Heat shock proteins (HSPs) are molecular chaperones that assist in protein synthesis, folding and degradation and prevent stress-induced protein aggregation. The present study examined the pattern of accumulation of HSP30 and HSP70 in cells recovering from heat shock as well as the effect of proteasome inhibition on cytoplasmic/nuclear and endoplasmic reticulum (ER) molecular chaperone accumulation, large multimeric HSP30 complexes, stress granule and aggresome formation in Xenopus laevis A6 kidney epithelial cells. Initial immunoblot analysis revealed the presence of elevated levels of HSP30 after 72 h of recovery. However, the relative levels of HSP70 declined to near control levels after 24 h. The relative levels of both hsp30 and hsp70 mRNA were reduced to low levels after 24 h of recovery from heat shock. Pretreatment of cells with cycloheximide, a translational inhibitor, produced a rapid decline in HSP70 but not HSP30. The cycloheximide-associated decline of HSP70 was blocked by the proteasomal inhibitor, MG132, but had little effect on the relative level of HSP30. Also, treatment of cells with the phosphorylation inhibitor, SB203580, in addition to cycloheximide treatment enhanced the stability of HSP30 compared to cycloheximide alone. Immunocytochemical studies detected the presence of HSP30 accumulation in a granular pattern in the cytoplasm of recovering cells and its association with aggresome-like structures, which was enhanced in the presence of SB203580. To verify if proteasome inhibition in A6 cells induced the formation of similar HSP30 granules, immunoblot and immunocytochemical analyses were performed. MG132, celastrol and withaferin A enhanced ubiquitinated proteins, inhibited chymotrypsin-like activity of the proteasome and induced the accumulation of cytoplasmic/nuclear HSPs, HSP30 and HSP70 as well as ER chaperones, BiP and GRP94 and heme oxygenase-1. Northern blot experiments determined that proteasome inhibitors induced an accumulation in hsp30, hsp70 and bip mRNA but not eIF1α. The final part of this study demonstrated that treatment of A6 cells with proteasome inhibitors or sodium arsenite or cadmium chloride induced HSP30 multimeric complex formation primarily in the cytoplasm. Moreover, these stressors also induced the formation of RNA stress granules, pre-stalled translational complexes, which were detected via TIA1 and polyA binding protein (PABP), which are known stress granule markers. These stress granules, however, did not co-localize with large HSP30 multimeric complexes. In comparison, proteasome inhibition or treatment with sodium arsenite or cadmium chloride also induced the formation of aggresome-like structures, which are proteinaceous inclusion bodies formed as a result of an abundance of aggregated protein. Aggresome formation was identified by monitoring the presence of vimentin and γ-tubulin, both of which are cytoskeletal proteins and serve as markers of aggresome detection. Aggresome formation, which was also verified using the ProteoStat assay, co-localized with large HSP30 multimeric complexes. Co-immunoprecipitation experiments revealed that HSP30 associated with γ-tubulin and β-actin in cells treated with proteasome inhibitors or sodium arsenite or cadmium chloride suggesting a possible role in aggresome formation. In conclusion, this study has shown that the relative levels of heat shock-induced HSP30 persist during recovery in contrast to HSP70. While HSP70 is degraded by the ubiquitin-proteasome system, it is likely that the presence of HSP30 multimeric complexes that are known to associate with unfolded protein as well as its association with aggresome-like structures may delay its degradation. Finally, proteasome inhibition, sodium arsenite and cadmium chloride treatment of A6 cells induced cytoplasmic/nuclear and ER chaperones as well as resulting in the formation stress granules and aggresome-like structures which associated with large HSP30 multimeric complexes.

