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Doprinos mišićno-intrinzične toksičnosti ALS-mutiranog FUS-a motornoj neurodegeneraciji u ALS-u
No full textAmyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by loss of motor neurons. Most commonly exhibited symptoms include muscular weakness and atrophy. Clinical manifestations begin in adult age and the disease progresses until eventually, death occurs due to respiratory system failure, on average around 3-5 years after clinical onset. The majority of ALS cases are sporadic, with no previously recorded family history of the disease, while a minor proportion of cases are familial. Genes most commonly implicated in ALS are C9orf72, SOD1, FUS and TDP-43. FUS is an RNA/DNA binding protein involved in multiple cellular processes, including transcription regulation, splicing, RNA transport and degradation. Most ALS mutations in FUS are found in the nuclear localization signal, a domain responsible for import of FUS into the nucleus. Mutations in the NLS disrupt FUS import into the nucleus and cause FUS to aggregate within the cytoplasm. ALS is considered to be a distal axonopathy, and the dying-back hypothesis proposes that it originates in the neuromuscular junctions and progresses retrogradely, implicating the involvement of skeletal muscle in disease pathogenesis. The FusΔNLS mutant mice are an adequate model for FUS-ALS as their mutation mimicks those in ALS patients by preventing the transcription of the NLS. The mutation can be fully reversed to wild-type, and its reversal in just the skeletal muscle cells allows the study of non-cell-autonomous contribution of muscle tissue to ALS. FusΔNLS mice exhibit mild but progressive motor deficits, motor axon loss as well as muscular atrophy. Number and area of muscle fibers are slightly reduced, and signs of denervation in motor neurons are also present, as indicated by the reduction in axon numbers. Reversal of the mutation in skeletal muscle partially rescues some phenotypes. The findings suggest that ALS does indeed arise at least in part due to non-cell-autonomous mechanisms of skeletal muscle.Amiotrofična lateralna skleroza (ALS) je progresivna neurodegenerativna bolest karakterizirana gubitkom motornih neurona. Najčešći simptomi uključuju slabost i atrofiju mišića. Kliničke manifestacije započinju u odrasloj dobi i bolest napreduje dok naposljetku ne nastupi smrt uslijed zatajenja respiratornog sustava, u prosjeku 3-5 godina nakon nastupanja kliničkih znakova. Većina slučajeva ALS-a jest sporadično, bez prethodno zabilježene obiteljske povijesti bolesti, s manjim udjelom familijarnih slučajeva. Najčešći geni koji imaju ulogu u ALS-u su C9orf72, SOD1, FUS i TDP-43. FUS je protein koji veže RNA i DNA, i koji je uključen u mnoge stanične procese, poput regulacije transkripcije, „splicinga“, te transporta i razgradnje RNA. Većina ALS mutacija u FUS genu događa se u nuklearnom lokalizacijskom signalu, domeni odgovornoj za unos FUS-a u jezgru. Mutacije u NLS-u ometaju unos FUS-a u jezgru i uzrokuju agregaciju FUS-a unutar citoplazme. ALS se smatra distalnom aksonopatijom, i „dying-back“ hipoteza postulira da bolest započinje u neuromuskularnim spojevima te da napreduje retrogradno, sugerirajući na upletenost skeletnih mišića u patogenezi bolesti. FusΔNLS miševi su prikladan model za FUS-ALS budući da njihova mutacija oponaša mutacije zapažene kod ALS pacijenata time što sprječava transkripciju NLS-a. Mutacija se može u potpunosti poništiti preobraćanjem na divlji genotip, a preobraćanje mutacije samo u stanicama skeletnih mišića omogućava proučavanje ne-stanično-autonomnog doprinosa mišićnog tkiva ALS-u. FusΔNLS miševi iskazuju blage ali progresivne motorne defekte, gubitak motornih aksona, kao i atrofiju mišića. Broj i povšina mišićnih vlakana su blago umanjeni, i znakovi denervacije u motornim neuronima su također prisutni, što je dokazano smanjenjem broja aksona. Preobraćanje mutacije u skeletnim mišićima djelomično spašava neke fenotipe. Ova otkrića sugeriraju da se ALS razvija barem djelomično zbog ne-stanično-autonomnih mehanizama skeletnih mišića
Uloga PI(4,5)P2 i PI4P u sazrijevanju megakariocita, stvaranju protrombocita i funkciji trombocita
No full textDuring megakaryocyte (MK) maturation there is an extensive formation of the demarcation membrane system (DMS) which serves as a membrane reservoir for future platelets (PLTs). During the DMS formation, there is an active transport of vesicles from the Golgi apparatus to the DMS suggesting that lipids and proteins that are necessary for DMS growth originate from the Golgi apparatus. Once mature, MKs release PLTs into the bloodstream where upon encounter with a vessel wall injury PLTs adhere, activate, and aggregate which results in clot formation. The most important lipid that regulates the anterograde Golgi trafficking is the phosphatidylinositol-4-monophosphate (PI4P) and its levels at the Golgi apparatus are controlled to a great extent by the SACM1L phosphatase. In this study, we show that PI4P localizes to the Golgi apparatus and the plasma membrane (PM) of immature MKs while in mature MKs is mostly localized to the PM. We demonstrate that the Golgi pool of PI4P that is controlled by the SACM1L phosphatase is necessary for MK maturation and proplatelet formation because the exogenous expression of wild-type, but not catalytically inactivated SACM1L, retains the dispersion of the Golgi apparatus during MK maturation and results in a decrease of proplatelet formation. In addition, we show that the PM pool of PI4P that is controlled by the PI4KIIIα is also necessary for proplatelet formation since pharmacological inhibition of this kinase decreases proplatelet formation.
