristeći se in silico metodom predikcije te ih analizirali in vitro. Na temelju priloženih rezultata, predlažemo in vitro SUMOilaciju unutar “S” regije DISC1 proteina. Uz to, daljnjim smo istraživanjem ukazali na postojanje dodatnog SUMOilacijskog mjesta unutar DISC1 sekvence. Također, analizom podataka uočili smo kako i TRIOBP-1 podliježe SUMOilaciji. Ovo je, prema našim saznanjima, prva implikacija TRIOBP-1 proteina kao mete SUMOilacije. Nasuprot tome, dysbindin 1A nije se pokazao metom SUMOilacije. Sveukupno, ovi rezultati predstavljaju dobar temelj za buduća istraživanja povezanosti SUMOilacije s patologijom mentalnih bolesti.Post-translational modifications have long been implicated in a range of diseases, including chronic mental illnesses. SUMOylation is a reversible, covalent post-translational modification that involves attachment of small ubiquitin-related modifiers (SUMOs) to lysine residues of numerous target proteins. It has a role in a wide array of cellular processes, including, but not limited to brain development, synapse formation and neuronal maturation. Disrupted in Schizophrenia 1 (DISC1) is a known SUMOylation target. It is encoded by the DISC1 gene, a major risk factor in numerous mental illnesses including schizophrenia, bipolar disorder and major depression. However, the mechanism by which it is regulated is still largely unknown, partly due to a lack of knowledge of the structure of DISC1. Recently, four novel DISC1 regions labelled “D”, “I”, “S” and “C” have been discovered. Here, we set out to confirm DISC1 SUMOylation in vitro, taking its recently discovered domain structure into consideration. Furthermore, we investigated two additional SUMOylation targets, TRIO F-actin-binding protein 1 (TRIOBP-1) and dysbindin 1A, both of which are implicated in pathology of mental illnesses. We used a recombinant SUMOylation assay to express novel DISC1 regions containing the previously reported SUMOylation site at lysine residue 643 (K643). Furthermore, we employed in silico methods to predict possible SUMOylation sites in TRIOBP-1 and dysbindin 1A, then tested them in vitro. In this study, we suggest in vitro DISC1 SUMOylation of K643 within the “S” region. However, our data also implicates the existence of another SUMOylation site within the DISC1 sequence. Furthermore, our results suggest that TRIOBP-1 is prone to SUMOylation, which is, to our knowledge, the first report of such occurrence. Finally, we report negative results on the SUMOylation of dysbindin 1A. Altogether, these results present a solid base for future research of the involvement of SUMOylation in the pathology of mental illnesses
Analiza LC3-II kao markera autofagije u različitim razvojnim stadijima megakariocita
Get PDFAutofagija je unutarstanični proces uključen u razgradnju neželjenih citoplazmatskih komponenti poput proteina, lipida, nukleotida i sl., ali i staničnih organela s ciljem ponovnog korištenja degradiranog sadržaja u biosintetskim procesima ili za stvaranje energije. Upravo zbog toga autofagija se smatra jednim od glavnih mehanizama adaptacije stanice na nepovoljne unutarstanične uvjete kao što su deprivacija hranjivih tvari, hipoksija, signali urođene imunosti i brojni drugi. Osim fizioloških signala, proces autofagije može biti ometen korištenjem različitih farmakoloških agensa koji potiču njenu aktivnost, ili pak inhibiciju. Poznate su njene brojne fiziološke uloge, a jedna od najvažnijih je ona u razvoju i diferencijaciji. Budući da između ostalog ima utjecaj i na megakariopoezu i trombopoezu, u ovom radu Western blot analizom i korištenjem protutijela na LC3-II specifični marker autofagije, pratili smo njenu razinu u različitim razvojnim stadijima primarnih mišjih megakariocita i trombocita, te u ljudskoj megakariocitnoj staničnoj liniji DAMI. Nadalje, razinu autofagije analizirali smo i u megakariocitima iz koštane srži tretiranim induktorom ili inhibitorima autofagije. U konačnici, LC3 protein u DAMI stanicama pratili smo njihovom transfekcijom mRFP-GFP-LC3 plazmidom s ciljem analize autofagijskog fluksa. Prisutstvo LC3 proteina vidljivo je u obliku fluorescentno obojenih vezikula koje koreliraju s autofagijskim strukturama unutar stanice. Na temelju dobivenih