Truncated HSPB1 causes axonal neuropathy and impairs tolerance to unfolded protein stress

Peer reviewe

Redox regulation of GRPEL2 nucleotide exchange factor for mitochondrial HSP70 chaperone

Mitochondria are central organelles to cellular metabolism. Their function relies largely on nuclear-encoded proteins that must be imported from the cytosol, and thus the protein import pathways are important for the maintenance of mitochondrial proteostasis. Mitochondrial HSP70 (mtHsp70) is a key component in facilitating the translocation of proteins through the inner membrane into the mitochondrial matrix. Its protein folding cycle is regulated by the nucleotide-exchange factor GrpE, which triggers the release of folded proteins by ATP rebinding. Vertebrates have two mitochondrial GrpE paralogs, GRPEL1 and 2, but without clearly defined roles. Using BioID proximity labeling to identify potential binding partners of the GRPELs in the mitochondrial matrix, we obtained results supporting a model where both GRPELs regulate mtHsp70 as homodimers. We show that GRPEL2 is not essential in human cultured cells, and its absence does not prevent mitochondrial protein import. Instead we find that GRPEL2 is redox regulated in oxidative stress. In the presence of hydrogen peroxide, GRPEL2 forms dimers through intermolecular disulfide bonds in which Cys87 is the thiol switch. We propose that the dimerization of GRPEL2 may activate the folding machinery responsible for protein import into mitochondrial matrix or enhance the chaperone activity of mtHSP70, thus protecting mitochondrial proteostasis in oxidative stress.Peer reviewe

A patient with pontocerebellar hypoplasia type 6 : Novel RARS2 mutations, comparison to previously published patients and clinical distinction from PEHO syndrome

Pontocerebellar hypoplasia type 6 (PCH6) is a rare infantile-onset progressive encephalopathy caused by biallelic mutations in RARS2 that encodes the mitochondrial arginine-tRNA synthetase enzyme (mtArgRS). The clinical presentation overlaps that of PEHO syndrome (Progressive Encephalopathy with edema, Hypsarrhythmia and Optic atrophy). The proband presented with severe intellectual disability, epilepsy with varying seizure types, optic atrophy, axial hypotonia, acquired microcephaly, dysmorphic features and progressive cerebral and cerebellar atrophy and delayed myelination on MRI. The presentation had resemblance to PEHO syndrome but sequencing of ZNHIT3 did not identify pathogenic variants. Subsequent whole genome sequencing revealed novel compound heterozygous variants in RARS2, a missense variant affecting a highly conserved amino acid and a frameshift variant with consequent degradation of the transcript resulting in decreased mtArgRS protein level confirming the diagnosis of PCH6. Features distinguishing the proband's phenotype from PEHO syndrome were later appearance of hypotonia and elevated lactate levels in blood and cerebrospinal fluid. On MRI the proband presented with more severe supratentorial atrophy and lesser degree of abnormal myelination than PEHO syndrome patients. The study highlights the challenges in clinical diagnosis of patients with neonatal and early infantile encephalopathies with overlapping clinical features and brain MRI findings.Peer reviewe

Instability of the mitochondrial alanyl-tRNA synthetase underlies fatal infantile-onset cardiomyopathy