PLT activation is accompanied by massive shape change, and an important role in actin reorganization has phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] that is, among other enzymes, regulated by the OCRL phosphatase. Mutations in OCRL cause Lowe syndrome (LS) and it has been shown that, among other symptoms, LS patients can have bleeding problems. Here, we firstly show that for the visualization of PI4P and PI(4,5)P2 in PLTs, a modification of the existing staining protocol is needed and that the best staining of the PM pool of these lipids is obtained when permeabilizing the cells with 0.5% saponin for 5 minutes. Next, we show that the pharmacological inhibition of OCRL in human PLTs leads to an impaired spreading on three different matrices (glass, fibrinogen, and collagen) and that OCRL-inhibited PLTs spread in fibrinogen instead of actin stress fibres, form actin nodules. These actin nodules colocalize with proteins important for actin dynamics (ARP2/3 complex, vinculin, SNX9) and are sites of active signalling. The impaired actin reorganization is mediated by the myosin light chain (MLC) signalling since OCRL-inhibited PLTs have decreased MLC phosphorylation. Furthermore, OCRL-inhibited PLTs have impaired microtubular reorganization, shown by a retained microtubular coil during PLT activation which is accompanied by higher levels of acetylated tubulin. Interestingly, OCRL inhibition does not affect PLT degranulation or integrin activation. Finally, we show that
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the OCRL KO mice also have impaired PLT spreading shown by an increased number of PLTs forming filopodia, and a decreased number of PLTs forming lamellipodia.
Taken together, these results contribute to a better understanding of the role of PI4P during MK maturation and PI(4,5)P2 during PLT activation. They also show the importance of different phosphoinositide pools in MK maturation and proplatelet formation, and they give an insight into the molecular mechanism of impaired PLT activation in LS patients.Megakariociti (MK) prilikom sazrijevanja stvaranju demarkacijski sustav membrana (DMS) koji služi kao rezervoar membrana za buduće trombocite. Za vrijeme stvaranja DMS-a, vezikule iz Golgijevog aparata konstantno putuju prema DMS-u, navodeći na zaključak da lipidi i proteini koji su potrebni za rast DMS-a nastaju u Golgijevom aparatu. Kad MK sazriju, u krvotok otpuštaju trombocite. Trombociti, u kontaktu s oštećenim endotelom krvne žile, adheriraju na mjesto ozljede, aktiviraju se te agregiraju što dovodi do stvaranja krvnog ugruška i zaustavljanja krvarenja. Jedan od najvažnijih lipida koji reguliraju anterogradni transport vezikula od Golgijevog aparata je fosfatidilinozitol-4-monofosfat (PI4P) te su njegove razine u Golgijevom aparatu u velikoj mjeri regulirane djelovanjem SACM1L fosfataze. U ovom istraživanju, pokazali smo da se PI4P u nezrelim MK nalazi u Golgijevom aparatu i na plazma membrani (PM) dok se u zrelim MK uglavnom nalazi na PM. Pokazali smo da PI4P koji se nalazi na Golgijevom aparatu, a čije su razine regulirane djelovanjem SACM1L fosfataze, je potreban za sazrijevanje MK i stvaranje protrombocita jer egzogena ekspresija divljeg tipa, ali ne katalitički ne-aktivnog tipa SACM1L fosfataze ne dozvoljava raspršenje Golgijevog aparata prilikom sazrijevanja MK i dovodi do smanjenog stvaranja protrombocita. Također, pokazali smo da PI4P koji se nalazi na PM, a čije je stvaranje regulirano djelovanjem PI4KIIIα je isto nužan za stvaranje protrombocita jer farmakološka inhibicija ove kinaze smanjuje postotak MK koji stvaraju protrombocite.
Prilikom aktivacije, trombociti mijenjaju svoj oblik, a u procesu reorganizacije aktina važnu ulogu ima fosfatidilinozitol-4,5-bisfosfat [PI(4,5)P2] kojeg, između ostalih enzima, regulira OCRL fosfataza. Mutacije OCRL fosfataze dovode do Lowe sindroma (LS) te je pokazano da, između ostalih simptoma, LS pacijenti mogu imati problema s krvarenjem. U ovom istraživanju smo pokazali da je za vizualizaciju PI4P i PI(4,5)P2 u trombocitima potrebno modificirati postojeći protokol te da je za optimalnu vizualizaciju ovih lipida na PM za permeabilizaciju potrebno koristiti 0.5% saponin 5 minuta. Nadalje, pokazali smo da farmakološka inhibicija OCRL-a u ljudskim trombocitima dovodi do smanjene aktivacije trombocita na različitim matricama (staklo, fibrinogen i kolagen) te da OCRL inhibirani trombociti aktivirani na fibrinogenu umjesto aktinskih stres niti stvaraju aktinske nodule. Ovi aktinski noduli kolokaliziraju s proteinima koji su važni za dinamiku aktina (ARP2/3 kompleks, vinculin, SNX9) te su mjesta aktivne signalizacije. Poremećena reorganizacija aktina je posredovana putem signalizacije lakog lanca miozina (MLC) jer OCRL inhibirani trombociti imaju smanjenu fosforilaciju MLC. Također, OCRL inhibirani trombociti ne reorganiziraju mikrotubule prilikom aktivacije već zadržavaju mikrotubularnu zavojnicu te imaju povišene razine acetiliranog tubulina. Unatoč nepotpunoj aktivaciji, OCRL inhibicija ne utječe na degranulaciju i aktivaciju integrina. Konačno, pokazali smo da se trombociti
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OCRL KO miševa također ne aktiviraju u potpunosti, odnosno da stvaraju više filopodija, a manje lamelipodija.