rezultata, utvrdili smo da razina LC3-II raste s diferencijacijom megakariocita, te da je autofagijski fluks veći u nezrelijim stanicama.Autophagy is an intracellular process involved in degradation of both unwanted cytoplasmic components such as proteins, lipids or nucleotides, and different cellular organelles. Its goal is to reuse degraded content in biosynthetic processes or to generate energy. Because of that, autophagy is considered to be one of the main mechanisms for cell adaptation to unfavorable intracellular conditions such as nutrient starvation, hypoxia, innate immune signals etc. In addition to physiological signals, the autophagy process can be disrupted by the use of various pharmacological agents that stimulate its activity or inhibition. There are plenty of autophagy physiological roles, but one of the most important is in the development and differentiation. Because it also affects megakaryopoiesis and thrombopoiesis, in this study, using Western blot analysis and antibody against LC3-II autophagy marker, we examined its levels in different developmental stages of primary mouse megakaryocytes and platelets, and also in the human cell line DAMI. Furthermore, we analyzed the level of autophagy in the bone marrow derived megakaryocytes that were treated with inducer or inhibitors of autophagy. Finally, LC3 was also monitored by transfecting DAMI cells with mRFP-GFP-LC3 plasmid, in order to assess autophagic flux. The presence of LC3 was visualised in the form of punctate structures that correlate with autophagy structures within the cells. Based on the results obtained, we determined that LC3-II levels increase with differentiation of megakaryocytes and that autophagy flux is higher in less mature cells
Molecular dynamics studies of the steps in the catalytic cycle of gamma-secretase: searching for novel drug-design strategies for Alzheimer's disease
No full textBackground: Alzheimer’s disease is the most common type of dementia, and currently the biggest financial burden for health care providers in developed countries. γ-secretase is a large membrane embedded protease and a major target in drug development efforts based on over 250 mutations in its structure that can cause the disease. We used all-atom and coarse-grained molecular dynamic studies to describe the structural changes in the catalytic cycle of γ-secretase.
Results: Adaptive Poisson-Boltzmann calculations showed that γ-secretase is a highly polarized enzyme that can attract N-terminal and C-terminal parts of its protein substrate. The presenilin subunit of γ-secretase has a cluster of positive residues that can stabilize the negatively charged C-terminal of the catalytic intermediates. The nicastrin subunit can close above the catalytic site like a lid or rotate above the enzyme. The 6 Å distance between the active site aspartates showed that the enzyme structure from the Protein Data Bank represents an inactive enzyme. In silico mutations showed that 4.5 Å distance between the active site aspartates is optimal for catalysis. Asparagines in place of the active site aspartates can be used to mimic the active enzyme in structural studies of substrate binding steps. The cholesterol-rich membrane supports the optimal distance between the active site aspartates, what is consistent with experimental results which showed that cholesterol is crucial for the enzyme activity. Disease causing mutation G384A can increase the distance between the active site aspartates, what can explain experimentally observed pathogenic decrease in the activity of mutated enzyme.
Conclusion: Molecular dynamic studies can be used for the development of drug candidates that can specifically target changes in γ-secretase structure that support the pathogenic changes in the enzyme activity.Pozadina: Alzheimerova bolest je najčešći oblik demencije i trenutno najveći financijski teret za zdravstvene sustave u razvijenim zemljama. γ-sekretaza je velika membranska proteaza koja je postala važna meta u pokušajima razvoja lijekova zbog preko 250 mutacija u njenoj strukturi, koje mogu izazvati bolest. U ovom radu, pomoću coarse-grained i all-atom studija molekularne dinamike opisali smo strukturne promjene u katalitičkom ciklusu γ-sekretaze.
