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  • 1.
    Andreasson, Claes
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Schick, Anna J.
    Pfeiffer, Susanne M.
    Sarov, Mihail
    Stewart, Francis
    Wurst, Wolfgang
    Schick, Joel A.
    Direct Cloning of Isogenic Murine DNA in Yeast and Relevance of Isogenicity for Targeting in Embryonic Stem Cells2013In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, no 9, p. e74207-Article in journal (Refereed)
    Abstract [en]

    Efficient gene targeting in embryonic stem cells requires that modifying DNA sequences are identical to those in the targeted chromosomal locus. Yet, there is a paucity of isogenic genomic clones for human cell lines and PCR amplification cannot be used in many mutation-sensitive applications. Here, we describe a novel method for the direct cloning of genomic DNA into a targeting vector, pRTVIR, using oligonucleotide-directed homologous recombination in yeast. We demonstrate the applicability of the method by constructing functional targeting vectors for mammalian genes Uhrf1 and Gfap. Whereas the isogenic targeting of the gene Uhrf1 showed a substantial increase in targeting efficiency compared to non-isogenic DNA in mouse E14 cells, E14-derived DNA performed better than the isogenic DNA in JM8 cells for both Uhrf1 and Gfap. Analysis of 70 C57BL/6-derived targeting vectors electroporated in JM8 and E14 cell lines in parallel showed a clear dependence on isogenicity for targeting, but for three genes isogenic DNA was found to be inhibitory. In summary, this study provides a straightforward methodological approach for the direct generation of isogenic gene targeting vectors.

  • 2.
    Andréasson, Claes
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Ott, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Büttner, Sabrina
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute. University of Graz, Austria.
    Mitochondria orchestrate proteostatic and metabolic stress responses2019In: EMBO Reports, ISSN 1469-221X, E-ISSN 1469-3178, Vol. 20, no 10, article id e47865Article in journal (Refereed)
    Abstract [en]

    The eukaryotic cell is morphologically and functionally organized as an interconnected network of organelles that responds to stress and aging. Organelles communicate via dedicated signal transduction pathways and the transfer of information in form of metabolites and energy levels. Recent data suggest that the communication between organellar proteostasis systems is a cornerstone of cellular stress responses in eukaryotic cells. Here, we discuss the integration of proteostasis and energy fluxes in the regulation of cellular stress and aging. We emphasize the molecular architecture of the regulatory transcriptional pathways that both sense and control metabolism and proteostasis. A special focus is placed on mechanistic insights gained from the model organism budding yeast in signaling from mitochondria to the nucleus and how this shapes cellular fitness.

  • 3.
    Andréasson, Claes
    et al.
    Stockholm University, Faculty of Science, The Wenner-Gren Institute , Cell Biology.
    Rampelt, Heike
    Fiaux, Jocelyne
    Druffel-Augustin, Silke
    Bukau, Bernd
    The endoplasmic reticulum Grp170 acts as a nucleotide exchange factor of Hsp70 via a mechanism similar to that of the cytosolic Hsp112010In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 285, no 16, p. 12445-53Article in journal (Refereed)
    Abstract [en]

    Grp170 and Hsp110 proteins constitute two evolutionary distinct branches of the Hsp70 family that share the ability to function as nucleotide exchange factors (NEFs) for canonical Hsp70s. Although the NEF mechanism of the cytoplasmic Hsp110s is well understood, little is known regarding the mechanism used by Grp170s in the endoplasmic reticulum. In this study, we compare the yeast Grp170 Lhs1 with the yeast Hsp110 Sse1. We find that residues important for Sse1 NEF activity are conserved in Lhs1 and that mutations in these residues in Lhs1 compromise NEF activity. As previously reported for Sse1, Lhs1 requires ATP to trigger nucleotide exchange in its cognate Hsp70 partner Kar2. Using site-specific cross-linking, we show that the nucleotide-binding domain (NBD) of Lhs1 interacts with the NBD of Kar2 face to face, and that Lhs1 contacts the side of the Kar2 NBD via its protruding C-terminal alpha-helical domain. To directly address the mechanism of nucleotide exchange, we have compared the hydrogen-exchange characteristics of a yeast Hsp70 NBD (Ssa1) in complex with either Sse1 or Lhs1. We find that Lhs1 and Sse1 induce very similar changes in the conformational dynamics in the Hsp70. Thus, our findings demonstrate that despite some differences between Hsp110 and Grp170 proteins, they use a similar mechanism to trigger nucleotide exchange.