Zajedno, ovi rezultati doprinose boljem razumijevanju uloge PI4P-a tijekom sazrijevanja MK i PI(4,5)P2 tijekom aktivacije trombocita. Također, ovi rezultati ukazuju na važnost različitih izvora fosfoinozitida tijekom sazrijevanja MK i stvaranja protrombocita i daju uvid u molekularni mehanizam nepotpune aktivacije trombocita u LS pacijenata
Analiza proteinskih karakteristika i patogenosti varijanti optineurina u amiotrofičnoj lateralnoj sklerozi i glaukomu primjenom bioinformatike
No full textAmyotrophic lateral sclerosis (ALS) is a severe, rapidly progressive neurodegenerative disease that affects upper and lower motor neurons. ALS is characterized by high genetic heterogeneity – so far 17 confirmed or “gold standard” genes and 100 potential risk factors - have been linked to this disease. One of the confirmed genes is OPTN, which encodes for optineurin, a multifunctional, ubiquitin binding adaptor protein proposed to act in various biological processes. OPTN mutations have also been found in glaucoma, another neurodegenerative disease, encompassing a group of eye conditions in which the optic nerve is damaged. The aim of this work was to examine optineurin protein features and to compare OPTN variants present in ALS and glaucoma with naturally occurring variants through different in silico programs. We also examined if the mutations were more frequent in the evolutionary conserved regions of optineurin. The OPTN variants and subsequent protein changes were compiled by expert literature curation and taken from UniProt, OMIM, ClinVar and gnomAD databases. In silico species alignment analysis of optineurin domain conservation was done using ClustalOmega. Our results showed that OPTN variants in both ALS and glaucoma predominantly affect coiled-coil regions of optineurin, but this is comparable to their length. However, ALS variants were enriched in the zinc finger (ZF) domain. They were curiously not enriched in one of the main functional domains of optineurin, the ubiquitin-binding region of ABIN and NEMO (UBAN), but clustered around it. Pathogenicity of OPTN mutations patient mutations was predicted using Polyphen-2, SIFT and PROVEAN programs. Although prediction of pathogenicity differed between the programs for variants present in both ALS and glaucoma, they were consistent in assigning K59N, R83C, Q314L, M447R, E478G, K557T, D564H, L568S, H571Q as damaging protein changes present in ALS, and in assigning E50K as damaging in glaucoma. Eight different species representing major vertebrate genera were taken for in silico species alignment analysis of the optineurin conservation. This analysis showed that 89% of predicted pathogenic variants reported in ALS patients mapped to the conserved regions of OPTN, suggesting that they are more likely to be pathogenic. The highest degree of the conservation, both total and partial, was observed in the UBAN and ZF regions of optineurin. The optineurin E50K glaucoma-linked variant mapped to conserved regions as well. Although variant E322K is located on the conserved position of the optineurin protein, it is predicted to be benign according to Polyphen-2, SIFT and PROVEAN analysis. This in silico research of OPTN variants represents the basis for the further in vivo and in vitro investigations and could possibly help conceive future experimental directions relevant for uncovering optineurin function in neurodegenerative diseases.Amiotrofična lateralna skleroza (ALS) je teška, brzo progresivna neurodegenerativna bolest koja zahvaća gornje i donje motoričke neurone. ALS karakterizira visoka genetska heterogenost s do sada 17 potvrđenih ili ''gena zlatnog standarda'' te >100 potencijalnih faktora rizika povezanih s ovom bolešću. OPTN gen kodira za optineurin, višenamjenski, adaptorski protein koji veže ubikvitin te za koji se pretpostavlja da djeluje u raznim biološkim procesima. Mutacije OPTN gena također su pronađene u još jednoj skupini neurodegenerativnih bolesti – glaukomu – u kojima dolazi do oštećenja i gubitka neurona vidnog živca. Cilj ovog rada bio je usporediti značajke varijanti proteina optineurina i OPTN gena prisutnih u ALS-u i glaukomu s prirodnim varijantama putem različitih in silico programa. Također smo ispitali jesu li mutacije pronađene u pacijentima bile češće u evolucijski očuvanim regijama optineurina. OPTN varijante navedene su stručnim pregledom literature i preuzete iz baza podataka UniProt, OMIM, ClinVar i gnomAD. In silico analiza sličnosti vrsta i očuvanje domene optineurina među vrstama analizirana je pomoću programa ClustalOmega. Naši rezultati pokazuju da OPTN varijante i kod ALS-a i glaukoma pretežno zahvaćaju zavijene zavojnice (Coiled-coil) optineurina, ali to je usporedivo s njihovom duljinom. Međutim, ALS varijante obogaćene su u domeni cinkova prsta (engl. Zinc Finger, ZF). Suprotno očekivanjima, varijante nisu bile obogaćene u ubikvitin-vezujućoj regiji ABIN i NEMO proteina (engl. ubiquitin-binding region of ABIN and NEMO, UBAN), koja predstavlja glavnu funkcionalnu domenu optineurina, ali su bile grupirani oko nje. Patogenost OPTN mutacija je predviđena pomoću programa Polyphen-2, SIFT i PROVEAN. Iako se predviđanje patogenosti djelomično razlikovalo među programima i za varijante u ALS-u i glaukomu, programi su bili dosljedni u označavanju K59N, R83C, Q314L, M447R, E478G, K557T, D564H, L568S, H571Q kao štetne promjene proteina u ALS-u te E50K kao štetne u glaukomu. Osam različitih vrsta koje predstavljaju glavne rodove kralježnjaka uzeto je za in silico analizu sličnosti između vrsta te za ispitivanje očuvanja optineurina i utjecaja konzerviranosti na pojavu mutacija. Analiza sličnosti između vrsta pokazala je da je 89% predviđeno patogenih varijanti optineurina prisutnih u bolesnika s ALS-om mapirano na očuvane regije, što sugerira njihovu patogenost. Najviši stupanj očuvanosti (potpuni i djelomični) uočen je u UBAN i ZF regijama optineurina. Optineurin E50K varijanta, pronađena kod pacijenata s glaukomom, također je smještena u očuvanim regijama. Iako se varijanta E322K nalazi na očuvanom položaju proteina optineurina, prema Polyphen-2, SIFT i PROVEAN analizi je benigna. Zaključno, ovo in silico istraživanje OPTN varijanti predstavlja temelj za daljnja in vivo i in vitro istraživanja i isto bi moglo pomoći u osmišljavanju budućih eksperimenata relevantnih za razumijevanje funkcije optineurina u neurodegenerativnim bolestima
Analysis of NK cells in CD16 deficient animals
