Tissue-specific effects of temperature on proteasome function

Variation in ambient growth temperature can cause changes in normal animal physiology and cellular functions such as control of protein homeostasis. A key mechanism for maintaining proteostasis is the selective degradation of polyubiquitinated proteins, mediated by the ubiquitin-proteasome system (UPS). It is still largely unsolved how temperature changes affect the UPS at the organismal level. Caenorhabditis elegans nematodes are normally bred at 20 °C, but for some experimental conditions, 25 °C is often used. We studied the effect of 25 °C on C. elegans UPS by measuring proteasome activity and polyubiquitinated proteins both in vitro in whole animal lysates and in vivo in tissue-specific transgenic reporter strains. Our results show that an ambient temperature shift from 20 to 25 °C increases the UPS activity in the intestine, but not in the body wall muscle tissue, where a concomitant accumulation of polyubiquitinated proteins occurs. These changes in the UPS activity and levels of polyubiquitinated proteins were not detectable in whole animal lysates. The exposure of transgenic animals to 25 °C also induced ER stress reporter fluorescence, but not the fluorescence of a heat shock responsive reporter, albeit detection of a mild induction in hsp-16.2 mRNA levels. In conclusion, C. elegans exhibits tissue-specific responses of the UPS as an organismal strategy to cope with a rise in ambient temperature.Peer reviewe

The chromatin remodeling factor ISW-1 integrates organismal responses against nuclear and mitochondrial stress

Age-associated changes in chromatin structure have a major impact on organismal longevity. Despite being a central part of the ageing process, the organismal responses to the changes in chromatin organization remain unclear. Here we show that moderate disturbance of histone balance during C. elegans development alters histone levels and triggers a stress response associated with increased expression of cytosolic small heat-shock proteins. This stress response is dependent on the transcription factor, HSF-1, and the chromatin remodeling factor, ISW-1. In addition, we show that mitochondrial stress during developmental stages also modulates histone levels, thereby activating a cytosolic stress response similar to that caused by changes in histone balance. These data indicate that histone and mitochondrial perturbations are both monitored through chromatin remodeling and involve the activation of a cytosolic response that affects organismal longevity. HSF-1 and ISW-1 hence emerge as a central mediator of this multi-compartment proteostatic response regulating longevity.Peer reviewe

Insulin/IGF-1 Signaling Regulates Proteasome Activity through the Deubiquitinating Enzyme UBH-4

Peer reviewe

Loss of muscleblind splicing factor shortens Caenorhabditis elegans lifespan by reducing the activity of p38 MAPK/PMK-1 and transcription factors ATF-7 and Nrf/SKN-1

Muscleblind-like splicing regulators (MBNLs) are RNA-binding factors that have an important role in developmental processes. Dysfunction of these factors is a key contributor of different neuromuscular degenerative disorders, including Myotonic Dystrophy type 1 (DM1). Since DM1 is a multisystemic disease characterized by symptoms resembling accelerated aging, we asked which cellular processes do MBNLs regulate that make them necessary for normal lifespan. By utilizing the model organism Caenorhabditis elegans, we found that loss of MBL-1 (the sole ortholog of mammalian MBNLs), which is known to be required for normal lifespan, shortens lifespan by decreasing the activity of p38 MAPK/PM K-1 as well as the function of transcription factors ATF-7 and SKN-1. Furthermore, we show that mitochondrial stress caused by the knockdown of mitochondrial electron transport chain components promotes the longevity of mbl-1 mutants in a partially PMK-1-dependent manner. Together, the data establish a mechanism of how DM1-associated loss of muscleblind affects lifespan. Furthermore, this study suggests that mitochondrial stress could alleviate symptoms caused by the dysfunction of muscleblind splicing factor, creating a potential approach to investigate for therapy.Peer reviewe

Proteasome Activity Influences UV-Mediated Subnuclear Localization Changes of NPM

Peer reviewe

Specific SKN-1/Nrf Stress Responses to Perturbations in Translation Elongation and Proteasome Activity

Peer reviewe

Suppression of RNAi by dsRNA-Degrading RNaseIII Enzymes of Viruses in Animals and Plants