  • 4.
    Fritzell, Kajsa
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Xu, Li-Di
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Otrocka, Magdalena
    Andréasson, Claes
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Öhman, Marie
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Sensitive ADAR editing reporter in cancer cells enables high-throughput screening of small molecule libraries2019In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 47, no 4, article id e22Article in journal (Refereed)
    Abstract [en]

    Adenosine to inosine editing is common in the human transcriptome and changes of this essential activity is associated with disease. Children with ADAR1 mutations develop fatal Aicardi-Goutieres syndrome characterized by aberrant interferon expression. In contrast, ADAR1 overexpression is associated with increased malignancy of breast, lung and liver cancer. ADAR1 silencing in breast cancer cells leads to increased apoptosis, suggesting an anti-apoptotic function that promotes cancer progression. Yet, suitable high-throughput editing assays are needed to efficiently screen chemical libraries for modifiers of ADAR1 activity. We describe the development of a bioluminescent reporter system that facilitates rapid and accurate determination of endogenous editing activity. The system is based on the highly sensitive and quantitative Nanoluciferase that is conditionally expressed upon reporter-transcript editing. Stably introduced into cancer cell lines, the system reports on elevated endogenous ADAR1 editing activity induced by interferon as well as knockdown of ADAR1 and ADAR2. In a single-well setup we used the reporter in HeLa cells to screen a small molecule library of 33 000 compounds. This yielded a primary hit rate of 0.9% at 70% inhibition of editing. Thus, we provide a key tool for high-throughput identification of modifiers of A-to-I editing activity in cancer cells.

  • 5.
    Gestin, Maxime
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Falato, Luca
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Ciccarelli, Michela
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Andréasson, Claes
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Transfection of Heat Shock Protein70kDa (HSP70)Manuscript (preprint) (Other academic)
    Abstract [en]

    Heat shock protein 70kDa (HSP70) is a major protein family in the cell protections against stress-induced denaturation and aggregation and in the folding of nascent proteins. It is a highly conserved protein that can be found in most organisms and is strongly connected to several intracellular pathways such as protein folding and refolding, protein degradation and regulation, and protection against intense stress. Cellular delivery of HSP70 would be of high impact for clarification of its role in these cellular processes. 

    PepFect14 is a cell-penetrating peptide known to be able to mediate the transfection of various oligonucleotides to multiple cell lines with a higher efficacy than most commercially available transfection agents and without inducing significant toxic effects. 

    In this study we demonstrated that PepFect14 was able to form a complex with HSP70 and to deliver it inside cells in the same fashion with oligonucleotide delivery. The delivered HSP70 showed an effect in the cell regulation indicating that the protein was biologically available in the cytoplasm and the interactions with PepFect14 did not impeach its active sites once the plasma barrier crossed. This study reports the first successful delivery of HSP70 to our knowledge and the first protein transfection mediated by PepFect14. It opens new fields of research for both PepFect14 as a delivery agent and HSP70 as a therapeutic agent; with potential in peptide aggregation caused diseases such as Parkinson’s and Alzheimer’s diseases.

  • 6.
    Gowda, Naveen K. C.
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Kaimal, Jayasankar M.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Kityk, Roman
    Daniel, Chammiran
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Liebau, Jobst
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Öhman, Marie
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Mayer, Matthias P.
    Andréasson, Claes
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Nucleotide exchange factors Fes1 and HspBP1 mimic substrate to release misfolded proteins from Hsp702018In: Nature Structural & Molecular Biology, ISSN 1545-9993, E-ISSN 1545-9985, Vol. 25, no 1, p. 83-+Article in journal (Refereed)
    Abstract [en]