No full textPrirodnoubilačke ili stanice NK (engl. natural killer cells, NK cells) važna su sastavnica urođenog imunološkog sustava zbog svoje sposobnosti brzog prepoznavanja i eliminacije tumorskih i virusom zaraženih stanica. Njihova aktivacija ovisi o ravnoteži signala primljenih preko aktivacijskih i inhibicijskih receptora. Jedan od aktivacijskih receptora izraženih na stanicama NK je i CD16 receptor, poznat po svojoj ulozi u staničnoj citotoksičnosti posredovanoj protutijelima (engl. antibody-dependent cellular cytotoxicity, ADCC). Međutim, uloga ovog receptora u drugim aspektima biologije stanica NK još uvijek nije dovoljno istražena. Stoga je naš glavni cilj bio ispitati utjecaj nedostatka izražaja CD16 receptora na stanice NK u različitim organima. Koristili smo genetski modificirane miševe kojima nedostaje izražaj CD16 receptora i usporedili ih s miševima divljeg soja. Proveli smo fenotipsku i funkcionalnu analizu stanica NK. Uočili smo da nedostatak izražaja CD16 receptora dovodi do promjene u sazrijevanju stanica NK, kao i do promjena u njihovom postotku i broju u koštanoj srži. Nedostatak izražaja ovog receptora nije doveo do promjene u izražaju većine analiziranih aktivacijskih i inhibicijskih receptora, izuzev NCR1 receptora čiji je izražaj smanjen kod CD16 deficitnih životinja. Također, CD16 deficitne stanice NK nisu pokazale razliku u sposobnosti produkcije citokina u odnosu na kontrolnu grupu. Rezultati ovoga rada ukazuju na dosad nepoznatu ulogu CD16 receptora u sazrijevanju stanica NK iako su potrebna dodatna istraživanja kako bi se ta uloga do kraja istražila.Natural killer (NK) cells are an important component of the innate immune system due to their ability to rapidly recognize and eliminate tumor cells and virus-infected cells. Their activation depends on a balance of signals received through activating and inhibitory receptors. One key activating receptor expressed on NK cells is the CD16 receptor, known for its role in antibody-dependent cellular cytotoxicity (ADCC). However, the role of this receptor in other aspects of NK cell biology remains largely unexplored. The main aim of this study was to investigate the impact of CD16 receptor deficiency on NK cells in different organs. We used genetically modified mice lacking CD16 expression and compared them to wild-type mice. We performed phenotypic and functional analyses of NK cells. We observed that the absence of CD16 expression leads to changes in NK cell maturation, as well as changes in their percentage and number in the bone marrow. The absence of this receptor did not result in changes in the expression of most analysed activating and inhibitory receptors, except for the NCR1 receptor, which showed reduced expression in CD16-deficient animals. Additionally, CD16-deficient NK cells did not show differences in cytokine production capacity compared to the control group. These findings indicate a previously unknown role of the CD16 receptor in NK cell maturation, although further studies are needed to fully elucidate this role
Contribution of different estrogen receptor alpha (ER) domains in ER-driven transcriptomic profile of mammary epithelial cells and characterization of the ER-PR proximity in ER+ breast cancer
No full textAim of the study: 70% of breast cancers (BC) are estrogen receptor alphapositive (ER+), and typically treated by targeting ER signaling. Previous research showed that membrane ER, as well as activation function 1 (AF-1) and activation function 2 (AF-2) domains are involved in the mammary gland and BC development. We aimed to characterize the contribution of each ER domain in ER regulated transcriptional profile in mammary epithelial cells (MEC). First, we researched whether ER-dependent gene
expression is domain-specific. We tested the hypothesis that pathways relevant for BC are ER domain-dependent, and aimed to characterize whether those pathways are regulated by the cooperation of all domains or by an individual domain. We focused on the dependences of the expression of ER target genes, such as Areg, Greb1, and Pgr on different ER domains. Finally, we aimed to characterize the levels of interactions between PR and ER in cancer cell lines MCF7 and T47D. Material and methods: In this
thesis, RNAseq data of GFP+ MECs from C57Bl6 mice lacking complete ER, membrane ER, AF-1 or AF-2 ER domains were analyzed in R software. MCF7 and T47D cell lines were treated with 100 nM progesterone (P4) to establish the conditions suitable for ER-PR interactions. The expression of Pgr was measured by qPCR and ER-PR interactions were analyzed by proximity ligation assay. Results and conclusions: Each ER domain strongly contributes to ER-regulated gene expression in MECs. To regulate pathways involved in carcinogenesis, cell proliferation, and immune response, ER domains act synergically, but also, in some pathways, an individual domain plays a regulatory role of the whole receptor. ER-PR direct interactions occur
in nuclei, but also in non-nuclear compartments in MCF7 and T47D cells with an increased number of interactions upon P4 treatment
MALDI-MS i LC-MS karakterizacija različitih nusprodukata tijekom sinteze antivirusnih peptid-porfirin konjugata na čvrstom nosaču
No full textThe latest discoveries of promising antiviral drug candidates have brought in
focus so-called peptide-drug conjugates (PDCs). PDCs are class of molecules
formed by linkage, either directly or through a linker, of a drug cargo to a
carrier peptide. Among the various classes of PDCs, the ones with antiviral
properties are drawing lot of attention due to their remarkable pharmacological
potential to fight various viral diseases – one of the biggest humankind health
concerns today. Herein we have performed solid-phase peptide synthesis
(SPPS) of one PDC, with proven in vitro antiviral activity against the ZIKA
virus: PPIX-P1 (VQQLTKRFSL-amide), and characterized the main impurities
in its, and another PDC’s of also proven in vitro anti-ZIKA virus activity: PPIX
P3 (AGILKRW-amide), final purified sample by using Matrix-Assisted Laser
Desorption Ionization – Mass Spectrometry (MALDI-MS) and Liquid
Chromatography – Mass Spectrometry (LC-MS) analytical techniques. First, we
have performed SPPS of peptide P1 and analyzed it by LC-MS and MALDI-MS
to
ensure the credibility of its synthesis. Secondly, conjugation of
protoporphyrin IX (PPIX) with the synthesized peptide at the peptide’s N
terminus was done and the newly synthesized conjugate also analyzed by LC
MS and MALDI-MS analytical techniques. Furthermore, the synthesized
conjugate was cleaved from the solid support and purified by preparative C18
high performance liquid chromatography (HPLC). Obtained pure samples of
PPIX-P1, and PPIX-P3; synthesized and purified in the same methodology,
were then characterized by LC-MS and LC-MS/MS analyses, along with their
impurities. By the MS/MS analysis of some impurities found in the PDCs’
purified samples (m/z 521.7 in case of PPIX-P1 and m/z 473.7 in case of PPIX
P3) we were able to conclude that: (i) peptide part of the conjugate is the
origin of the corresponding impurity and (ii) C-terminal part of the peptide is
more prone to modifications during the synthetic process of the conjugate.
