Evolucijska analiza dipeptidil peptidaze I

diplomsko delo

Nina Varda (Author), Marko Novinec (Mentor)

Abstract

Človeška dipeptidil peptidaza I (DPPI) oz. katepsin C je encim, ki ga uvrščamo v družino papainu podobnih cisteinskih peptidaz. Njegovi posebni lastnosti sta izključitvena domena, ki omogoča njegovo eksopeptidazno aktivnost in tetramerizacijo, ter odvisnost encimske aktivnosti od kloridnega iona. Na njegovo tetramerizacijo vpliva tudi prisotnost glikozilacije. Ločen evolucijski razvoj DPPI se je najverjetneje začel s pridružitvijo eksopeptidazne domene in tvorbe funkcionalne monomerne eksopeptidaze. Namen dela je bil raziskati evolucijo DPPI, pri čemer smo se osredotočili na lastnosti, ki omogočajo tetramerizacijo. Analizirali smo stične površine med podenotami DPPI in identificirali aminokislinske ostanke, ki tvorijo vodikove vezi. Nato smo izvedli filogenetsko analizo 41 zaporedij DPPI in njegovih homologov. Aminokislinski ostanki, ki vežejo kloridni ion, so v vseh vrstah zelo dobro ohranjeni, iz česar smo zaključili, da je vezava kloridnega iona evolucijsko dobro ohranjena lastnost. Iz ohranjenosti glikozilacijskih vzorcev in ohranjenosti ostankov, ki tvorijo vezi med podenotami, smo zaključili, da se je oligomerizacija najverjetneje pojavila v razvojni liniji vretenčarjev. Med izvajanjem analiz smo ugotovili, da je bočna interakcija med podenotami znatno šibkejša od interakcije glava-rep (angl. head-to-tail), zato smo v drugem delu naloge to povezavo želeli stabilizirati. V ta namen smo identificirali tri najbolj primerne pare ostankov na interakcijski površini, ki bi po zamenjavi s Cys ostankom lahko tvorili disulfidne mostičke in tako stabilizirali interakcijo. Ugotovitve smo želeli preveriti tudi eksperimentalno, a zaradi netopnosti mutante vpliva teh mutacij nismo uspeli potrditi.

Keywords

encimi;peptidaze;dipeptidil peptidaza I;oligomerizacija proteinov;evolucija;stabilizacija;filogenetska analiza;mutageneza DPPI;diplomska dela;

Data

Language:	Slovenian
Year of publishing:	2021
Typology:	2.11 - Undergraduate Thesis
Organization:	UL FKKT - Faculty of Chemistry and Chemical Technology
Publisher:	[N. Varda]
UDC:	577.15(043.2)
COBISS:	78758147
Views:	256
Downloads:	50
Average score:	0 (0 votes)
Metadata:

Other data

Secondary language:	English
Secondary title:	Evolutionary Analysis of Dipeptidyl-peptidase I
Secondary abstract:	Human dipeptidyl peptidase I (DPPI), also known as cathepsin C, is an enzyme belonging to the family of papain-like cysteine proteases. Its characteristic features are the exclusion domain, which enables its exopeptidase activity as well as tetramerization and its dependence on chloride ions for enzymatic activity. The evolution of DPPI most likely began with the addition of the exclusion domain to form a monomeric exopeptidase. The aim of this work was to investigate the evolution of DPPI with particular attention to structural features that enabled its oligomerization. By examining the subunit interfaces, we first determined the amino acid residues that form hydrogen bonds between the subunits. We then analyzed 41 sequences of DPPI and its homologs. The amino acid residues that bind the chloride ion are highly conserved in all species, leading us to conclude that chloride ion binding is an evolutionarily conserved feature of DPPI. From the conservation of glycosylation patterns and amino acid residues forming bonds between subunits, we concluded that oligomerization likely evolved with the emergence of vertebrates. In our analysis, we determined that the lateral interaction between the subunits was substantially weaker than the head-to-tail interaction, so we aimed to stabilize it in the second part of this work. We identified 3 most suitable positions on the interaction surface, that would be able to form disulfide bonds after their replacement by Cys residues and thereby stabilize the interaction. In the last part of the work, we wanted to verify these findings experimentally, but due to the insolubility of the produced mutant, we could not confirm the effect of these mutations.
Secondary keywords:	oligomerization;evolution;stabilization;
Type (COBISS):	Bachelor thesis/paper
Study programme:	1000371
Embargo end date (OpenAIRE):	1970-01-01
Thesis comment:	Univ. v Ljubljani, Fak. za kemijo in kemijsko tehnologijo, UNI Biokemija
Pages:	43 str.
ID:	13328265