    Protein quality control depends on the tight regulation of interactions between molecular chaperones and polypeptide substrates. Substrate release from the chaperone Hsp70 is triggered by nucleotide-exchange factors (NEFs) that control folding and degradation fates via poorly understood mechanisms. We found that the armadillo-type NEFs budding yeast Fes1 and its human homolog HspBP1 employ flexible N-terminal release domains (RDs) with substrate-mimicking properties to ensure the efficient release of persistent substrates from Hsp70. The RD contacts the substrate-binding domain of the chaperone, competes with peptide substrate for binding and is essential for proper function in yeast and mammalian cells. Thus, the armadillo domain engages Hsp70 to trigger nucleotide exchange, whereas the RD safeguards the release of substrates. Our findings provide fundamental mechanistic insight into the functional specialization of Hsp70 NEFs and have implications for the understanding of proteostasis-related disorders, including Marinesco-Sjögren syndrome.

  • 7.
    Gowda, Naveen Kumar C.
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Kaimal, Jayasankar Mohanakrishnan
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Kityk, Roman
    Daniel, Chammiran
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Liebau, Jobst
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Öhman, Marie
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Mayer, Matthias P.
    Andréasson, Claes
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Substrate-mimicking domain of nucleotide-exchange factor Fes1/HspBP1 ensures efficient release of persistent substrates from Hsp70Manuscript (preprint) (Other academic)
  • 8.
    Gowda, Naveen Kumar Chandappa
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Kaimal, Jayasankar Mohanakrishnan
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Masser, Anna E.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Kang, Wenjing
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Friedländer, Marc R.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Andréasson, Claes
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Cytosolic splice isoform of Hsp70 nucleotide exchange factor Fes1 is required for the degradation of misfolded proteins in yeast2016In: Molecular Biology of the Cell, ISSN 1059-1524, E-ISSN 1939-4586, Vol. 27, no 8, p. 1210-1219Article in journal (Refereed)
    Abstract [en]

    Cells maintain proteostasis by selectively recognizing and targeting misfolded proteins for degradation. In Saccharomyces cerevisiae, the Hsp70 nucleotide exchange factor Fes1 is essential for the degradation of chaperone-associated misfolded proteins by the ubiquitin-proteasome system. Here we show that the FES1 transcript undergoes unique 3' alternative splicing that results in two equally active isoforms with alternative C-termini, Fes1L and Fes1S. Fes1L is actively targeted to the nucleus and represents the first identified nuclear Hsp70 nucleotide exchange factor. In contrast, Fes1S localizes to the cytosol and is essential to maintain proteostasis. In the absence of Fes1S, the heat-shock response is constitutively induced at normally non-stressful conditions. Moreover, cells display severe growth defects when elevated temperatures, amino acid analogues or the ectopic expression of misfolded proteins, induce protein misfolding. Importantly, misfolded proteins are not targeted for degradation by the ubiquitin-proteasome system. These observations support the notion that cytosolic Fes1S maintains proteostasis by supporting the removal of toxic misfolded proteins by proteasomal degradation. This study provides key findings for the understanding of the organization of protein quality control mechanisms in the cytosol and nucleus.

  • 9.
    Gowda, Naveen Kumar Chandappa
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Kandasamy, Ganapathi
    Froehlich, Marceli S.
    Dohmen, R. Jürgen
    Andréasson, Claes
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Hsp70 nucleotide exchange factor Fes1 is essential for ubiquitin-dependent degradation of misfolded cytosolic proteins2013In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 110, no 15, p. 5975-5980Article in journal (Refereed)
    Abstract [en]

    Protein quality control systems protect cells against the accumulation of toxic misfolded proteins by promoting their selective degradation. Malfunctions of quality control systems are linked to aging and neurodegenerative disease. Folding of polypeptides is facilitated by the association of 70 kDa Heat shock protein (Hsp70) molecular chaperones. If folding cannot be achieved, Hsp70 interacts with ubiquitylation enzymes that promote the proteasomal degradation of the misfolded protein. However, the factors that direct Hsp70 substrates toward the degradation machinery have remained unknown. Here, we identify Fes1, an Hsp70 nucleotide exchange factor of hitherto unclear physiological function, as a cytosolic triaging factor that promotes proteasomal degradation of misfolded proteins. Fes1 selectively interacts with misfolded proteins bound by Hsp70 and triggers their release from the chaperone. In the absence of Fes1, misfolded proteins fail to undergo polyubiquitylation, aggregate, and induce a strong heat shock response. Our findings reveal that Hsp70 direct proteins toward either folding or degradation by using distinct nucleotide exchange factors.