Rezultati: Adaptivni Poisson-Boltzmann izračuni pokazali su da je γ-sekretaza vrlo polariziran enzim koji može privući N-terminalni i C-terminalni dio svog supstrata. Presenilinska podjedinica γ-sekretaze, sadrži klaster pozitivnih aminokiselina koje mogu stabilizirati negativno nabijeni C-terminalni dio katalitičkih međuprodukata. Nikastrinska podjedinica se može poput poklopca preklopiti iznad katalitičkog mjesta te rotirati iznad enzima. Udaljenost od 6 Å između aspartata u aktivnom mjestu pokazala je da struktura enzima dostupna na Protein Data Bank predstavlja inaktivni enzim. In silico mutacije pokazale su da je udaljenost od 4.5 Å između aspartata optimalna za katalizu. Asparagini umjesto aspartata u aktivnom mjestu mogu služiti za oponašanje aktivnog enzima kod istraživanja strukture u koracima vezanja supstrata. Optimalna udaljenost između aspartata u aktivnom mjestu je u membranama bogatim kolesterolom, što se slaže sa rezultatima eksperimenata koji su pokazali da je kolesterol presudan za enzimsku aktivnost. G384A mutacija, koja izaziva bolest, može povećati udaljenost između aspartata u aktivnom mjestu, što može objasniti eksperimentalno potvrđeno patogeno smanjenje aktivnosti mutiranog enzima.
Zaključak: Studije molekularne dinamike se mogu koristiti za razvoj potencijalnih lijekova koji će specifično ciljati na promjene u strukturi γ-sekretaze koje vode do patogenih promjena u enzimskoj aktivnosti
Proteomic and glycomic analyses of the potential protective role of zeolite on an osteoporotic rat model
Get PDFBone homeostasis is secured by a combined action of bone forming osteoblasts and bone resorbing osteoclasts. When this balance is impaired, osteopenia is induced, which in time develops into osteoporosis. Antibody glycosylation influences osteoclast differentiation. It is known that immunoglobulin G (IgG) immune complexes positively stimulate osteoclast differentiation, resulting in subsequent inflammatory bone loss. In order to further investigate this process, a method was developed to isolate IgG from serum and to investigate the changes in IgG glycosylation patterns in osteoporotic rats treated with clinoptilolite, a natural zeolite with great detoxification and ion exchange properties. An experimental rat model was set up, and following groups of animals were analysed: healthy control, sham control, ovariectomized control (OVX), OVX supplemented with synthetic zeolite, OVX supplemented with natural clinoptilolite and OVX supplemented with micro activate clinoptilolite. Using affinity chromatography by use of monolithic supports with immobilized immunoglobulin binding ligands as a robust tool for isolation of immunoglobulins, IgG can be purified from rat sera. Compared to proteins A and G, recombinant protein L binds by far the largest number of isoforms of all immunoglobulins. For this reason, this ligand immobilized on a monolithic column has been used in this work. To procure a highly enriched IgG preparation, the fraction with proteins eluted from the column was analysed on a 1D polyacrylamide gel, after which in-gel tryptic digestion of this protein was performed. The resulting peptides were successfully identified by MALDI-TOF/TOF mass spectrometry as parts of IgG heavy and light chains. In the next step, the IgG glycan structure was analysed by use of the same technique, after the glycans have been removed from the protein using deglycosylation enzyme PNGase F. Next to the IgG analysis, liver proteomes of healthy, sham operated and ovariectomized (OVX) rats treated with zeolites were fractionated according to hydrophobicity and each fraction was separately analysed by SDS PAGE. Finally, liver cryo slices were examined by Synchrotron radiation. Present results give us the evidence that the developed high throughput protocols for analysis of glycosylation of rat immunoglobulins, namely IgG, IgA and IgM, as well as the protocol for quantitative proteomic investigations of rat liver proteome, give us the fundament for further investigations by use of a larger number of experimental animals. We suggest that clinoptilolite positively affects bone status in osteoporotic rats as a consequence of signalling changes in the body, particularly those initiated by the liver and the systemic spread of IgG molecules.Rad ne sadrži sažetak na drugom jeziku
Exploring the sodium binding site in trasporters: the NLS1 paradigm
No full textMajor facilitator superfamily domain containing 2A (MFSD2A) gene encodes for a sodium-dependent lysophosphatidylcholine transporter, NLS1. It is essential for importing of fatty acids, like docosahexaenoic acid (DHA), into human brain [1]. Importing of DHA, is essential for normal human brain growth and cognitive function. Also, NLS1 is involved in microcephaly, a condition characterized by a smaller cerebral cortex, where brain weight is reduced. Because exact structure of NLS1 is still unknown, our research could help to get the insight into the molecular mechanism of NLS1.