Recessively inherited variants in AARS2 (NM_020745.2) encoding mitochondrial alanyl-tRNA synthetase (mt-AlaRS) were first described in patients presenting with fatal infantile cardiomyopathy and multiple oxidative phosphorylation defects. To date, all described patients with AARS2-related fatal infantile cardiomyopathy are united by either a homozygous or compound heterozygous c.1774C>T (p.Arg592Trp) missense founder mutation that is absent in patients with other AARS2-related phenotypes. We describe the clinical, biochemical and molecular investigations of two unrelated boys presenting with fatal infantile cardiomyopathy, lactic acidosis and respiratory failure. Oxidative histochemistry showed cytochrome c oxidase-deficient fibres in skeletal and cardiac muscle. Biochemical studies showed markedly decreased activities of mitochondrial respiratory chain complexes I and IV with a mild decrease of complex III activity in skeletal and cardiac muscle. Using next-generation sequencing, we identified a c.1738C>T (p.Arg580Trp) AARS2 variant shared by both patients that was in trans with a loss-of-function heterozygous AARS2 variant; a c.1008dupT (p.Asp337*) nonsense variant or an intragenic deletion encompassing AARS2 exons 5-7. Interestingly, our patients did not harbour the p.Arg592Trp AARS2 founder mutation. In silico modelling of the p.Arg580Trp substitution suggested a deleterious impact on protein stability and folding. We confirmed markedly decreased mt-AlaRS protein levels in patient fibroblasts, skeletal and cardiac muscle, although mitochondrial protein synthesis defects were confined to skeletal and cardiac muscle. In vitro data showed that the p.Arg580Trp variant had a minimal effect on activation, aminoacylation or misaminoacylation activities relative to wild-type mt-AlaRS, demonstrating that instability of mt-AlaRS is the biological mechanism underlying the fatal cardiomyopathy phenotype in our patients.Peer reviewe