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  • 10.
    Holmberg, Mats A.
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Gowda, Naveen Kumar Chandappa
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Andréasson, Claes
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    A versatile bacterial expression vector designed for single-step cloning of multiple DNA fragments using homologous recombination2014In: Protein Expression and Purification, ISSN 1046-5928, E-ISSN 1096-0279, Vol. 98, p. 38-45Article in journal (Refereed)
    Abstract [en]

    Production of recombinant proteins is the starting point for biochemical and biophysical analyses and requires methodology to efficiently proceed from gene sequence to purified protein. While optimized strategies for the efficient cloning of single-gene fragments for bacterial expression is available, efficient multiple DNA fragment cloning still presents a challenge. To facilitate this step, we have developed an efficient cloning strategy based on yeast homologous recombination cloning (YHRC) into the new pET-based bacterial expression vector pSUMO-YHRC. The vector supports cloning for untagged expression as well as fusions to His6-SUMO or His6 tags. We demonstrate that YHRC from single PCR products of 6 independent genes into the vector results in virtually no background. Importantly, in a quantitative assay for functional expression we find that single-step YHRC of 7 DNA fragments can be performed with very high cloning efficiencies. The method and reagents described in this paper significantly simplifies the construction of expression plasmids from multiple DNA fragments, including complex gene fusions, chimeric genes and polycistronic constructs.

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    A versatile bacterial expression vector designed for single-step cloning of multiple DNA fragments using homologous recombination
  • 11.
    Kaimal, Jayasankar Mohanakrishnan
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Habernig, Lukas
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Büttner, Sabrina
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Andréasson, Claes
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Nuclear targeting of Hsp110 modifies the proteostasis system by mobilizing latent Hsp70 chaperonesManuscript (preprint) (Other academic)
  • 12.
    Kaimal, Jayasankar Mohanakrishnan
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Kandasamy, Ganapathi
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Gasser, Fabian
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Andréasson, Claes
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Coordinated Hsp110 and Hsp104 Activities Power Protein Disaggregation in Saccharomyces cerevisiae2017In: Molecular and Cellular Biology, ISSN 0270-7306, E-ISSN 1098-5549, Vol. 37, no 11, article id e00027-17Article in journal (Refereed)
    Abstract [en]

    Protein aggregation is intimately associated with cellular stress and is accelerated during aging, disease, and cellular dysfunction. Yeast cells rely on the ATP-consuming chaperone Hsp104 to disaggregate proteins together with Hsp70. Hsp110s are ancient and abundant chaperones that form complexes with Hsp70. Here we provide in vivo data showing that the Saccharomyces cerevisiae Hsp110s Sse1 and Sse2 are essential for Hsp104-dependent protein disaggregation. Following heat shock, complexes of Hsp110 and Hsp70 are recruited to protein aggregates and function together with Hsp104 in the disaggregation process. In the absence of Hsp110, targeting of Hsp70 and Hsp104 to the aggregates is impaired, and the residual Hsp104 that still reaches the aggregates fails to disaggregate. Thus, coordinated activities of both Hsp104 and Hsp110 are required to reactivate aggregated proteins. These findings have important implications for the understanding of how eukaryotic cells manage misfolded and amyloid proteins.

