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Co-Aggregation and Parallel Aggregation of Specific Proteins in Major Mental Illness
No full textBackground: Disrupted proteostasis is an emerging area of research into major depressive disorder. Several proteins have been implicated as forming aggregates specifically in the brains of subsets of patients with psychiatric illnesses. These proteins include CRMP1, DISC1, NPAS3 and TRIOBP-1. It is unclear, however, whether these proteins normally aggregate together in the same individuals and, if so, whether each protein aggregates independently of each other (“parallel aggregation”) or if the proteins physically interact and aggregate together (“co-aggregation”). Materials and methods: Post mortem insular cortex samples from major depressive disorder and Alzheimer’s disease patients, suicide victims and control individuals had their insoluble fractions isolated and tested by Western blotting to determine which of these proteins are insoluble and, therefore, likely to be aggregating. The ability of the proteins to co-aggregate (directly interact and form common aggregate structures) was tested by systematic pairwise expression of the proteins in SH-SY5Y neuroblastoma cells, which were then examined by immunofluorescent microscopy. Results: Many individuals displayed multiple insoluble proteins in the brain, although not enough to imply interaction between the proteins. Cell culture analysis revealed that only a few of the proteins analyzed can consistently co-aggregate with each other: DISC1 with each of CRMP1 and TRIOBP-1. DISC1 was able to induce aggregation of full length TRIOBP-1, but not individual domains of TRIOBP-1 when they were expressed individually. Conclusions: While specific proteins are capable of co-aggregating, and appear to do so in the brains of individuals with mental illness and potentially also with suicidal tendency, it is more common for such proteins to aggregate in a parallel manner, through independent mechanisms. This information aids in understanding the distribution of protein aggregates among mental illness patients and is therefore important for any future diagnostic or therapeutic approaches based on this aspect of mental illness pathology
Symptom severity in schizophrenia patients with NPAS3, dysbindin-1 and/or TRIOBP protein pathology in their blood serum: a PANSS-based follow up study
No full textBackground: It has been proposed that aggregation of specific proteins in the brain may be a pathological element in schizophrenia and other chronic disorders. Multiple such aggregating proteins have now been implicated through post mortem investigation, including NPAS3 (Neuronal PAS domain protein 3), dysbindin-1 (encoded by the DTNBP1, Dystrobrevin Binding Protein 1, gene) and TRIOBP (Trio-Binding Protein, multiple isoforms). While the presence of protein aggregates in the brain is interesting in terms of understanding pathology, it is impractical as a biomarker. These proteins were therefore investigated recently in blood serum of schizophrenia patients and controls, showing patients to have higher levels of NPAS3 in their serum generally. TRIOBP-1 and dysbindin-1 were also found in an insoluble state, implying aggregation, but did not clearly corresponding to disease state. Subject and methods: We revisit 47 of the originally recruited 50 patients with schizophrenia, all of whom are Croatian and aged between 18 and 72. We assessed their symptom specificity and severity using PANSS (the Positive and Negative Symptoms Scale), comparing those with NPAS3, insoluble dysbindin-1 and/or insoluble TRIOBP-1 in their blood serum to those lacking any such protein dysregulation. Results: The frequency of each individual potential protein pathology among these patients was too low for meaningful statistical analysis, however the 11 patients that displayed one or more of these pathologies (NPAS3, dysbindin-1, TRIOBP-1 and/or TRIOBP-5/6) showed a subtle but significant increase in total PANSS scores compared to the 36 patients displaying none of the pathologies (p = 0.031), seemingly driven principally by increased scores on the general psychopathology scale. Conclusion: While the numbers of patients involved do not allow firm conclusions to be drawn at this time, this provides the first indication that disturbed proteostasis in blood serum, of proteins that aggregate in the brains of schizophrenia patients, may correlate with the severity of schizophrenia symptoms
O-Alkylation of (oxido)pyridylporphyrins
No full textFotodinamička terapija (PDT) je vrsta terapije tumora koja uključuje akumulaciju fotosenzibilizatora u tumorskom tkivu gdje nakon osvjetljavanja određenom valnom duljinom uz prisutnost kisika dolazi do izravne smrti tumorskih stanica, oštećenja mikrovaskulature i indukcije lokalne upalne reakcije. Spojevi porfirinskog tipa se zbog pogodnih fizikalnih i kemijskih svojstava često koriste kao fotosenzibilizatori u PDT-u. Prilikom odabira fotosenzibilizatora poželjne karakteristike uključuju netoksičnost bez zračenja, učinkovitu apsorpciju svjetlosti u vidljivom dijelu spektra, mogućnost generiranja singletnog kisika i drugih reaktivnih kisikovih vrsta, ali i optimalan omjer hidrofilnih i lipofilnih svojstava za djelotvornu intravenoznu primjenu i olakšano prodiranje u tumorska tkiva.