Therefore, in order to achieve higher conjugate quality, the future synthesis
should include some adjustments related to the synthesis of the conjugate’s
peptide part.Najnovija otkrića obećavajućih kandidata za nove antivirusne lijekove dovela
su nedavno u fokus takozvane peptid-lijek konjugate (eng. peptide- drug
conjugates, PDCs). Peptid-lijek konjugati klasa su molekula koje nastaju
povezivanjem, izravno ili preko spojnika (eng. linker), tereta lijeka na peptid
nosač. Među različitim klasama peptid-lijek konjugata, u posljednje vrijeme
veliku pažnju privlače oni s antivirusnim svojstvima zbog svog izvanrednog
farmakološkog potencijala u borbi protiv raznih virusnih bolesti – jedne od
najvećih zdravstvenih briga čovječanstva danas. U okviru ovih nedavnih
znanstvenih otkrića, izvršili smo sintezu peptida na čvrstoj fazi (eng. solid
phase peptide synthesis, SPPS) jednog peptid-lijek konjugata, s dokazanom in
vitro antivirusnom aktivnošću protiv ZIKA virusa: PPIX-P1 (VQQLTKRFSL
amid), te karakterizirali glavne nečistoće u njegovu, i još jednom peptid-lijek
konjugatu s također dokazanom in vitro anti-ZIKA virus aktivnošću: PPIX-P3
(AGILKRW-amid), konačnom pročišćenom uzorku korištenjem analitičkih
tehnika matriksom asistirana laserska desoprcija ionizacijom – masena
spektrometrija (eng. Matrix-Assisted Laser Desorption Ionization – Mass
Spectrometry, MALDI-MS) te tekućinska kromatografija – masena
spektrometrija (eng. Liquid- Chromatography – Mass Spectrometry, LC-MS).
Najprije smo izvršili sintezu peptida P1 na čvrstom nosaču i analizirali ga
MALDI-MS i LC-MS analitičkim tehnikama kako bismo potvrdili vjerodostojnost
njegove sinteze. Potom smo učinili konjugaciju protoporfirina IX (PPIX) sa
sintetiziranim peptidom na njegovom N-terminusu, a novosintetizirani
konjugat također zatim analizirali pomoću LC-MS i MALDI-MS analitičkih
tehnika. Nadalje, sintetizirani konjugat bio je skinut sa čvrstog nosača i
pročišćen
preparativnom C18 tekućinskom kromatografijom visoke
učinkovitosti (eng. High Performance Liquid Chromatography, HPLC). Dobiveni
čisti uzorak peptid-porfirin konjugata PPIX-P1 te PPIX-P3 ( sintetiziranog i
pročišćenog istom metodologijom) te nečistoće u njima karakterizirali smo
potom LC-MS i tekućinska kromatografija – tandemna masena spektrometrija
(eng. Liquid Chromatography - Tandem Mass Spectrometry, LC-MS/MS)
analizama. Temeljem rezultata analiza nečistoća u uzorcima peptid-porfirin
konjugata (m/z 521.7 u slučaju PPIX-P1 i m/z 473.7 u slučaju PPIX-P3)
tandemnom masenom spektrometrijom (eng. Tandem Mass Spectrometry,
MS/MS) mogli smo zaključiti da je: (i) peptidni dio konjugata porijeklo
odgovarajuće nečistoće i (ii) C-terminalni dio peptida skloniji modifikacijama
tijekom sintetskog procesa konjugata. Stoga, kako bi se postigla njihova veća
kvaliteta, potrebno je uvesti određene prilagodbe u peptidnom dijelu sinteze
peptid-porfirin konjugata
Stanično-specifična uloga optineurina u TLR4 ili ER stresom posredovanoj aktivaciji NF-κB puta
No full textAmyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease, which leads to motor neuron death in the brain and the spinal cord. In 2010 the first optineurin mutations were found in the ALS patients, and the initial biochemical analyses in cell lines suggested that optineurin truncation or mutation in the ubiquitin binding region result in exaggerated nuclear factor κB (NF-κB) activation. Such excessive NF-κB activation and subsequent inflammation were reported to result in neuronal cell death. However, several follow-up studies in primary innate immune cells from optineurin deficiency or insufficiency models could not reproduce these findings. To further test this issue, we decided to induce NF-κB activation with paclitaxel [Toll-like receptor 4 (TLR4) agonist] and tunicamycin [endoplasmic reticulum (ER) stress inducer] to observe if optineurin will exert its role as a negative regulator. These stimuli could mimic some of the stresses that central nervous system (CNS) cells are exposed to during the neurodegenerative process. Microglial (BV2) and neuronal (N2A) optineurin wild-type (WT) and knock-out (KO) cells were used in this research. Optineurin KO BV2 cells exhibited a diminished p65 phosphorylation and TNF-α secretion upon paclitaxel treatment but stronger inhibitor of κB-α (IκB-α) degradation, while N2A KO cells exhibited slightly higher NF-κB activation as detected by p65 phosphorylation. Tunicamycin treated optineurin KO BV2 cells also revealed slightly higher IκB-α degradation and diminished p65 phosphorylation, but TNF-α secretion was not different. Notably, optineurin KO N2A cells also exhibited a slight tendency of stronger NF-κB activation as evidenced by p65 phosphorylation. Results obtained from N2A cells are in correlation with previous studies done on neuronal cell lines, while the ones obtained in BV2 cells differ from results done on primary microglia. Taken together, we propose that optineurin has cell-specific effects, acting as a negative regulator of NF-κB activation in BV2 and N2A cells in response to paclitaxel. It must be noted that these results are obtained in cell lines so they should be used as a preliminary information for