  • 13.
    Kandasamy, Ganapathi
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Andréasson, Claes
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Hsp70-Hsp110 chaperones deliver ubiquitin-dependent and -independent substrates to the 26S proteasome for proteolysis in yeast2018In: Journal of Cell Science, ISSN 0021-9533, E-ISSN 1477-9137, Vol. 131, no 6, article id jcs210948Article in journal (Refereed)
    Abstract [en]

    During protein quality control, proteotoxic misfolded proteins are recognized by molecular chaperones, ubiquitylated by dedicated quality control ligases and delivered to the 26S proteasome for degradation. Proteins belonging to the Hsp70 chaperone and Hsp110 (the Hsp70 nucleotide exchange factor) families function in the degradation of misfolded proteins by the ubiquitin-proteasome system via poorly understood mechanisms. Here, we report that the Saccharomyces cerevisiae Hsp110 proteins (Sse1 and Sse2) function in the degradation of Hsp70-associated ubiquitin conjugates at the post-ubiquitylation step and are also required for ubiquitin-independent proteasomal degradation. Hsp110 associates with the 19S regulatory particle of the 26S proteasome and interacts with Hsp70 to f acilitate the delivery of Hsp70 substrates for proteasomal degradation. By using a highly defined ubiquitin-independent proteasome substrate, we show that the mere introduction of a single Hsp70-binding site renders its degradation dependent on Hsp110. The findings define a dedicated and chaperone-dependent pathway for the efficient shuttling of cellular proteins to the proteasome with profound implications for understanding protein quality control and cellular stress management.

  • 14.
    Martins, António
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Pfirrmann, Thorsten
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Heessen, Stijn
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Sundqvist, Gustav
    Bulone, Vincent
    Andréasson, Claes
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Ljungdahl, Per O.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Ssy5 is a signaling serine protease that exhibits atypical biogenesis and marked S1 specificity2018In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 293, no 22, p. 8362-8378Article in journal (Refereed)
    Abstract [en]

    Ssy5 is a signaling endoprotease that plays a key role in regulating central metabolism, cellular aging, and morphological transitions important for growth and survival of yeast (Saccharomyces cerevisiae) cells. In response to extracellular amino acids, Ssy5 proteolytically activates the transcription factors Stp1 and Stp2, leading to enhanced Ssy1-Ptr3-Ssy5 (SPS) sensor-regulated gene expression. Ssy5 comprises a catalytic (Cat) domain and an extensive regulatory prodomain. Ssy5 is refractory to both broad-spectrum and serine protease-specific inhibitors, confounding its classification as a protease, and no information about Ssy5's cleavage-site preferences and its mechanism of substrate selection is available. Here, using mutational and inhibition experiments, we investigated the biogenesis and catalytic properties of Ssy5 and conclusively show that it is a serine protease. Atypical for the majority of serine proteases, Ssy5's prodomain was obligatorily required in cis during biogenesis for the maturation of the proteolytic activity of the Cat domain. Autolysis and Stp1 and Stp2 cleavage occurred between a cysteine (at the P1 site) and a serine or alanine (at the P1 site) and required residues with short side chains at the P1 site. Substitutions in the Cat domain affecting substrate specificity revealed that residues Phe-634, His-661, and Gly-671 in the S1-binding pocket of this domain are important for Ssy5 catalytic function. This study confirms that the signaling protease Ssy5 is a serine protease and provides a detailed understanding of the biogenesis and intrinsic properties of this key enzyme in yeast.

  • 15.
    Masser, Anna E.
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Andréasson, Claes
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    A co-translational folding reporter to monitor proteostasisManuscript (preprint) (Other academic)
  • 16.
    Masser, Anna E.
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Kandasamy, Ganapathi
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Kaimal, Jayasankar Mohanakrishnan
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Andréasson, Claes
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Luciferase NanoLuc as a reporter for gene expression and protein levels in Saccharomyces cerevisiae2016In: Yeast, ISSN 0749-503X, E-ISSN 1097-0061, Vol. 33, no 5, p. 191-200Article in journal (Refereed)
    Abstract [en]