Osim hidrofilnih i lipofilnih obilježja, struktura i naboj fotosenzibilizatora određuju njegova farmakokinetička i farmakodinamička svojstva. U odnosu na zwitterionske (oksido)piridilporfirine, kationski O-alkilirani (oksido)piridilporfirini imaju potencijal bolje topljivosti u biološkim otapalima te učinkovitijeg prodiranja i vezanja za tumorska tkiva.
O-Etilirani (oksido)piridilporfirini sintetizirani uz diklormetan kao otapalo i etil-tosilat kao alkilirajući reagens uspješno su izolirani. Sintetizirani 5-(4-acetamidofenil)-10,15,20-tris(1-etoksidopiridin-3-il)porfirin triklorid (porfirin 8) dokazao je bolju topljivost u biološkim medijima i viši intenzitet apsorpcijskih i emisijskih vrpci spektara u usporedbi s porfirinom 4 te nižu IC50 vrijednost na stanicama raka dojke (MDA-MB-231) nakon osvjetljavanja lampom crvenog osvjetljenja (λ = 645 nm, 3,6 J/cm2) u usporedbi sa svojim prekursorima (porfirin 4 i porfirin P).Photodynamic therapy (PDT) is a type of tumor therapy that involves the accumulation of photosensitizers in the tumor tissue where, after illumination with a certain wavelength in the presence of oxygen leads to the direct death of the tumor cell, damage to the microvasculature and the induction of a local inflammatory reaction. Due to their favorable physical and chemical properties, porphyrin-type compounds are often used as photosensitizers in PDT. When choosing photosensitizers, desired characteristics include non-toxicity without radiation, efficient absorption of the light in the visible spectrum, generation of singlet oxygen and other reactive oxygen species, but also an optimal ratio of hydrophilic and lipophilic properties for effective intravenous administration and facilitated penetration into the tumor tissue.
In addition to hydrophilic and lipophilic properties, the structure and the charge of the photosensitizer determine its pharmacokinetic and pharmacodynamic properties. Compared to zwitterionic (oxido)pyridylporphyrins, cationic O-alkylated (oxido)pyridylporphyrins have the potential for better solubility in biological solvents and more effective penetration and binding to tumor tissues.
O-Ethylated (oxido)pyridylporphyrins synthesized with dichloromethane as solvent and ethyl tosylate as alkylating reagent were successfully isolated. Synthesized 5-(4-acetamidophenyl)-10,15,20-tris(1-ethoxydopyridin-3-yl)porphyrin trichloride (porphyrin 8) showed a better solubility in the biological medium and higher intensity of the absorption and emission spectrum compared with porphyrin 4 and lower IC50 values on breast cancer cells (MDA-MB-231) after illumination with a red light lamp (λ = 645 nm, 3,6 J/cm2 ) compared to their precursors (porphyrin 4 and porphyrin P)
Therapeutic inhibition of ribosome biogenesis in cancer therapy
No full textRibosomi su stanični strojevi za sintezu bjelančevina. Proizvodnja novih
ribosoma neophodna je za povećanje kapaciteta sinteze bjelančevina tijekom
rasta, diobe i diferencijacije stanica. Povećanje sinteze ribosoma dovodi do
neograničenog rasta i proliferacije stanica, a brojna istraživanja pokazala su
da uslijed prekomjerne sinteze ribosoma dolazi do maligne transformacije.
S druge strane, heterozigotne mutacije velikog broja specifičnih ribosomskih
proteina, prikazuju prijelaz iz ranih simptoma, uzrokovanih
hipoproliferacijom stanica, u rak, bolest hiperproliferacije. Pogreške u sintezi
ribosoma, ključni su događaj tijekom zloćudne preobrazbe normalnih
stanica, a aktivacija supresora tumora p53 prepreka je za nastanak tumora
uslijed pogrešaka u sintezi ribosoma. Nejasno je kako gubitak funkcije
supresora tumora p53 doprinosi zloćudnoj preobrazbi putem poremećaja
funkcije jezgrice, ključnog mjesta sinteze ribosoma. Razumijevanje tih
mehanizama neophodno je za razvoj učinkovitih protutumorskih lijekova.
