izpis_h1_title_alt

Znotrajcelična lokalizacija, izločanje in privzem cistatina F v človeški mikrogliji ter njegov vpliv na delovanje mikroglije
Drofenik, Lea (Author), Kos, Janko (Mentor) More about this mentor... This link opens in a new window, Perišić Nanut, Milica (Co-mentor)

.pdfPDF - Presentation file, Download (2,50 MB)
MD5: 1E36EF814333A59C52EE87E91B2098CB

Abstract
Cistatin F je zaviralec cisteinskih peptidaz in član skupine tipa II cistatinske družine. Sintetizira se kot 145 aminokislin (AK) dolg neaktiven dimer. N-vezani sladkorji mu omogočajo prenos v endosome/lizosome, kjer se aktivira. Monomerizacijo in aktivacijo olajša proteolitična cepitev 15 AK na N-končnem delu, ki tudi spremeni inhibitorne lastnosti monomerne oblike. N-končno skrajšan cistatin F postane močan zaviralec katepsina C. Izločena neaktivna dimerna oblika cistatina F se lahko privzame tudi v okoliške celice in se znotraj njih aktivira. Cistatin F se specifično izraža v imunskih celicah, v patoloških stanjih pa je njegovo izražanje lahko prisotno tudi v drugih celicah. Tako se pri multipli sklerozi med procesom demielinizacije izraža v mikrogliji. Povišano izražanje gena Cst7, ki kodira cistatin F, je pokazano tudi v mikrogliji, na mišjem modelu Alzheimerjeve bolezni. Mikroglije so makrofagom podobne celice centralno živčnega sistema (CŽS). Namen magistrske naloge je bil določiti znotrajcelično lokalizacijo cistatina F v človeški mikrogliji, njegovo izločanje iz celic in privzem v okoliške celice mikroglije. Glavne tarče inhibitornega delovanja cistatina F smo določili z merjenjem aktivnosti katepsinov. V ta namen smo hTERT celice transfecirali s plazmidi, ki v svojem nukleotidnem zaporedju nosijo zapis za dimerno (divji tip), aktivno N-končno skrajšano ali neglikozilirano obliko cistatina F. Proteinske vrednosti cistatina F v mikrogliji, njegovo izločanje v gojišče in privzem v okoliške celice smo določili s prenosom western. Lokalizacijo posameznih oblik cistatina F v celičnih predelkih pa z uporabo fluorescenčne konfokalne mikroskopije. Določili smo, da se tako dimerni (divji tip) cistatin F kot tudi aktiven N-končno skrajšan cistatin F privzemata iz zunajceličnega prostora in preneseta v endosome/lizosome. Neglikozilirana oblika cistatina F pa se zaradi odsotnosti N-vezanih sladkorjev ne privzame v netransfecirane celice in je tudi v monomerni obliki ne najdemo v endosomih/lizosomih. Odsotnost N-vezanih sladkorjev torej onemogoči zaviranje cisteinskih katepsinov in delovanje cistatina F in trans v človeški mikrogliji. Izražanje cistatina F v mikrogliji nekoliko zmanjša aktivnost katepsina C, bolj pa aktivnost katepsina L.

Language:Slovenian
Keywords:cistatin F, mikroglija, privzem proteina, cisteinski katepsini
Work type:Master's thesis/paper (mb22)
Organization:FFA - Faculty of Pharmacy
Year:2021
Views:187
Downloads:53
Metadata:XML RDF-CHPDL DC-XML DC-RDF
 
Average score:(0 votes)
Your score:Voting is allowed only to logged in users.
:
Share:AddThis
AddThis uses cookies that require your consent. Edit consent...

Secondary language

Language:English
Title:Intracellular localization, secretion and uptake of cistatin F in human microglia and its impact on microglial functioning
Abstract:
Cystatin F is a cysteine peptidase inhibitor and a member of the type II cystatin family. It is synthesized as an 145 amino acid (AA) long inactive dimer. N-linked glycans alow it to transfer to endosomes/lysosomes, where it is activated. Monomerization and activation of cystatin F is facilitated by the proteolytic cleavege of the 15 AA at the N-terminal part. The proteolytic cleavage also alters the inhibitory profile of the monomeric form. Consequently, the N-terminal truncated cystatin F becomes a strong inhibitor of cathepsin C. The secreted inactive dimeric form of cystatin F can also be taken up by bystander cells and activated within them. Cystatin F is specifically expressed in immune cells, however, in pathological conditions its expression can be present in different cell types. In multiple sclerosis, during the phase of demyelination it is expressed in microglia. Elevated expression of the Cst 7 gene, which encodes the cystatin F, has also been shown in a mouse model of Alzheimerˊs disease. Microglia are macrophage-like cells of the central nervous system (CNS). The aim of our study was to determine the intracellular localization of cistatin F in human microglia, its secretion and uptake into the bystander cells. Main targets of cystatin F in microglia were determined by measuring the activity of cysteine proteases. For this purpose, h-TERT cells were transfected with plasmids containing dimeric (wild type), active N-terminally truncated or non-glycosylated form of cistatin F. The expression of cystatin F in microglia, its secretion from cells and uptake into bystander cells were determined by using western blot. Localization of individual forms of cystatin F within the cells were determined by using fluorescence confocal microscopy. Both dimeric (wild type) and active N-terminally truncated cystatin F were found to be internalized and transfered to endosomes/lysosomes of microglia. However, due to the absence of N-linked glycans, the non-glycosylated form of cystatin F was not taken up by recipient cells and did not enter the endo/lysosomal system. The absence of N-linked glycans thus inhibits the inhibition of cysteine cathepsins and the action of cystatin F in trans in human microglia. The expression of cystatin F in microglia reduces the activity of cathepsin L wereas its effects on cathepsin C activity were negligible.

Keywords:cystatin F, microglia, protein uptake, cystein cathepsins

Similar documents

Similar works from RUL:
Similar works from other Slovenian collections:

Comments

Leave comment

You have to log in to leave a comment.

Comments (0)
0 - 0 / 0
 
There are no comments!

Back