Certain RNA and DNA viruses that infect plants, insects, fish or poikilothermic animals encode Class 1 RNaseIII endoribonuclease-like proteins. dsRNA-specific endoribonuclease activity of the RNaseIII of rock bream iridovirus infecting fish and Sweet potato chlorotic stunt crinivirus (SPCSV) infecting plants has been shown. Suppression of the host antiviral RNA interference (RNAi) pathway has been documented with the RNaseIII of SPCSV and Heliothis virescens ascovirus infecting insects. Suppression of RNAi by the viral RNaseIIIs in non-host organisms of different kingdoms is not known. Here we expressed PPR3, the RNaseIII of Pike-perch iridovirus, in the non-hosts Nicotiana benthamiana (plant) and Caenorhabditis elegans (nematode) and found that it cleaves double-stranded small interfering RNA (ds-siRNA) molecules that are pivotal in the host RNA interference (RNAi) pathway and thereby suppresses RNAi in non-host tissues. In N. benthamiana, PPR3 enhanced accumulation of Tobacco rattle tobravirus RNA1 replicon lacking the 16K RNAi suppressor. Furthermore, PPR3 suppressed single-stranded RNA (ssRNA)–mediated RNAi and rescued replication of Flock House virus RNA1 replicon lacking the B2 RNAi suppressor in C. elegans. Suppression of RNAi was debilitated with the catalytically compromised mutant PPR3-Ala. However, the RNaseIII (CSR3) produced by SPCSV, which cleaves ds-siRNA and counteracts antiviral RNAi in plants, failed to suppress ssRNA-mediated RNAi in C. elegans. In leaves of N. benthamiana, PPR3 suppressed RNAi induced by ssRNA and dsRNA and reversed silencing; CSR3, however, suppressed only RNAi induced by ssRNA and was unable to reverse silencing. Neither PPR3 nor CSR3 suppressed antisense-mediated RNAi in Drosophila melanogaster. These results show that the RNaseIII enzymes of RNA and DNA viruses suppress RNAi, which requires catalytic activities of RNaseIII. In contrast to other viral silencing suppression proteins, the RNaseIII enzymes are homologous in unrelated RNA and DNA viruses and can be detected in viral genomes using gene modeling and protein structure prediction programs.Peer reviewe

Active transcriptomic and proteomic reprogramming in the C-elegans nucleotide excision repair mutant xpa-1

Peer reviewe

Reconstruction and Validation of RefRec: A Global Model for the Yeast Molecular Interaction Network

Molecular interaction networks establish all cell biological processes. The networks are under intensive research that is facilitated by new high-throughput measurement techniques for the detection, quantification, and characterization of molecules and their physical interactions. For the common model organism yeast Saccharomyces cerevisiae, public databases store a significant part of the accumulated information and, on the way to better understanding of the cellular processes, there is a need to integrate this information into a consistent reconstruction of the molecular interaction network. This work presents and validates RefRec, the most comprehensive molecular interaction network reconstruction currently available for yeast. The reconstruction integrates protein synthesis pathways, a metabolic network, and a protein-protein interaction network from major biological databases. The core of the reconstruction is based on a reference object approach in which genes, transcripts, and proteins are identified using their primary sequences. This enables their unambiguous identification and non-redundant integration. The obtained total number of different molecular species and their connecting interactions is ∼67,000. In order to demonstrate the capacity of RefRec for functional predictions, it was used for simulating the gene knockout damage propagation in the molecular interaction network in ∼590,000 experimentally validated mutant strains. Based on the simulation results, a statistical classifier was subsequently able to correctly predict the viability of most of the strains. The results also showed that the usage of different types of molecular species in the reconstruction is important for accurate phenotype prediction. In general, the findings demonstrate the benefits of global reconstructions of molecular interaction networks. With all the molecular species and their physical interactions explicitly modeled, our reconstruction is able to serve as a valuable resource in additional analyses involving objects from multiple molecular -omes. For that purpose, RefRec is freely available in the Systems Biology Markup Language format