Danas se razvijaju selektivni inhibitori Pol I transkripcije koji imaju prednost
nad klasičnim kemoterapeuticima te su vrlo učinkoviti u liječenju raka.
Tumorske stanice su vrlo osjetljive na lijekove koji inhibiraju prepisivanje ili
sazrijevanje rRNA te je njihova primjena vrlo važna u terapiji raka.A ribosome is the cellular machinery responsible for making proteins. The
production of new ribosomes is necessary to increase the capacity of protein
synthesis during cell growth, division and differentiation. An increase in
ribosome biogenesis leads to unlimited growth and proliferation of cells, and
numerous studies have shown that increased ribosome biogenesis rate leads
to malignant transformation. On the other hand, heterozygous mutations of
a large number of distinct ribosomal protein genes show the transition from
early symptoms, caused by hypoproliferation of cells, to a hyperproliferation
which is a characteristic of cancer cells. Errors in ribosome biogenesis are a
key event during the malignant transformation of normal cells. Activation of
the p53 tumor suppressor when an error in ribosome biogenesis is present
may actually constitute an important barrier to tumorigenesis. It is unclear
how loss of function of the p53 tumor suppressor contributes to malignant
transformation by disrupting the function of the nucleolus, a key site of
ribosome biogenesis. Understanding these mechanisms is necessary for the
development of effective anticancer therapy. Today, selective inhibitors of
Pol I transcription are being developed, which have an advantage over
classical chemotherapeutics and are very effective in the treatment of
cancer. Cancer cells are very sensitive to drugs that inhibit the transcription
or maturation of rRNA, and their use is very important in cancer therapy
Analiza strukture i funkcije DNA metiltransferaze DNMT1 i razvoj nekovalentnih inhibitora koristeći računalne metode
No full textDNA methyltransferase 1 (DNMT1) is an enzyme that plays an essential role in
epigenetic regulation of mammalian genetic expression. Dysregulation of
methylation processes can lead to various pathological conditions, which in
recent years ,lead to extensive study of structure and function of DNMT1.
DNMT1 has multiple forms dependant of the stage of DNA methylation, which
posses limitations in both discovery and modification of new drugs. With use of
in silico methods we sought to identify drug like compounds capable of binding
to, and inhibiting the function of DNMT1.
Analysis of molecular dynamics (MD) simulations showed stronger binding of
SAH to all three DNMT1 proteins (4WXX, 3PTA and 4DA4), compared to tested
compounds. Two compounds moved from the SAH binding site during initial
simulations, for which they were excluded from further research.
Tested compounds adhere to the Lipinski rule but lack the formation of
hydrogen bonds with DNMT1, compared to SAH. Future research should use
the potential of π- π stacking interaction with residues inside SAH binding
pocket.DNA metiltransferaza 1 (DNMT1) je enzim koji ima ključnu ulogu u
epigenetskoj regulaciji genske ekspresije kod sisavaca. Disregulacija procesa
metilacije može dovesti do raznih patoloških stanja, što je posljednjih godina
dovelo do opsežnog proučavanja strukture I funkcije DNMT1. DNMT1 postoji u
više oblika ovisno o stupnju metilacije DNA, što ograničava mogućnost u
otkrivanju i modifikaciji novih lijekova. Korištenjem in silico metoda nastojali
smo identificirati spojeve slične lijekovima koji se mogu vezati I inhibirati
funkciju DNMT1.
Analiza simulacija molekularne dinamike (MD pokazale su jače vezanje SAH na
sva tri proteina DNMT1 (4WXX, 3PTA i 4DA4), u usporedbi s testiranim
spojevima. Dva spoja pomaknula su se s veznog mjesta SAH tijekom početnih
simulacija, zbog čega su isključena iz daljnjeg istraživanja.
Ispitani spojevi pridržavaju se Lipinskog pravila, ali stvaraju manje vodikovih
veza s DNMT1, u usporedbi sa SAH. Buduća bi istraživanja trebala iskoristiti
potencijal π-π interakcija s aminokiselinskim ostacima unutar SAH veznog
džepa
Učinak PIKfyve i PI4KIIIβ inhibitora na adheziju trombocita
No full textPlatelets are small anucleated blood cells whose primary role is hemostasis.