Ebola virus (EBOV) causes severe disease in humans characterized by high case fatality rates and significant immune dysfunction. A hallmark of EBOV infection is the formation of viral inclusions in the cytoplasm of infected cells. These inclusions contain the EBOV nucleocapsids and are sites of viral replication and nucleocapsid maturation. Although there is growing evidence that viral inclusions create a protected environment that fosters EBOV gene expression and genome replication, little is known about their role in the host response to infection. The cellular stress response is an antiviral strategy that leads to stress granule (SG) formation and translational arrest mediated by the phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eIF2α). Related to this response is the post-transcriptional regulation of RNA mediated by stability elements called AU-rich elements (AREs) and their associated binding proteins (ARE-BPs), many of which are found in SGs. Because these processes have antiviral implications, many viruses have evolved strategies to interfere with SG formation, or appropriate ARE-BPs to benefit viral replication. However, it is unknown if EBOV interacts with these cellular systems. Here, we show that SG proteins were sequestered within EBOV inclusions where they formed distinct granules that colocalized with viral RNA. The inclusion-bound aggregates were not canonical SGs, and did not lead to translational arrest in infected cells. EBOV did not induce cytoplasmic SGs at any time post infection, but was unable to overcome SG formation induced by additional stressors. Despite the sequestration of SG proteins, canonical SGs did not form within inclusions. At high levels of expression, viral protein 35 (VP35), the viral polymerase co-factor that also mediates various immune evasion functions, disrupted SGs formation independently of eIF2α phosphorylation. Finally, we found that the cellular ARE-BP tristetraprolin (TTP) specifically targeted the 3’untranslated region (UTR) of the viral nucleoprotein (NP) mRNA and promoted its degradation. Interestingly, TTP was not found within viral inclusions, leading us to speculate that inclusions might serve to prevent viral RNA from encountering TTP. These results indicate that EBOV interacts with the cellular stress response and associated RNA regulatory proteins in ways that promote viral replication.

Cytoplasmic mRNA-containing granules in eukaryotic cells play key roles inthe storage, localisation and degradation of mRNA. In yeast, depletion of glucoseleads to the rapid inhibition of translation initiation and consequent appearance of Pbodiesand EGP-bodies. P-bodies contain factors of the mRNA decay pathway andtherefore, are likely to be sites in which mRNAs targeted for degradation arelocalised. In contrast, EGP-bodies lack decay components and contain onlytranslation initiation factors and RNA binding proteins. Thus EGP-bodies have beensuggested to be storage repositories for mRNAs that need to be rapidly translatedfollowing glucose readdition. In this study we utilised the m-TAG system to investigate the localisation ofendogenous MS2-tagged mRNAs with P-bodies and EGP-bodies. A triplefluorescent labelled system developed show that a class of unregulated mRNAslocalised into P-bodies following glucose starvation. It was also observed that thesespecific abundant classes of mRNAs can be found in aggregates prior to any cellularstress and upon glucose starvation these aggregates coalesce into larger granules thatcolocalise with P-body components. This coalescence of mRNA aggregatesfollowing glucose starvation does not rely upon the recruitment of mRNA decayfactors and appears to precede this event. Indeed mRNAs in mutants deficient in Pbodyformation still develop large aggregates following glucose stress. In unstressedcells it appears that the mRNA granules are implicated in high-level translation ofthese specific abundant mRNAs. Following the inhibition of translation initiation inresponse to stress, these granules nucleate P-body formation via aggregation and therecruitment of mRNA decay factors.