subsequent studies on primary cells or in vivo models.Amiotrofična lateralna skleroza (ALS) je fatalna neurodegenerativna bolest koja dovodi do smrti motoričkih neurona u mozgu i leđnoj moždini. Prve mutacije optineurina pronađene su u pacijenata oboljelih od ALS-a 2010. godine. Prvotne biokemijske analize u staničnim linijama pokazale su da trunkacija ili mutacija ubikvitin vežuće regije optineurina dovodi do pojačane aktivacije puta nuklearnog čimbenika κB (NF-κB). Pretjerana NF-κB aktivacija te posljedična upala mogu dovesti do neuralne stanične smrti. Međutim, nekoliko studija napravljenih na primarnim stanicama urođene imunosti izoliranih iz modela deficijencije ili insuficijencije optineurina nisu mogle reproducirati rezultate dobivene na staničnim linijama. U svrhu ispitivanja tih kontradikcija, odlučili smo inducirati NF-κB aktivaciju s paklitakselom [agoinst receptora sličnom Toll-u (TLR4)] i tunikamicinom [izaziva stres endoplazmatskog retikuluma (ER)] kako bismo ispitali hoće li optineurin djelovati kao negativan regulator. Navedeni stimulusi mogu imitirati stresore kojem su stanice središnjeg živčanog sustava izložene prilikom procesa neurodegeneracije. U ovom istraživanju koristili smo migroglijalne (BV2) i neuronalne (N2A) stanične linije divljeg tipa (eng. wild-type, WT) i optineurin deficijentne (eng. knock-out, KO). BV2 KO stanice su pokazale smanjenu fosforilaciju p65 aktivaciju i sekreciju TNF-α, no jaču razgradnju inhibitora κB-α (IκB-α) nakon tretmana s paklitakselom. U N2A KO stanicama tretman paklitakselom rezultirao je blago pojačanom NF-κB aktivacijom što je zabilježeno fosforilacijom p65. BV2 KO stanice tretirane s tunikamicinom su pokazale jaču razgradnju IκB-α i smanjenu fosforilaciju p65, no razlika u izlučivanju TNF-α nije zabilježena. N2A KO stanice pokazale su tendenciju blago pojačane NF-κB aktivacije nakon tretmana tunikamicinom što je praćeno fosforilacijom p65. Rezultati dobiveni na N2A stanicama se slažu s prethodnim studijama provedenim na neuronalnim staničnim linijama. Međutim, rezultati dobiveni na BV2 stanicama se ne slažu s onim dobivenim na primarnoj mikrogliji. Uzimajući sve rezultate u obzir, predlažemo da optineurin ima stanično-specifični, te da ima potencijalnu ulogu kao negativan regulator NF-κB aktivacije u BV2 i N2A stanicama. Moralo bi se uzeti u obzir kako su ovi rezultati dobiveni iz staničnih linija te bi se pritom trebali koristiti kao preliminarna informacija za buduće studije koje bi se provele na primarnim stanicama ili u in vivo modelima
Effect of optineurin deficiency on IFN-β-induced autophagy
No full textUčinci različitih neurodegenerativnih bolesti na živčane stanice međusobno se bitno razlikuju, međutim nakupljanje unutarstaničnih, toksičnih agregata krivo smotanih proteina i posljedično gubitak funkcije stanica, zajednička su obilježja svih neurodegenerativnih bolesti. Osnovni razlog prisutnosti agregata nije poznat u velikoj većini slučajeva, ali dio mutacija u genima povezanim s neurodegenerativnim bolestima veže se uz autofagiju. Autofagija je evolucijski očuvani proces kojim se putem lizosoma razgrađuje citoplazmatski sadržaj poput proteinskih agregata, patogena i oštećenih organela. Pritom adaptori autofagije prepoznaju označeni sadržaj za razgradnju i dostavljaju ga do vezikula s dvostrukom membranom koje se zovu autofagosomi. Autofagosomi se spajaju s lizosomima što dovodi do stvaranja autolizosoma gdje se sadržaj razgrađuje. U adaptore autofagije ubrajaju se i dva proteina čije su mutacije pronađene u bolesnika s neurodegenerativnom bolešću amiotrofičnom lateralnom sklerozom (ALS), optineurin (OPTN) i sekvestosom 1 (SQSTM1 ili p62). Za OPTN je poznato da osim kao adaptor autofagije, sudjeluje u još dva koraka autofagije: inicijaciji autofagije i sazrijevanju autofagosoma. Stoga smo u ovom radu htjeli ispitati utjecaj OPTN-a na autofagiju u staničnoj liniji neuroblastoma N2a, koristeći N2a stanice u kojima je OPTN uklonjen CRISPR-Cas9 metodom. Analizirali smo bazalnu autofagiju i autofagiju induciranu s interferonom (IFN)-β, citokinom za koji je nedavno pokazano da regulira autofagiju u neuronima. Koristili smo standardne testove za praćenje autofagije poput mjerenja količine markera autofagosoma LC3-II (engl. microtubule associated protein light chain 3), adaptora p62, i OPTN-a Western blot metodom, te brojanje LC3 pozitivnih vezikula imunofluorescencijom ili fluorescentno označenim LC3 konstruktom. Iz naših rezultata zaključujemo da u OPTN-deficijentnim stanicama postoji djelomični blok autofagije u bazalnim uvjetima kojeg vidimo kao povećani broj i veličinu autofagosoma te povećano nakupljanje LC3-II. IFN-β je u N2a stanicama inducirao autofagiju, ali nismo uočili nakupljanje OPTN i p62 što može značiti da nisu adaptori autofagije potaknute s IFN-β te da djeluju na autofagiju u nekom drugom koraku. Nadalje, moguće je i da su razgrađeni prije naše analize te da pokuse treba modificirati. Zbog djelomičnog bloka u autofagiji pri nedostatku OPTN-a, moguće je da iz tog razloga dolazi do nakupljanja proteinskih agregata u ALS-u. U budućim pokusima važno je stoga dokazati da opaženi poremećaj autofagije dovodi do agregacije proteina.The effects