Lately, platelets have been associated with other functions like their
involvement in innate immunity, regulation of tumor growth and viral
infection, gaining increasing scientific interest. In addition, molecular
mechanisms that lead to platelet activation and downstream changes are
still not completely understood. After activation, platelets adhere to the
damaged vessel wall or activable surface, undergo cytoskeletal
reorganization, and degranulation, which leads to their complete spreading.
These processes are mediated by diverse signaling molecules, among
others, also with small lipids called phosphoinositides (PIs).
Phosphatidylinositol 3, 5-bisphosphate [PI(3,5)P2] produced by
phosphatidylinositol-3-phosphate 5-kinase (PIKfyve) regulates membrane
trafficking. Phosphatidylinositol 4-phosphate (PI4P) produced by
phosphatidylinositol-4-kinase-III-beta (PI4KIIIβ) and other PI4Ks is a
source of a key signaling molecule, PI(4,5)P2, that also mediates actin
cytoskeletal dynamics. Previous transcriptomic and proteomic data show
the expression of PIKfyve and PI4KIIIβ in both mouse and human platelets.
In this work, we aimed to decipher if pharmacological inhibition of PIKfyve
(inhibitor YM201636) or PI4KIIIβ (IN10) prevents human platelet
spreading. In addition, we analyzed downstream signaling pathways in the
presence of the inhibitors after activating collagen receptor (GPVI), namely
phosphorylation of Syk kinase and acetylation of tubulin. Also, we evaluated
the localization of PIKfyve and PI4KIIβ (another PI4K) in human platelets
in resting and activated conditions by immunostaining. We conclude that
PI4KIIIβ is potentially involved in platelet spreading by modulating the
cytoskeleton, however, additional studies are needed to confirm this
observation
Analysis of the rat serume IgG N-glycome
No full textImunoglobulin gama (IgG) je najzastupljenije protutijelo u serumu. Njegove imunološke funkcije regulirane su posttranslacijskim modifikacijama glikozilacijama. N-glikozilacije IgG se odnose na šećerne skupine vezane za IgG preko aminokiseline asparagin na poziciji 297 u polipeptidnom lancu. Šećerne skupine fukoza, galaktoza ili sijalinska kiselina važne su za regulaciju protuupalnih ili proupalnih svojstava IgG tako što utječu na vezanje regije Fc IgG na aktivirajuće ili inhibirajuće receptore Fc gama na imunološkim stanicama. Fiziološki status te stres mogu utjecati na N-glikozilaciju IgG zbog čega istraživanje profila N-glikoma može omogućiti otkriće novih biomarkera. Uz miševe, štakori su drugi najčešće korišteni modelni organizam u znanstvenim istraživanjima. Karakterizacija N-glikoma IgG zdravog štakora može se stoga koristiti u istraživanjima patogeneze bolesti ili za analize mehanizama djelovanja lijekova. Analiza N-glikoma je tehnički vrlo zahtjevna i ovisi o metodama za izolaciju, pročišćavanje i identifikaciju N-glikana. Izolacija IgG iz seruma štakora u ovom je radu provedena uz pomoć afinitetne visokoprotočne monolitne kromatografije. Prednost korištenja monolitnih kolona je u kraćem vremenu analize, većoj rezolucijite boljoj stabilnosti kolone pri većim tlakovima. Kao ligand za vezanje IgG je korišten imobilizirani protein L zbog visokog afiniteta za kapa lake lance IgG. Potvrda izolacije IgG provedena je uz pomoć MALDI-TOF masene spektrometrije (MS). U radu su također uspoređeni protokoli za tripsinsku digestiju u gelu iz literature s protokolom razvijenim na Odjelu za biotehnologiju, tzv. in-house protokol. Usporedba je pokazala da je s protokolom iz literature identifikacija IgG uz pomoć MALDI-TOF MS bila pouzdanija . Identifikacija N-glikana provedena je također uz pomoć MALDI TOF MS nakon derivatizacije glikana čime se omogućila identifikacija glikana koji sadrže ostatak α2,6-NeuAc.