Le gène suppresseur de tumeur LKB1 code une sérine/thréonine kinase qui régule le métabolisme énergétique et la polarité cellulaire. Son action biologique s'exerce en partie via la protéine kinase activée par l'AMP (AMPK), substrat de LKB1 dont la phosphorylation stimule l'activité catalytique. Nous avons récemment mis en évidence une interaction entre LKB1 et la déacétylase HDAC6. HDAC6 régule principalement l'état d'acétylation de protéines localisées dans le cytoplasme telles que la molécule chaperon HSP90, la tubuline α, et la cortactine. HDAC6 contrôle la stabilité des protéines liées à HSP90 mais agit aussi sur la polarité et l'adhérence des cellules. De plus, HDAC6 répond à différentes situations de stress cellulaire en favorisant le transport des protéines polyubiquitinées vers les aggrésomes, où celles ci sont dégradées, et en promouvant la formation des granules de stress, complexes ribonucléoprotéiques participant au stockage des ARNm et au blocage de la traduction. Mon projet de recherche a porté sur les conséquences fonctionnelles de l'interaction entre LKB1 et HDAC6. J'ai ainsi pu montrer que la formation de ce complexe est renforcée en condition de stress oxydatif et thermique. Dans cette situation biologique, LKB1 interfère avec la capacité de HDAC6 à fixer les protéines ubiquitinylées, et par conséquent prévient la formation des aggrésomes et des granules de stress. A l'inverse, LKB1 stimule l'activité déacétylase de HDAC6, et cette action de LKB1 est requise pour la migration orientée des cellules ainsi que pour la polarisation apico-basale dans un modèle de culture d'entérocytes. Ce travail nous a ainsi permis d'identifier un nouvel effecteur de LKB1 qui intervient dans la réponse au stress et dans la polarisation cellulaire. Il s'agit aussi de la première mise en évidence d'une régulation de l'activité de liaison à l'ubiquitine de HDAC6. Ces données suggèrent que LKB1, via son effet sur HDAC6, pourrait limiter la réponse adaptative des cellules soumises à des stress exogènes et endogènes, comme ceux que les cellules en voie de transformation rencontrent dans leur microenvironnement, une propriété qui pourrait s'avérer essentielle pour son activité de suppresseur de tumeur / The tumor suppressor LKB1 is a serine-threonine kinase that acts as a critical regulator of energy homeostasis and cell polarity 1,2. LKB1 relays its intracellular signal through the AMP-activated protein kinase (AMPK) as well as twelve additional members of the AMPK sub-family 3-5. However, despite the identification of these LKB1 effectors, the mechanisms that underlie LKB1-mediated biological effects remain incompletely understood. We now report that LKB1 interacts with and phosphorylates HDAC6, a deacetylase that protects cells against extrinsic insults through its ability to ligate polyubiquinated misfolded proteins and to dynamically associate with both the microtubule and the actin cytoskeleton networks 6. We further found that the formation of the LKB1-HDAC6 complex was promoted in response to diverse stressful stimuli. As a consequence, HDAC6 ubiquitin-binding activity was inhibited, thus impeding the formation of aggresomes and stress granules, two transient cellular structures that, respectively, prevent the accumulation of aggregated proteins 7 and remodel messenger ribonucleoprotein complexes following stresses that block translation 8. Collectively, these data identify HDAC6 as a key downstream component of the LKB1 signalling pathway. Our findings further suggest that LKB1, via its inhibitory effect on HDAC6 ubiquitin-binding activity, limits the cellular adaptive response to a protracted stress, a distinctive biological property that is likely to contribute to its tumor-suppressive function

No description available.

RACK1 (Receptor for activated C kinase 1) is an evolutionary conserved protein which has essential role in most studied eukaryotic organisms, except for yeast. Although RACK1 was originally described as a binding partner of protein kinase C, later studies re- vealed its significant role in other cellular signalizations such as MAPK, Src or FAK. Thanks to this, RACK1 participates in the regulation of key cellular processes including migration, apoptosis or translation. As a binding partner of a small ribosomal subunit, RACK1 contributes to transla- tion regulation by integrating signals from different cellular pathways and several transla- tional components such as PKC and eIF6. Moreover, RACK1 has a role in translation regu- lation during stress. Under stress conditions there is a global reduction of translation, in- creased expression of specific mRNAs important for cellular stress response and formation of cytosolic foci called stress granules (SGs). SGs play an important role in protection of mRNAs and translation components against degradation. SGs also function in prevention of apoptosis. RACK1 has been identified as one of many components of SGs and its localization into SGs leads to inhibition of RACK1-mediated pro-apoptotic pathways. Aim of this diploma thesis was to elucidate the role of...