of different neurodegenerative diseases on neurons substantially differ, but the accumulation of intracellular toxic aggregates of misfolded proteins and the consequent loss of cell function are common features of all neurodegenerative diseases. The underlying reason for the presence of aggregates is not known in the vast majority of cases, but some genes mutated in neurodegenerative diseases are linked to autophagy. Autophagy is an evolutionarily conserved process by which noxious cytoplasmic contents such as protein aggregates, pathogens, and damaged organelles are broken down in lysosomes. Autophagy adaptors recognize the cargo labeled for degradation and deliver it to double-membrane vesicles called autophagosomes. Autophagosomes fuse with lysosomes leading to the formation of autolysosomes where the cargo is degraded. Autophagy adapters also include two proteins whose mutations have been found in patients with the neurodegenerative disease amyotrophic lateral sclerosis (ALS), optineurin (OPTN) and sequestosome 1 (SQSTM1 or p62). In addition to its adaptor role, OPTN has also been described to participate in two other autophagy steps: autophagy initiation and autophagosome maturation. Therefore, in this paper, we examined the effect of OPTN on autophagy in the N2a neuroblastoma cell line, using N2a cells in which OPTN was removed by the CRISPR-Cas9 approach. We analyzed basal autophagy and autophagy induced by interferon (IFN)-, a cytokine recently shown to regulate autophagy in neurons. We used standard tests to monitor autophagy such as measuring the amount of LC3-II (microtubule associated protein light chain 3), p62 adaptor, and OPTN by Western blotting, and counting LC3 positive vesicles by immunofluorescence or via a fluorescently labeled LC3 construct. We observed a partial block of basal autophagy in the OPTN-deficient cells manifested as an increased number and size of autophagosomes and increased accumulation of LC3-II. IFN-β induced autophagy in N2a cells, but we did not observe the accumulation of OPTN and p62 which may mean that they do not serve as autophagy adaptors upon IFN-β-treatment or that they act on autophagy in some other step(s). Furthermore, it is possible that they were degraded before our analysis and that our experimental approach needs to be modified. Due to the partial block in autophagy in the absence of OPTN, it is possible that this leads to an accumulation of protein aggregates in ALS. In future experiments, it is therefore important to test if this observed autophagy dysregulation in OPTN-deficient cells leads to protein aggregation
Infection of astrocytes with mouse cytomegalovirus
No full textCitomegalovirus (CMV) je široko rasprostranjen virus u ljudi čija je karakteristika uspostava cjeloživotne latencije. Kod zdravih osoba infekcija je uobičajeno asimptomatska, ali u osoba s oslabljenim i nezrelim imunosnim sustavom može uzrokovati po život opasne infekcije. Kongenitalna infekcija CMV-om je najčešća transplacentarno prenosiva prirođena infekcija koja može utjecati na razvoj mozga i uzrokovati trajne neurološke poremećaje. Dosad je poznato da in vitro CMV može inficirati većinu staničnih vrsta mozga do određene razine, međutim, nije jasno vrijedi li to in vivo i je li ta infekcija produktivna. Glavni cilj ovog diplomskog rada bio je na mišjem modelu kongenitalne infekcije utvrditi inficira li CMV astrocite in vivo i podržavaju li astrociti uspješnu replikaciju virusa. U svrhu istraživanja korišteni su miševi i mišji citomegalovirus (MCMV) divljeg tipa, ali i inducibilni Cre/loxP reporterski sustav koji se sastoji od rekombinantnog MCMV-flox virusa te specifičnog transgeničnog soja miševa, u kojem je Cre rekombinaza aktivna samo u astrocitima. Nakon infekcije novorođenih miševa i prikupljanja organa u određenim vremenskim točkama, provedena je analiza infekcije astrocita imunohistokemijskom metodom, te virusna titracija mozgova pomoću metode određivanja virusnih čistina (plakova). Dobiveni rezultati su pokazali da MCMV uspješno inficira astrocite in vivo. Štoviše, na vrhuncu infekcije u mozgu, trećina inficiranih stanica su bili upravo astrociti. Osim toga, dokazano je da je infekcija astrocita in vivo produktivna te da astrociti doprinose širenju CMV infekcije u mišjem mozgu. Ova saznanja doprinose boljem razumijevanju neuropatogeneze kongenitalne CMV infekcije i otvaraju nova pitanja o utjecaju infekcije na funkciju astrocita i o ulozi ostalih staničnih vrsta u mozgu tijekom infekcije.Cytomegalovirus (CMV) is a widespread virus, characterized by the establishment of lifelong latency. In healthy individuals the infection is typically asymptomatic, however, in immunocompromised persons and in congenitally infected children CMV infection can lead to a life-threatening condition. Congenital CMV infection is the most common intrauterine infection that can affect brain development and cause permanent neurological disabilities. So far, it was clear that CMV can infect most brain cell types in vitro to a certain level, but whether this is also true in vivo and whether the infection is productive remains unknown. Thus, the main objective of this thesis was to determine whether