In this research we constructed a tridimensional model for hNLS1 using sequences of two proteins, melibiose and NLS1. Using computational biology tools, we were able to build and analyze protein, especially at the sodium binding site level. The apo NLS1 (without sodium in the binding site) allowed the binding of three different sodium ions during a 300 ns all atom molecular dynamics (MD) simulation. The sodium-bound MD simulation with a sodium placed between Asp 93 and Asp 97 (named Asp67 and Asp71 in this work) remained stable throughout the 300 ns simulation.
Root Mean Square Deviation values of equilibration and production show that molecule has stabilized with time. Using Visual Molecular Dynamics (VMD) and the Bendix algorithm we were able to represent helical torsions and bending of specific helices derived from the MD simulation. To speed up the analysis of results we used the R-based Bio3D suite, which allowed us to get RMSD, PCA, DCCM and RMSF plots.
Using computational biology tools, we have been able to validate the sodium binding site in NLS1. This study yields a robust three-dimensional model, which satisfies, so far, the experimental data available on NLS1. In structural biology, this model is a very useful tool to design mutants for
NLS1 in order to obtain a three-dimensional structure of a functional protein.Glavna domena facilitatora superfamilije koja sadrži 2A (MFSD2A) gen kodira za natrij-ovisni lizofosfatidilkolinski transporter, NLS1. Važan je za unos masnih kiselina, poput doksosaheksaenoične kiseline (DHA), u ljudski mozak [1]. Unos DHA je neophodno za normalan rast i kognitivnu funkciju ljudskog mozga. Također, NLS1 je uključen u mikrocefaliju, stanje karakterizirano manjim cerebralnim korteksom, smanjenom masom mozga. Budući da je točna struktura NLS1 još uvijek nepoznata, naše istraživanje moglo bi pomoći da dobijete uvid u molekularni mehanizam NLS1.
U ovom istraživanju napravili smo trodimenzionalni model hNLS1 pomoću sekvenci dvaju proteina, melibiose i NLS1. Pomoću računalnih bioloških alata uspjeli smo izgraditi i analizirati bjelančevine, osobito na razini veznog mjesta za natrij. Apo NLS1 (bez natrija u veznom mjestu) omogućilo je vezanje tri različita iona natrija tijekom all atom simulacije molekularne dinamike (MD) u trajanju od 300 ns. Natrij-veznom MD simulacijom s natrijem postavljenim između Asp 93 i Asp 97 (nazvano Asp67 i Asp71 u ovom radu) ostao je stabilan kroz simulaciju od 300 ns.
RMSD, odstupanje vrijednosti ekvilibriranja i produkcije, pokazuje da se molekula stabilizira s vremenom. Pomoću VMDa i Bendix algoritma uspjeli smo prikazati spiralne torzije i savijanje specifičnih heliksa izvedenih iz MD simulacije. Da bismo ubrzali analizu rezultata koristili smo R-based Bio3D paket, koji nam je omogućio da dobijemo RMSD, PCA, DCCM i RMSF grafove.