9 ABSTRACT For proper function proteins should have a native conformation. If their conformation is impaired due to environmental stress or genetic mutation, proteins become prone to aggregation. There exist various types of protein aggregates. Stable non-membraneous inclusions can form which can serve for clearance of aberrant proteins from place where they can interfere with essential cellular processes. Another type of aggregates can serve as transient deposits of proteins thus protecting them from stress conditions. Stress granules (SG) are a such example of transient granules. Their formation is induced by heat shock for example. SGs contain mRNA, components of translation machinery, and other proteins. One of these proteins is Mmi1, small highly conserved protein with unknown function. Association of Mmi1 with stress granules and partial co-localization with chaperon Cdc48 and proteasom indicates Mmi1 can mediate heat stress damaged protein degradation. We have uncovered that yeast prion protein Sup35 is a component of stress granules as well. With regard to its aggregation capability there existed an assumption that prion domain of Sup35 could serve as scaffold for SG assembly. However as we show deletion of prion domain of Sup35 protein does not affect stress granules formation dynamics. Yeast...

Indiana University-Purdue University Indianapolis (IUPUI) / More than 20 million people have died of AIDS since the early eighties, while nearly 34 millions are currently infected with the HIV. Anti-retroviral therapy (ART) directed at key viral enzymes has changed AIDS from uniformly fatal to a manageable chronic disease. However, ART-associated drug resistance and toxicity have posed a great challenge for long-term management of the disease and have called for development of new therapeutics. In this study, we focused on the viral factor Nef and the host factor Sam68. Nef is a major pathogenic viral determinant for HIV-1, and no therapeutics have been targeted to this factor. Sam68 is indispensible for HIV-1 propagation. We revealed that Sam68 variants were very potent in preventing Nef expression. We found that these effects were associated with their ability to form a macromolecular structure called stress granules (SG). In addition, we demonstrated that these variants bound to nef mRNA in a sequence-specific manner. Furthermore, we showed that these variants co-localized with nef mRNA in SG. Importantly, we validated these findings in the context of HIV-1 infection of its natural target cells and found significant loss of Nef function in these cells. Taken together, these results demonstrate that SG induction and nef mRNA sequestration account for translational suppression of Nef expression and offer a new strategy for development of anti-HIV therapeutics. Sam68 is implicated in a variety of other important cellular processes. Our findings that Sam68 variants were able to induce SG formation prompted us to investigate whether wild-type Sam68 was also recruited to SG. We found that Sam68 was increasingly recruited into SG under oxidative stress, and that its specific domains were involved. However, Sam68 knockdown had no effects on SG assembly, suggesting that Sam68 is not a constitutive component of SG assembly. Lastly, we demonstrated that Sam68 complexed with TIA-1, an essential SG component. Taken together, these results provide direct evidence for the first time that Sam68 is recruited into SG through complexing with TIA-1, and suggest that SG recruitment of Sam68 and ensuing changes in Sam68 physiological functions are part of the host response to external stressful conditions.

No description available.