Molecular Consequences of Transfer-RNA Charging Defects

Proteins, consisting of amino acids, work as building blocks in the cells. In addition, they carry out vast amounts of cellular functions. Accurate protein synthesis is thus crucial for the normal function of cells. The first step of protein synthesis is the charging of transfer-RNAs (tRNAs) with their cognate amino acids. Evolutionarily conserved and extremely old proteins, aminoacyl-tRNA synthetases (aaRSs), carry out this step and each amino acid-tRNA pair has its own synthetase for the task. However, in some cases the amino acids are so similar in size and structure that they cannot be separated well enough by the aaRSs. To avoid mischarging and the subsequent protein misfolding, some of the synthetases have an editing domain, which recognizes and hydrolyses incorrect amino acid-tRNA pairs. In addition, cells have other important quality control mechanisms to ensure protein homeostasis, the capacity of cells to maintain internal stability of the proteome. Patient mutations in genes connected to protein synthesis and quality control are found to cause different diseases, the mechanisms of which are not yet, however, well known. Research on these topics is thus important. The aim of this thesis was to study the molecular mechanisms of different tRNA-charging defects and protein quality control mechanisms in both protein-translating compartments of a eukaryotic cell, cytosol and mitochondria. The first part of the thesis describes the molecular mechanism and the clinical phenotype of a special cytosolic tRNA charging defect caused by mutations in SEPSECS gene. The corresponding protein is involved in charging of the 21st amino acid, selenocysteine, to its tRNA. We identified mutations in this gene and showed them to lead to a decreased amount of selenocysteine-containing proteins, selenoproteins, in the brain of a patient with a severe early onset encephalopathy. Our study also indicated increased protein oxidation in the patient brain. This study extends the clinical phenotypes connected to SEPSECS mutations, and indicates that selenoprotein synthesis defect can resemble mitochondrial disease with lactate elevation. In the second part of this thesis, the potential of an amino acid analogue of arginine, canavanine, to induce protein misfolding in mitochondria was studied. The results demonstrated that mitochondrial protein translation machinery does not distinguish canavanine from arginine. The amino acid analog was incorporated into mitochondrially encoded proteins causing protein instability and formation of aberrant polypeptides. Surprisingly, however, canavanine did not induce mitochondrial unfolded protein response (UPRmt), a previously described signalling pathway induced by accumulation of misfolded proteins inside mitochondria. The study showed that none of the protein quality control mechanisms were able to solve protein misfolding caused by canavanine, which led to a severe respiratory chain defect. Canavanine has been used previously in a large number of studies to induce cytosolic protein misfolding, but the impact of canavanine for mitochondrial function has been largely ignored. Canavanine can be used in future as a tool to study further the consequences of protein misfolding in mitochondria, and for studying how mitochondria solve stalled ribosomes, which was also detected to be a consequence of canavanine in our study. The goal of the third part of the thesis was to study UPRmt in an animal model. The purpose was to generate a mouse model that has a mutation in the editing domain of mitochondrial alanyl-tRNA synthetase, leading to amino acid mischarging and formation of unfolded proteins inside mitochondria in vivo. The result of the study indicated for the first time the importance of amino acid editing by a tRNA synthetase as an essential quality control mechanism in mammalian mitochondria. The work presented in this thesis provides new information concerning the mechanisms of different tRNA charging defects and their consequences for the cell and organism. Special emphasis was on mitochondrial function.Proteiinit muodostuvat aminohapoista, jotka on liitetty toisiinsa peptidisidoksin. Proteiinit toimivat sekä solujen rakennuspalikoina, että valtavassa määrässä solujen reaktioketjuja. Virheetön proteiinisynteesi on täten elintärkeää solujen toiminnalle. Ensimmäinen vaihe proteiinisynteesissä on siirtäjä-RNA:iden (tRNA) ja niitä vastaavien aminohappojen yhteen liittäminen. Evolutiivisesti erittäin konservoituneet proteiinit, aminoasyyli-tRNA syntetaasit (aaRS), vastaavat tästä liitoksesta, ja jokaisella aminohappo-tRNA parilla on oma aaRS-entsyyminsä tähän tehtävään. Jotkin aminohapot ovat niin samanlaisia kooltaan ja rakenteeltaan, että osa aaRS:sta ei pysty erottamaan niitä toisistaan. Välttääkseen väärien aminohappojen latauksen tRNA:ihin ja siitä seuraavan proteiinien väärin laskostumisen, osalla aaRS:sta on rakenteessaan oikolukuosa, joka tunnistaa väärin ladatut aminohappo-tRNA parit ja hydrolysoi ne. Sen lisäksi soluilla on muita laaduntarkkailumekanismeja, jotka vastaavat proteiinien homeostaasista eli solujen kapasiteetista ylläpitää proteomin tasapainoa. Monista proteiinisynteesiin tai laaduntarkkailumekanismeihin liittyvistä geeneistä on löydetty tautimutaatioita, jotka aiheuttavat hyvin erilaisten sairauksien ilmenemisen ihmisellä. Näiden sairauksien mekanismeja ei kuitenkaan tunneta tarkkaan, joten niiden molekyylimekanismien tutkiminen on tärkeää. Tämän väitöskirjan tavoitteena oli tutkia erilaisia tRNA:iden latausvirheitä ja proteiinien laaduntarkkailumekanismeja kahdessa solujen proteiineja tuottavassa osassa, sytosolissa ja mitokondrioissa. Väitöskirjan ensimmäisessä osatyössä kuvataan sytoplasmisen tRNA:n lataushäiriön molekulaarinen mekanismi perinnöllisessä aivosairaudessa. Häiriö aiheutuu mutaatioista SEPSECS geenissä, jota tarvitaan 21. aminohapon, selenokysteiinin, lataukseen sen tRNA:han. Potilailla tämän geenin mutaatiot johtivat selenokysteiiniä sisältävien proteiinien, selenoproteiinien, määrän laskuun aivoissa, mitä seurasi potilailla vakavan aivosairauden kehittyminen. Tutkimuksemme osoitti lisäksi potilaiden aivonäytteissä lisääntynyttä proteiinien hapettumista. Tutkimuksemme laajensi SEPSECS-geenivirheisiin liitettyjen sairauksien kliinistä kuvaa, sillä osoitimme potilailla aiemmin kuvaamattomia mitokondriosairauksille tyypillisistä oireista kuten veren kohonneet laktaattiarvot. Väitöskirjan toisessa osatyössä tutkittiin arginiini-aminohapon analogin, kanavaniinin, kykyä indusoida proteiinien väärinlaskostumista mitokondrioissa. Tutkimus osoitti, ettei mitokondrioiden translaatiokoneisto kykene erottamaan kanavaniinia arginiinista, joten se päätyy mitokondrioissa tuotettaviin proteiineihin arginiinin sijaan aiheuttaen näiden proteiinien poikkeavaa laskostumista. Yllättäen kanavaniini ei kuitenkaan indusoinut aiemmin kuvattua mitokondriaalisten, väärinlaskostuneiden proteiinien aiheuttamaa signaalikaskadia. Tutkimus osoitti, etteivät mitokondrion laaduntarkkailumekanismit kykene korjaamaan kanavaniinin aiheuttamaa proteiinien väärinlaskoutumista ja siitä aiheutuvaa hengitysketjupuutosta mitokondrioissa. Kanavaniinia on käytetty monissa sytosolisten proteiinien väärinlaskoutumisen seurauksia selvittävissä tutkimuksissa, mutta niissä kanavaniinin vaikutuksia mitokondrioiden toiminnalle ei ole otettu aikaisemmin huomioon. Tulostemme perusteella kanavaniinia voidaan käyttää jatkossa työkaluna tutkittaessa väärinlaskostuneiden proteiinien vaikutuksia mitokondrioiden toiminnalle. Väitöskirjan kolmannessa osatyössä haluttiin tutkia aikaisemmin kuvattua väärinlaskostuneiden proteiinien aiheuttamaa signaalikaskadia mitokondrioissa hiirimallin avulla. Tämän projektin tarkoituksena oli luoda hiirimalli, jonka mitokondriaalisessa alanyyli-tRNA syntetaasin oikolukuosassa on mutaatio, joka johtaa oikolukutoiminnon häiriöön, väärin ladattuihin tRNA ja siitä seuraavaan proteiinien väärin laskostumiseen mitokondrioissa. Väitöskirjan kolmas osatyö osoitti ensimmäistä kertaa, että aminohappojen oikoluku on elintärkeää nisäkkäiden mitokondrioissa. Tämä väitöskirja on tuottanut uutta tietoa tRNA:iden lataushäiriöistä ja niiden seurauksista solun ja organismin toiminnalle. Väitöskirjassa keskityttiin erityisesti mitokondrioiden toimintaan