Tehnička reproducibilnost identifikcije N-glikana u serumu štakora nije bila zadovoljavajuća te je protokol za pripremu uzorka potrebno dalje optimizirati.Immunoglobulin gamma is the most abundant antibody present in serum. Its functions are regulated by glycosylation which is a type of posttranslational protein modification. IgG N-glycosylation describes oligosaccharides linked to the polypeptide chain through the asparagine residue 297. Sugar moieties such as fucose, galactose or sialic acid mediate IgG’s anti-inflammatory or pro-inflammatory properties by affecting the binding of the Fc region on IgG to activating or inhibitory Fc gamma receptors located on immune cells. Due to the fact that the physiological state and stress have a great impact on IgG N-glycosylation, research of N-glycome profiles can result with discovering novel biomarkers. After mice, rats are the second most used animal model in scientific research. Characterization of IgG N-glycome of a healthy rat can be compared to physiologically altered states such as disease, or it can be used to analyze drug mechanisms of action. N-glycome analyzes are technically very challenging and therefore strongly depend on implementing precise and sensitive methods for isolation, purification and identification of N linked glycans. In this thesis, isolation of IgG from rat serum was performed by affinity monolithic chromatography. Advantages of using monolithic columns include shorter time of analysis, narrower detection peak and, greater physical stability at higher pressures. Immobilized protein L was used as a ligand due to high affinity for IgG kappa light chains. Confirmation of IgG isolation was performed by MALDI-TOF mass spectrometry. A comparison between protocols for in-gel tryptic digestion was carried out wherein a protocol developed by Shevchenko et al.. was compared with an in-house protocol developed at the Department of Biotechnology. The comparison showed that the protocol developed Shevchenko et al. achieved more reliable results compared to the results obtained through the in-house developed protocol. N-glycan identification was performed using MALDI-TOF MS. Identification of glycans largely depends on the derivatization process. Derivatization protocols used in this thesis have not achieved the desired technical reproducibility for all samples and glycans from only two samples were successfully identified. In the future, better protocol optimization could identify the complete N-glycan profile of IgG
Individual and combined effects of mycotoxins (ochratoxin a and citrinin) and resveratrol antioxidant on the expression of organic anion transporters in rat kidneys
No full textDugotrajna izloženost niskim dozama mikotoksina predstavlja rizik za pojavu
bubrežnih oboljenja. U javnozdravstvenom smislu najznačajniji su mikotoksini koje
proizvode plijesni rodova Aspergillus, Penicillium i Fusarium, koji kontaminiraju
namirnice za prehranu ljudi i životinja te uzrokuju pojavu mikotoksikoza. Ulazak
mikotoksina u epitelne stanice proksimalnih kanalića (PK) bubrega posredovan je
membranskim prijenosnicima organskih aniona (Oat), a nakupljanje mikotoksina u
PK preduvjet je njihova toksičnog učinka. Kako su namirnice najčešće kontaminirane
različitim mikotoksinima, cilj ovog rada je istražiti pojedinačne i združene učinke
okratoksina A (OTA) i citrinina (CIT) nakon 21-dnevnog tretmana te mogući
protektivni/reparatorni učinak resveratrola (RSV) na ekspresiju housekeeping
(rNa/K-ATPaze i r-aktina) i Oat proteina (rOat1, rOat2, rOat3 i rOat5) u bubrezima
štakora metodama western i imunocitokemijske analize. OTA (0,125 mg/kg tj.m. ili
0,250 mg/kg tj.m.) je u ovisnosti o dozi smanjio ekspresiju rOat1, ali ne rOat3 i rOat5
proteina, dok se ekspresija proteina rOat2 smanjila višom dozom OTA (0,250 mg/kg
tj.m). Tretman CIT (20 mg/kg tj.m.) je smanjio je ekspresiju rOat2 i rOat5, ali ne rOat1
i rOat3 proteina. Smjesa mikotoksina (0,250 mg/kg tj.m. OTA+20 mg/kg tj.m. CIT)
smanjila je ekspresiju rOat1, rOat2 i rOat5, a povećala ekspresiju rOat3 proteina.