Pomoću računalnih bioloških alata uspjeli smo provjeriti vezno mjesto natrija u NLS1. Ova studija daje robusni trodimenzionalni model koji zadovoljava do sada eksperimentalne podatke dostupne o NLS1. U strukturnoj biologiji, ovaj model je vrlo koristan alat za dizajniranje mutanata za NLS1 kako bi se dobila trodimenzionalna struktura funkcionalnog proteina
Characterizing the role of optineurin in inflammatory signaling in microglial and neuronal cell lines
No full textMutations in the optineurin gene have recently been linked to amyotrophic lateral sclerosis (ALS), a progressive motor neuron degenerative disease. Optineurin has been implicated in the regulation of many cellular processes such as inflammation, autophagy, Golgi maintenance, and vesicular trafficking. However, the exact physiological function of optineurin remains unknown. Since motor neuron degeneration in ALS has been shown to be driven by microglia, the resident immune cells of the brain, we focused here on the role of optineurin in the regulation of inflammatory signaling. Optineurin deficiency has been proposed to drive neuronal degeneration by overactivation of pro-inflammatory NF-κB signaling and by the insufficient activation of protective TBK1 signaling. However, discrepancies in findings exist between cell lines and animal models and the reason behind it is unclear. In order to contribute to understanding the role of optineurin in these pathways, we established novel microglial (BV2) and neuronal (Neuro2A) optineurin knockout (Optn KO) cell lines by employing the CRISPR/Cas9 technique. We tested the early markers of NF-κB activation, IκB degradation and p65 phosphorylation upon LPS or TNF-α treatment and found no difference in Optn KO compared to WT in neither BV2 or Neuro2A cell lines. We also tested for TBK1 activation in BV2 after LPS treatment and found that TBK1 and IRF3 phosphorylation was substantially impaired in the absence of optineurin. Furthermore, we set up a flow-cytometry based phagocytic assay to test the phagocytic properties of Optn KO BV2 cell line. We found phagocytosis was mildly impaired in optineurin deficient conditions. TNF-α secretion was found to be severely impaired upon LPS treatment in optineurin KO BV2 as well. Our results suggest that optineurin deficiency potentially might drive the progression of ALS in more ways than previously described. In conclusion, in this study, we confirmed previous findings of the role of optineurin in TBK1 activation and opened a new
possibility that insufficient phagocytic clearance by microglia may contribute to motor neuron damage.Mutacije optineurina nedavno su pronađene u bolesnika s amiotrofičnom lateralnom sklerozom (ALS), fatalnom bolesti motornih neurona. Za optineurin se smatra da sudjeluje u regulaciji nekoliko staničnih procesa kao što su upala, autofagija, promet vezikula te održavanje strukture Golgijeva tjelešca. Međutim, točna fiziološka uloga optineurina još uvijek je nepoznata. Pokazano je da su mikroglija stanice, koje su glavne imunološke stanice u mozgu, važne u procesu degeneracije motornih neurona u ALS-u te smo se zbog toga u ovom radu usredotočili na ulogu optineurina u upalnoj signalizaciji. Zapaženo je da deficijencija optineurina prekomjerno aktivira upalni NF-κB put te istovremeno nepotpuno aktivira potencijalno protektivnu TBK1 signalizaciju. Međutim, rezultati dobiveni na staničnim linijama ne slažu se s onima dobivenima na primarnim stanicama te je nejasno zašto. Kako bismo pridonijeli razjašnjavanju uloge optineurina u ovim signalnim putevima, uspostavili smo nove, optineurin deficijentne (Optn KO) mikroglijalne (BV2) i neuronalne (Neuro2A) stanične linije pomoću CRISPR/Cas9 tehnologije. Testirali smo razine ranih markera nakon NF-κB aktivacije, poput degradacije IκB te fosforilacije p65 nakon tretmana TNF-om ili LPS-om, no nismo zamijetili razliku niti između neuronalnih niti mikroglijalnih Optn KO i WT linija. Također, testirali smo TBK1 aktivaciju u BV2 staničnoj liniji te smo zamijetili pad u TBK1 i IRF3 fosforilaciji u Optn KO u usporedbi s WT stanicama. Nadalje, uspostavili smo esej fagocitoze baziran na protočnoj citometriji kako bismo testirali fagocitičke sposobnosti BV2 KO stanične linije. Primijetili smo da je fagocitoza u uvjetima bez optineurina bila blago smanjena. Sekrecija citokina TNF-α nakon tretmana LPS-om također je bila smanjena u Optn KO BV2 stanicama. Naši rezultati sugeriraju da deficijencija optineurina potencijalno posreduje progresiju ALS-a na više načina nego što je do sada opisano. Zaključno, ovo istraživanje potvrđuje ulogu optineurina kao pozitivnog regulatora TBK1 aktivacije te otvara novu mogućnost da
nedostatna mikroglijalna fagocitoza staničnog otpada pridonosi oštećenju motornih neurona