Editing activity for eliminating mischarged tRNAs is essential in mammalian mitochondria

Accuracy of protein synthesis is enabled by the selection of amino acids for tRNA charging by aminoacyl-tRNA synthetases (ARSs), and further enhanced by the proofreading functions of some of these enzymes for eliminating tRNAs mischarged with noncognate amino acids. Mouse models of editing-defective cytoplasmic alanyl-tRNA synthetase (AlaRS) have previously demonstrated the importance of proofreading for cytoplasmic protein synthesis, with embryonic lethal and progressive neurodegeneration phenotypes. Mammalian mitochondria import their own set of nuclear-encoded ARSs for translating critical polypeptides of the oxidative phosphorylation system, but the importance of editing by the mitochondrial ARSs for mitochondrial proteostasis has not been known. We demonstrate here that the human mitochondrial AlaRS is capable of editing mischarged tRNAs in vitro, and that loss of the proofreading activity causes embryonic lethality in mice. These results indicate that tRNA proofreading is essential in mammalian mitochondria, and cannot be overcome by other quality control mechanisms.Peer reviewe

GTPBP8 is required for mitoribosomal biogenesis and mitochondrial translation

Mitochondrial translation occurs on the mitochondrial ribosome, also known as the mitoribosome. The assembly of mitoribosomes is a highly coordinated process. During mitoribosome biogenesis, various assembly factors transiently associate with the nascent ribosome, facilitating the accurate and efficient construction of the mitoribosome. However, the specific factors involved in the assembly process, the precise mechanisms, and the cellular compartments involved in this vital process are not yet fully understood. In this study, we discovered a crucial role for GTP-binding protein 8 (GTPBP8) in the assembly of the mitoribosomal large subunit (mt-LSU) and mitochondrial translation. GTPBP8 is identified as a novel GTPase located in the matrix and peripherally bound to the inner mitochondrial membrane. Importantly, GTPBP8 is specifically associated with the mt-LSU during its assembly. Depletion of GTPBP8 leads to an abnormal accumulation of mt-LSU, indicating that GTPBP8 is critical for proper mt-LSU assembly. Furthermore, the absence of GTPBP8 results in reduced levels of fully assembled 55S monosomes. This impaired assembly leads to compromised mitochondrial translation and, consequently, impaired mitochondrial function. The identification of GTPBP8 as an important player in these processes provides new insights into the molecular mechanisms underlying mitochondrial protein synthesis and its regulation.Peer reviewe