Tretman OTA ili CIT, kao i njihovom smjesom nisu mijenjali ekspresiju housekeeping
proteina, izuzev smjese mikotoksina s višom dozom OTA koja je smanjila ekspresiju
r-aktina. Tretman RSV (0,250 mg/kg tj.m. OTA+20 mg/kg CIT+20 mg/kg tj.m. RSV)
nije umanjio nefrotoksične učinke mikotoksina. Dobiveni podatci ukazuju na važnost
interakcija mikotoksina u regulaciji ekspresije proteina rOat u bubrezima štakora.Prolonged exposure to low doses of mycotoxins poses a risk for kidney disease. The
most important mycotoxins which contaminate foodstuffs for human and animal
consumption and cause mycotoxicosis are produced by molds of the genera
Aspergillus, Penicillium and Fusarium. The entry of mycotoxins into the renal
proximal tubules (PT) epithelial cells is mediated by organic anions transporters
(Oat), and the accumulation of mycotoxins in the PT is a prerequisite for their toxic
effect. The aim of this study was to investigate the individual and combined effects
of ochratoxin A (OTA) and citrinin (CIT) after 21 day exposure and the possible
protective/reparative effect of resveratrol (RSV) on expression pattern of
housekeeping (rNa / K- ATPase and rβ-actin) and Oat protein (rOat1, rOat2, rOat3
and rOat5) in rat kidneys by western and immunocytochemical analysis. OTA (0.125
mg/kg bw or 0.250 mg/kg bw) reduced the expression of rOat1 but not rOat3 and
rOat5 protein, depending on the dose, while the expression of rOat2 protein
decreased with a higher dose of OTA (0.250 mg/kg bw). CIT treatment (2 mg/kg bw)
reduced the expression of rOat2 and rOat5, but not rOat1 and rOat3 proteins. A
mixture of mycotoxins (0.250 mg/kg bw OTA + 2 mg/kg bw CIT) decreased rOat1,
rOat2 and rOat5 expression and increased rOat3 protein expression. Treatment with
OTA or CIT, as well as their mixture, did not alter the expression of housekeeping
protein, except for a mixture of mycotoxins with a higher dose of OTA that reduced
rβ-actin expression. RSV treatment (0.250 mg/kg bw OTA + 2 mg/kg CIT + 20 mg/kg
bw RSV) did not reduce the nephrotoxic effects of mycotoxins. The data obtained
indicate the importance of mycotoxin interactions in the regulation of rOat protein
expression in rat kidneys
Elucidating the role of a protease BACE1 in the pathogenesis of a rare neurodegenerative disorder Niemann-Pick type C
No full textBolest Niemann-Pick tipa C (NPC) je rijetka nasljedna neurodegenerativna bolest nakupljanja slobodnog kolesterola i drugih lipida uzrokovana mutacijama u genu NPC1 ili NPC2, koja pokazuje niz patoloških sličnosti s kompleksnom Alzheimerovom bolesti (AB). Naša prethodna istraživanja su utvrdila povišeno djelovanje ključnog enzima u AB, proteaze BACE1, u NPC modelima. Stoga je cilj ovog doktorskog rada bio istražiti molekularnu pozadinu uočenog efekta te ispitati učinak inhibicije BACE1 na patološke karakteristike bolesti. Također, cilj je bio analizirati lipidom kako bi se utvrdile najranije promjene lipida odgovorne za različitu vulnerabilnost moždanih regija i disfunkciju endosoma u bolesti NPC. Korištene su primarne kulture neurona, kulture organotipskih rezova mozgova te moždane regije hipokampusa i malog mozga miševa koji imaju spontanu mutaciju u genu NPC1 te ne sintetiziraju proteina NPC1 (miševi NPC1) i miševa divljeg tipa (wt, od engl. wild type). U neuronima miševa NPC1 u odnosu na wt detektirana je povećana proteoliza supstrata BACE1- Sez6L i Sez6. Imunocitokemijska analiza je pokazala nakupljanje ovih supstrata u ranim endosomima NPC1 neurona. Promijenjen smještaj Sez6 i Sez6L u ranim endosomima je potvrđen i frakcioniranjem endolizosoma u moždanim regijama hipokampusa i malog mozga miševa NPC1. Interesantno, tretman inhibitorom BACE1 je djelomično popravio patološke značajke bolesti NPC, uključujući smanjenje nakupljanja Sez6 i Sez6L, smanjenje veličine vezikula ranih endosoma te smanjenje ukupne razine proteina tau u neuronima NPC1, kao i smanjenje neuroinflamacije (aktivacije astrocita i mikroglija) u kortiko-hipokamplanim rezovima miševa NPC1. Nadalje, analiza lipidoma je utvrdila da već u asimptomatskoj fazi bolesti postoji značajna razlika u distribuciji i razini određenih lipida u malom mozgu i hipokampusu, te u ranim endosomima miševa NPC1 u odnosu na wt. Ovo istraživanje je utvrdilo disfunkciju proteaze BACE1 i njenih supstrata te njihovu potencijalnu ulogu u patogenezi bolesti NPC. Uočene promjene