Biokemijska karakterizacija od kalcija neodvisne metakaspaze tipa II iz vrste bombaževca Gossypium raimondii

Jenko, Anže (author), Klemenčič, Marina (mentor)

Regulirana celična smrt (RCD) je evolucijsko ohranjen mehanizem, ki je pomemben tako pri odzivu na stres kot tudi pri rasti in razvoju organizma. Pri poteku RCD pri živalskih celicah so ključne cisteinske endopeptidaze kaspaze, ki pa jih pri preostalih organizmih ni. Na morfološki ravni so RCD opazili tudi pri višjih rastlinah. Za to naj bi bila odgovorna kompleksna signalna pot, kjer pride do usklajenega delovanja različnih proteaz. Med proteazami, ki bi lahko bile ključne pri tem procesu, naj bi bile tudi metakaspaze, ki so strukturno homologne živalskim kaspazam. Kljub enakemu tipu zvitja, se kaspaze in metakaspaze v nekaterih funkcijskih lastnostih razlikujejo. Kaspaze za svojo aktivacijo potrebujejo dimerizacijo ter cepijo svoje substrate zgolj za kislimi aminokislinskimi ostanki, medtem ko metakaspaze cepijo substrate za pozitivno nabitimi aminokislinskimi ostanki ter so aktivne v monomerni obliki, za svojo aktivacijo pa v splošnem potrebujejo kalcijeve ione. Izjema so nekatere izmed metakaspaz tipa II, ki kalcija za svoje delovanje ne potrebujejo ter so aktivne zgolj pri vrednostih pH v rahlo kislem območju. Predmet našega raziskovanja je bila metakaspaza GrMCA-IId iz vrste bombaževca Gossypium raimondii. Ta metakaspaza je strukturno atipična, saj je njeno delovanje od kalcija neodvisno, poleg tega pa je do sedaj prvo opažena metakaspaza, ki jo sestavljata dva segmenta s kaspaznim/hemoglobinaznim zvitjem in je torej nekakšen približek dimera, ki je sicer značilen za kaspaze. Glavni cilj tega dela je bil izraziti, očistiti in okarakterizirati metakaspazo GrMCA-IId ter oba ločena segmenta. Zaradi nezadostno dobljene količine GrMCA-IId celotne dolžine smo za karakterizacijo uporabili le rekombinantno izražena posamezna segmenta oz. polovici; GrMCA-IId (P1) ter GrMCA-IId (P2). Z različnimi encimskimi testi smo ugotovili, da sta obe polovici optimalno katalitično aktivni pri rahlo kislem pH. Obe proteazi pri takšnem pH uspešno cepita tako proteinski substrat β-kazein kot tudi majhna sintetična substrata Z-FR-AMC in Z-RR-AMC, kjer je vrednost Michaelisove konstante v splošnem nižja pri uporabi substrata Z-RR-AMC. Poleg tega smo pri obeh polovicah opazili, da ob izpostavitvi kislemu okolju poteče specifična avtoproteolitska cepitev znotraj posamezne polipeptidne verige, vendar pa ta proces pri GrMCA-IId (P2) poteče precej počasneje kot pri GrMCA-IId (P1). Zanimalo nas je tudi, če se obe polovici spontano povežeta v kompleks preko nekovalentnih interakcij, vendar pa tega z izvedbo gelske filtracije pri izbranih pogojih nismo zaznali. Na podlagi opažanj iz uporabljenih encimskih testov, smo ugotovili, da si obe polovici med seboj katalitično nista zelo podobni, saj je GrMCA-IId (P2) precej slabše aktivna od GrMCA-IId (P1). S poravnavo zaporedij kot tudi pri pripravi modelov struktur v programu Alphafold nismo opazili konkretnih razlik, ki bi lahko bile odgovorne za takšno razliko v delovanju obeh polovic, seveda pa obstaja odprto vprašanje, kako delujeta in sodelujeta obe polovici v GrMCA-IId celotne dolžine

• Language: Slovenian
• Keywords: metakaspaze tipa II, cisteinske proteaze, Gossypium raimondii, regulirana celična smrt
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: FKKT - Faculty of Chemistry and Chemical Technology
• Year: 2022
• PID: 20.500.12556/RUL-141513
• COBISS.SI-ID: 133104899
• Publication date in RUL: 30.09.2022

Secondary language

• Language: English
• Title: Biochemical characterization of calcium-independent metacaspase type II from cotton plant species Gossypium raimondii

Abstract

Regulated cell death (RCD) is an evolutionarily conserved mechanism, which has a vital role in stress response as well as in the development and growth of organisms. In animal cells cysteine endopeptidases - caspases play a crucial role during RCD. However, these enzymes are not present in other organisms.RCD was already reported in higher plants at the morphological level. There is a common belief that this is a consequence of a complex signaling cascade in which different proteases participate in an organized manner. Type of proteases that could be crucial in this mechanism are metacaspases which are structural homologs of caspases from animals. Despite having the same fold there are substantial differences between caspases and metacaspases at a functional level. In caspases, dimerization is crucial for their activation. Caspases also cleave their substrates after acidic amino acid residues whereas metacaspases cleave after positively charged amino acid residues and is active in the form of monomer therefore its activation is generally dependent on the presence of calcium ions. However, there are some exceptions within type II metacaspase that are calcium-independent and become active only under mildly acidic conditions. During our research, we focused on metacaspase GrMCA-IId from cotton plant species Gossypium raimondii. This metacaspase is structurally atypical since it is calcium-independent. It is also the first observed metacasapse to this date that contains two segments with a caspase/hemoglobinase fold, in that sense it resembles dimerization that is otherwise present in caspases. Our main objective was to recombinantly express, purify and characterize metacaspase GrMCA-IId and both separated segments. Since we were not able to produce enough full-legth GrMCA-IId, we performed enzyme assays using recombinantly expressed segments – halves GrMCA-IId (P1) and GrMCA-IId (P2). Using different enzyme assays we discovered that both halves are catalytically active and have a pH optimum at mildly acidic conditions. Under such conditions, both halves successfully cleave protein substrate β-casein as well as small synthetic substrates Z-FR-AMC in Z-RR-AMC. The value of Michaelis constant is generally lower when using Z-RR-AMC. We also noticed that both halves undergo specific autocatalytic cleavage inside each polypeptide chain in mildly acidic conditions. However, this process is considerably slower for GrMCA-IId (P2) than for GrMCA-IId (P1). We were also interested in whether both halves spontaneously form a protein complex through non-covalent interactions. However, we did not notice such interaction under testing conditions using gel filtration. Based on all the tests that we performed we uncovered that both halves are not catalytically similar since GrMCA-IId (P2) is less active than GrMCA-IId (P1). Using sequence alignment coupled with structural models that we prepared with the program Alphafold we did not notice any apparent differences that could be responsible for such differences in enzymatic properties of both halves. However, the question on how both halves work and interact in full-length GrMCA-IId remains open.

• Keywords: metacaspase type II, cysteine proteases, Gossypium raimondii, regulated cell death