Regulation of proteasome activity : the role of insulin/IGF-1 signaling

The ubiquitin-proteasome system (UPS) is the major pathway for controlled protein degradation in the cell. Substrates destined for degradation are polyubiquitinated by ubiquitin ligating enzymes and degraded by the proteasome, which is a complex multisubunit enzyme of over 2.5 MDa. Due to its essential role in maintenance of the cellular protein homeostasis, dysfunctions in proteasome activity have been linked to many severe disorders, such as Alzheimer s, Parkinson s and Huntington s disease and some cancers. Although the proteasome takes part in almost all cellular processes, it is still unclear which factors contribute to regulation of proteasome function. The aim of this thesis was to elucidate mechanisms of how proteasome activity is regulated in a multicellular organism. To enable monitoring and quantification of proteasome activity in vivo, a photoconvertible reporter system was developed to live image proteasome activity in C. elegans. By using this system, it was uncovered that proteasome activity varies between different tissues, as GABAergic and dopaminergic neurons showed faster reporter degradation than muscle cells, and that aging affects proteasome activity in a tissue-dependent manner. Subsequently, it was investigated whether signaling pathways regulating aging also affect proteasome activity. Insulin/IGF-1 signaling (IIS) is an evolutionarily conserved signaling pathway regulating lifespan in worms, flies, rodents and possibly humans. By using C. elegans as a model organism, this thesis shows that IIS regulates proteasome activity through the FOXO/DAF-16 and Nrf/SKN-1 transcription factors. Moreover, it was demonstrated that DAF-16, which is activated by reduced IIS, inhibits the expression of the proteasome-associated deubiquitinating enzyme UBH-4, which was shown to function as a tissue-specific proteasome inhibitor. The role of UBH-4 appears to be well-conserved, as downregulation of its mammalian ortholog, UCHL-5, increases proteasome activity and degradation of proteotoxic proteins in mammalian cells. Taken together, this thesis provides tools to study proteasome activity in vivo, and establishes a molecular mechanism linking IIS to efficiency of proteasomal degradation. These results can be utilized when designing new therapies for proteasome-associated diseases, especially for disorders that may be alleviated with tissue-specific modulation of proteasome activity.Ubikitiini-proteasomi järjestelmä (UPJ) vastaa solun hallitusta proteiininhajotuksesta. UPJ:ssä hajotettava proteiini merkataan ubikitiinimolekyyleillä, jonka jälkeen se hajotetaan proteasomin toimesta. Proteasomi on suuri, molekyylipainoltaan yli 2,5 MDa oleva proteiinikompleksi, jolla on erittäin tärkeä rooli solun proteomin ylläpitäjänä. Tämän vuoksi sen toimintahäiriöiden on epäilty olevan osittain syynä useisiin vakaviin sairauksien, kuten Alzheimerin-, Parkinsonin- ja Huntingtonin tautiin, sekä joihinkin syöpiin. Vaikka proteasomin tiedetään olevan solulle välttämätön, sen toiminnan säätelystä tiedetään suhteellisen vähän. Tämän väitöstutkimuksen tavoitteena oli selvittää kuinka proteasomin aktiivisuutta säädellään monisoluisessa eläimessä. Projektin ensimmäinen vaihe oli kehittää fluoresenssimikroskopiaan perustuva menetelmä, jolla proteasomin aktiivisuutta voidaan mitata elävässä C. elegans sukkulamadossa. Tämän menetelmän avulla havaittiin proteasomin aktiivisuuden vaihtelevan kudoksesta riippuen: GABAergisissä- ja dopaminergisissä hermosoluissa proteasomin toiminta oli lihassoluja tehokkaampaa. Hieman yllättäen havaittiin myös kuinka ikääntyminen, jonka yleisesti uskotaan hidastavan proteasomin toimintaa, ei vaikuta proteasomin aktiivisuuteen samalla tavalla kaikissa kudoksissa. Ikääntymisen ja proteasomin välisen vuorovaikutuksen selvittämistä jatkettiin tutkimalla ikääntymiseen liittyvien signaalivälitysreittien ja proteasomin välistä yhteyttä. Insuliini/IGF-1 signalointi (IIS) on konservoitunut signaalinvälitysreitti, joka vaikuttaa ikääntymiseen monissa eri organismeissa, mahdollisesti myös ihmisessä. Käyttäen C. elegans malliorganismia, IIS:n havaittiin säätelevän proteasomin aktiivisuutta FOXO/DAF-16 ja Nrf/SKN-1 transkriptiotekiöjöiden kautta. Tämän lisäksi, DAF-16:sta osoitettiin estävän proteasomin yhteydessä toimivan UBH-4 deubikitinoivan entsyymin ilmentymistä, jonka puolestaan todettiin toimivan kudoskohtaisesti proteasomin toiminnan estäjänä. Ihmisen UCHL5:llä, joka on ortologinen UBH-4:lle, havaittiin myös olevan kyky estää proteasomin toimintaa, sillä sen ilmentymisen estämisen havaittiin lisäävän proteasomin aktiivisuutta ja edesauttavan soluille haitallisten proteiinien hajotusta. Tämä väitöstutkimus on luonut uusia menetelmiä proteasomitutkimusta varten. Lisäksi tämä tutkimus osoittaa kuinka IIS säätelee proteasomin aktiivisuutta. Näitä tuloksia voidaan hyödyntää kehitettäessä uusia hoitomuotoja sairauksiin, joissa proteasomin toiminnan on todettu olevan heikentynyt