CMV infects astrocytes in vivo and whether astrocytes support successful viral replication, using a mouse model of congenital infection. In this research we have used wild-type mice and mouse cytomegalovirus (MCMV), but also a conditional Cre/loxP reporter system, consisting of recombinant MCMV-flox virus and transgenic mouse strain in which Cre recombinase is active only in astrocytes. After MCMV infection of newborn mice and organ harvesting at certain time points, analysis of astrocyte infection was performed using immunohistochemical staining, as well as determination of brain virus titers by plaque assay. The results obtained from this study confirmed that MCMV successfully infects astrocytes in vivo, moreover, at the peak of infection in the brain, a third of the infected cells were astrocytes. In addition, it has been shown that astrocytes support productive CMV infection in vivo and that they contribute to virus spread throughout the brain. These findings contribute to a better understanding of the neuropathogenesis of congenital CMV infection and are raising new questions about the impact of infection on astrocyte function and the role of other brain cell types during infection
Functional Hydrogels Based on Fmoc-Amino Acids
No full textHidrogelatori male molekulske mase temeljeni na Fmoc-amino kiselinama predstavljaju obećavajuće molekule, nudeći brojne prednosti poput jednostavne pripreme hidrogelova, prilagodljivih svojstava i biokompatibilnosti. Ovo istraživanje usmjereno je na dizajn i karakterizaciju hidrogelova temeljenih na Fmoc-histidinu i Fmoc-cisteinu, kao gradivnim jedinicama koje su sposobne formirati supramolekularne strukture putem nekovalentnih interakcija kao što su vodikove veze i π-π slaganje. Histidin, polarna aminokiselina, često se koristi za katalizu u kombinaciji s cisteinom, neutralnom aminokiselinom poznatom po svojoj sklonosti stvaranju disulfidnih mostova oksidacijom. U ovoj studiji, hidrogelovi su dobiveni pri koncentracijama od 10 mM i 15 mM Fmoc-Cys u PBS-u, a kristali su dobiveni pri 10 mM Fmoc-His na temelju samoorganizacije pojedinačnih Fmoc-amino kiselina. Nadalje, njihova ko-samoorganizacija pri koncentracijama od 20 mM i 30 mM rezultirala je prozirnim hidrogelovima. Procijenjena je stabilnost hidrogelova, strukturna svojstva i katalitička aktivnost. Fmoc-His se samoorganizirao u kristale, a njihova struktura je riješena korištenjem rendgenske difrakcije. Stabilnost gela Fmoc-Cys i Fmoc-Cys:Fmoc-His ispitana je pomoću testa inverzije bočice, termoreverzibilnog testa i reologije. Osim toga, provedene su LC-MS analiza i Ellmanov test kako bi se istražila oksidacija Fmoc-Cys, dok je fluorescencijska analiza otkrila kritične koncentracije agregacije (CAC) Fmoc-amino kiselina. p-NPA test korišten je za procjenu katalitičke aktivnosti hidrogelova. Rezultati sugeriraju da ko-samoorganizirani hidrogeli Fmoc-Cys:Fmoc-His pokazuju superiornu katalitičku aktivnost u usporedbi sa samoorganiziranim Fmoc-Cys. Niža aktivnost Fmoc-Cys može se pripisati formiranju disulfidnih mostova putem oksidacijskih procesa, što smanjuje njegovu nukleofilnu reaktivnost. Ova studija doprinosi razumijevanju hidrogelatora male molekulska mase i njihovih potencijalnih primjena kao minimalističkih katalizatora.Low molecular weight hydrogelators based on Fmoc-amino acids are promising molecules, offering numerous advantages such as ease of hydrogel preparation, tunable properties and biocompatibility. This research focuses on the design and characterization of hydrogels based on Fmoc-Histidine and Fmoc-Cysteine, as building blocks capable of forming structures supramolecular through non-covalent interactions such as hydrogen bonding and π-π stacking. Histidine, a polar amino acid, is frequently exploited for catalysis in combination with cysteine, a neutral amino acid known for its propensity to create disulfide bridges through oxidation. In this study, hydrogels are obtained at 10 mM and 15 mM of Fmoc-Cys in PBS and crystals are obtained at 10 mM of Fmoc-His based on the self-assembly of individual Fmoc-amino acids. Furthermore, their co-assembly at 20 mM and 30 mM resulted in transparent hydrogels. Hydrogel stability, structural properties and catalytic activity were assessed. Fmoc-His self-assembled into crystals and its structure was solved using X-ray diffraction. Gel stability of Fmoc-Cys and Fmoc-Cys:Fmoc-His was conducted using a vial inversion test, the thermoreversibility test and rheology. Moreover, LC-MS analysis and Ellman’s assay were performed to investigate the Fmoc-Cys oxidation, while fluorescence analysis to reveal critical aggregation concentrations (CAC) of Fmoc-amino acids. The p-NPA assay was used to evaluate the catalytic activity of hydrogels. The results suggest that Fmoc-Cys:Fmoc-His co-assembled hydrogels exhibit superior catalytic activity compared to self-assembled Fmoc-Cys. The lower activity of Fmoc-Cys might be attributed to the formation of disulfide bridges via the oxidation processes, thereby hindering its nucleophilic reactivity. This study contributes to the understanding of low molecular weight hydrogelators and their potential applications as minimalistic catalysts