sastava lipida moždanih regija i ranih endosoma mogu doprinijeti razvoju patoloških promjena u bolesti NPC.Niemann-Pick disease type C (NPC) is a rare hereditary neurodegenerative disorder, caused by mutations in the NPC1 or NPC2 gene. It is characterised by accumulation of free cholesterol and other lipids and has a number of pathological similarities with complex Alzheimer's disease (AD). In our previous studies, increased activity of BACE1, the key enzyme of AD, was found in NPC mouse models. Therefore, the aim of this dissertation was to explore the molecular background of the observed effect and examine the effect of BACE1 inhibition on the pathological characteristics of the disease. In addition, the goal was to determine the earliest lipid changes responsible for the differences in brain region vulnerability and endosomal dysfunction in NPC disease by lipidome analysis. Primary neuronal cell cultures, organotipic brain slice cultures, and hippocampus and cerebellum of mice that have a spontaneous mutation in NPC1 gene and do not synthesize the NPC1 protein (NPC1 mice) and wild type mice (wt) were used. NPC1 neurons showed increased proteolysis of BACE1 substrates- Sez6L and Sez6 compared with wt neurons. Immunocytochemical analysis demonstrated accumulation of the aforementioned substrates in early endosomes of NPC1 neurons. The altered localization of Sez6 and Sez6L in early endosomes was confirmed by endolysosome fractionation in the hippocampus and cerebellum of NPC1 mice. Interestingly, treatment with a BACE1 inhibitor partially ameliorated the pathological features of NPC disease, including reduction in the accumulation of Sez6 and Sez6L, reduction in the size of early endosome vesicles, reduction in total tau protein level in NPC1 neurons, and reduction in neuroinflammation (activation of astrocytes and microglia) in cortico-hippocampal brain slices from NPC1 mice. Lipidome analysis revealed that even in the asymptomatic phase of the disease, there are significant differences in the distribution and content of certain lipids between NPC1 and wt mice in the cerebellum and hippocampus, and in early endosomes. These studies provide evidence for a possible role of the BACE1 protease and its substrates in the pathogenesis of NPC disease. The observed changes in the lipid composition of brain regions and early endosomes may contribute to the development of the pathological features of NPC disease
Structural Modifications Introduced by NS2B Cofactor Binding to the NS3 Protease of the Kyasanur Forest Disease Virus
No full textKyasanur Forest Disease virus (KFDV), a neglected human pathogenic virus, is a Flavivirus that causes severe hemorrhagic fever in humans. KFDV is transmitted to humans by the bite of the hard tick (Haemaphysalis spinigera), which acts as a reservoir of KFDV. The recent expansion of the endemic area of KFDV is of concern and requires the development of new preventive measures against KFDV. Currently, there is no antiviral therapy against KFDV, and the existing vaccine has limited efficacy. To develop a new antiviral therapy against KFDV, we focused on the nonstructural proteins NS2B and NS3 of KFDV, which are responsible for serine protease activity. Viral proteases have shown to be suitable therapeutic targets in the development of antiviral drugs against many diseases. However, success has been limited in flaviviruses, mainly because of the important features of the active site, which is flat and highly charged. In this context, the present study focuses on the dynamics of NS2B and NS3 to identify potential allosteric sites in the NS2B/NS3 protease of KDFV. To our knowledge, there are no reports on the dynamics of NS2B and NS3 in KFDV, and the crystal structure of the NS2B/NS3 protease of KFDV has not yet been solved. Overall, we created the structure of the NS2B/NS3 protease of KFDV using AlphaFold and performed molecular dynamics simulations with and without NS2B cofactor to investigate structural rearrangements due to cofactor binding and to identify alternative allosteric sites. The identified allosteric site is promising due to its geometric and physicochemical properties and druggability and can be used for new drug development. The applicability of the proposed allosteric binding sites was verified for the best-hit molecules from the virtual screening and MD simulations