Cells exposed to heat stress experience an increase in the amount of misfolded and aggregated proteins. Cells respond to this threat through coordinated and finely tuned ad- justments in gene expression. When the ambient temperature increases, cells activate the heat stress response (HSR), a process in which the transcription of mRNAs encoding heat shock proteins (Hsps) is upregulated. During severe heat stress, cells also downregulate the synthesis of misfolding-prone housekeeping proteins while the synthesis of Hsps takes precedence. Consequently, the amount of misfolded and aggregated proteins is reduced by Hsps. While the transcriptional HSR has been studied in depth over the last 50 years, our understanding of protein translation regulation during heat stress remains limited. Biomolecular condensates have been proposed as a new way to regulate cellular functions. In budding yeast exposed to severe heat stress, the repression in the synthesis of housekeep- ing proteins coincides with the formation of condensates called heat stress granules (HSGs). HSGs are enriched for translation factors and translationally-repressed mRNAs and they have been implicated in translation regulation. However, if and how HSGs regulate translation during severe heat stress has remained elusive. Using in vitro reconstitution assays, I demonstrate that the heat-induced condensation of translation factors together with mRNA is an adaptive mechanism to regulate protein synthesis during severe heat stress. My thesis work focused on the translation initiation factor complex eIF4F from Saccharomyces cerevisiae. eIF4F was previously shown to promote global translation of capped mRNAs. One subunit of eIF4F is eIF4G, an RNA-binding and scaffold protein that interacts with numerous translation initiation factors. Two other subunits of eIF4F are the RNA helicase eIF4A and the mRNA cap-binding protein eIF4E. eIF4G also interacts with the poly(A) binding protein (Pab1p) and the RNA helicase Ded1p, which like eIF4F, are crucial in translation initiation. Importantly, eIF4G, eIF4E, Pab1p and Ded1p condense into HSGs in yeast upon severe heat stress, while eIF4A remains soluble in the cytosol. To investigate the function of these translation factors in regulating translation, I purified eIF4F, Pab1p and Ded1p. Using purified eIF4F, nanoluciferase-encoding reporter mRNAsand an in vitro translation assay, I showed that eIF4F enhances general protein synthesis. Together with Pab1p and Ded1p, eIF4F enhances the translation of reporter mRNAs with 5’ UTRs of housekeeping transcripts to a greater extent than reporter mRNAs with 5’ UTRs of Hsp-encoding genes. These findings suggest important differences in translation regulation at physiological temperatures and that efficient translation of housekeeping mRNAs requires synergy between eIF4F, Pab1p and Ded1p. Next, I reconstituted eIF4G condensates in vitro using biochemical approaches. I found that eIF4G forms condensates with mRNA. The condensation of eIF4G-mRNA is promoted by heat-induced structural rearrangements and interaction valences between eIF4G RNAbinding domains (RBDs). eIF4G has three RBDs, where the removal of either RBDs did not affect the RNA binding affinity but repressed condensation. Thus, eIF4G-mRNA condensation requires cooperativity between the three RBDs. Critically, I found that the mechanism of heat-induced condensation is conserved and adapted in eIF4G orthologues from yeast species that thrive in colder or warmer temperatures. Using multi-component in vitro assays, I found that heated eIF4G-mRNA condensates recruit eIF4E and Pab1p. In agreement with the fact that eIF4A does not assemble into HSGs in cells, eIF4A did not partition into eIF4G-mRNA condensates, which is likely due to a heat-induced weakening of interactions with eIF4G. I next characterized eIF4G variants with targeted mutations in the eIF4E- and Pab1-binding sites of eIF4G. This allowed me to demonstrate that the recruitment of eIF4E into eIF4G-mRNA condensates is driven by protein-mediated interactions. Furthermore, I found that heterotypic interactions between eIF4G, Pab1 and the poly(A) tail of mRNA promote the solidification of heated condensates. This is consistent with previous observations reporting solid-like properties of HSGs. Finally, I investigated the translation activity of heated translation factor condensates in yeast cell-free extracts. Solid-like eIF4F-mRNA condensates with Pab1p or Ded1p resulted in a pronounced repression of translation. This coincided with the recruitment of reporter mRNAs into condensates. Based on these findings, I thus propose that the repression in translation of housekeeping mRNAs during severe heat stress in yeast is a consequence of the formation of solid-like translation factor and mRNA condensates. Further analyses revealed that mRNA outside of condensates are translated in an eIF4A-dependent manner. This is because eIF4A is not recruited to the condensates and remains active upon heating. In summary, I propose that heat stress promotes the condensation of mRNA with eIF4G, eIF4E, Pab1p and Ded1p into solid-like condensates. In vitro assays suggest that translation factors inside of condensates are inactive while the mRNA is translationally repressed. This model highlights a mechanism for the downregulation in the synthesis of housekeeping proteins during severe heat stress in yeast. My findings also suggest that the preferential translation of mRNAs encoding Hsps occurs independently of the condensate-forming translation factors and may be mediated by eIF4A, which does not localise into HSGs. I thus conclude that translation regulation during severe heat stress is achieved by specific translation initiation factors that form inactive and solid-like condensates with mRNA.