    Reporter proteins are essential tools in the study of biological processes and are employed to monitor changes in gene expression and protein levels. Luciferases are reporter proteins that enable rapid and highly sensitive detection with an outstanding dynamic range. Here we evaluated the usefulness of the 19 kDa luciferase NanoLuc (Nluc), derived from the deep sea shrimp Oplophorus gracilirostris, as a reporter protein in yeast. Cassettes with codon-optimized genes expressing yeast Nluc (yNluc) or its destabilized derivative yNlucPEST have been assembled in the context of the dominant drug resistance marker kanMX. The reporter proteins do not impair the growth of yeast cells and exhibit half-lives of 40 and 5 min, respectively. The commercial substrate Nano-Glo (R) is compatible with detection of yNluc bioluminescence in < 50 cells. Using the unstable yNlucPEST to report on the rapid and transient expression of a heat-shock promoter (PCYC1-HSE), we found a close match between the intensity of the bioluminescent signal and mRNA levels during both induction and decay. We demonstrated that the bioluminescence of yNluc fused to the C-terminus of a temperature-sensitive protein reports on its protein levels. In conclusion, yNluc and yNlucPEST are valuable new reporter proteins suitable for experiments with yeast using standard commercial substrate.

  • 17.
    Masser, Anna E.
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Kang, Wenjing
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Friedländer, Marc R.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Andréasson, Claes
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Cytoplasmic protein misfolding titrates nuclear Hsp70 to unleash active Hsf1Manuscript (preprint) (Other academic)
  • 18.
    Omnus, Deike J.
    et al.
    Stockholm University, Faculty of Science, The Wenner-Gren Institute, Cell Biology.
    Pfirrmann, Thorsten
    Stockholm University, Faculty of Science, The Wenner-Gren Institute, Cell Biology.
    Andréasson, Claes
    Stockholm University, Faculty of Science, The Wenner-Gren Institute, Cell Biology.
    Ljungdahl, Per O.
    Stockholm University, Faculty of Science, The Wenner-Gren Institute, Cell Biology.
    A phosphodegron controls nutrient-induced proteasomal activation of the signaling protease Ssy52011In: Molecular Biology of the Cell, ISSN 1059-1524, E-ISSN 1939-4586, Vol. 22, no 15, p. 2754-2765Article in journal (Refereed)
    Abstract [en]

    Regulated proteolysis serves as a mechanism to control cellular processes. The SPS (Ssy1-Ptr3-Ssy5) sensor in yeast responds to extracellular amino acids by endoproteolytically activating transcription factors Stp1 and Stp2 (Stp1/2). The processing endoprotease Ssy5 is regulated via proteasomal degradation of its noncovalently associated N-terminal prodomain. We find that degradation of the prodomain requires a conserved phosphodegron comprising phosphoacceptor sites and ubiquitin-accepting lysine residues. Upon amino acid induction, the phosphodegron is modified in a series of linked events by a set of general regulatory factors involved in diverse signaling pathways. First, an amino acid-induced conformational change triggers phosphodegron phosphorylation by the constitutively active plasma membrane-localized casein kinase I (Yck1/2). Next the prodomain becomes a substrate for polyubiquitylation by the Skp1/Cullin/Grr1 E3 ubiquitin ligase complex (SCF(Grr1)). Finally, the modified prodomain is concomitantly degraded by the 26S proteasome. These integrated events are requisite for unfettering the Ssy5 endoprotease, and thus Stp1/2 processing. The Ssy5 phosphoacceptor motif resembles the Yck1/2- and Grr1-dependent degrons of regulators in the Snf3/Rgt2 glucose-sensing pathway. Our work defines a novel proteolytic activation cascade that regulates an intracellular signaling protease and illustrates how general signaling components are recruited to distinct pathways that achieve conditional and specific signaling outputs.

  • 19.
    Pfirrmann, Thorsten
    et al.
    Stockholm University, Faculty of Science, The Wenner-Gren Institute, Cell Biology.
    Heessen, Stijn
    Stockholm University, Faculty of Science, The Wenner-Gren Institute, Cell Biology.
    Omnus, Deike J.
    Stockholm University, Faculty of Science, The Wenner-Gren Institute, Cell Biology.
    Andréasson, Claes
    Stockholm University, Faculty of Science, The Wenner-Gren Institute, Cell Biology.
    Ljungdahl, Per O.
    Stockholm University, Faculty of Science, The Wenner-Gren Institute, Cell Biology.
    The prodomain of Ssy5 protease controls receptor-activated proteolysis of transcription factor Stp12010In: Molecular and Cellular Biology, ISSN 0270-7306, E-ISSN 1098-5549, Vol. 30, no 13, p. 3299-309Article in journal (Refereed)
    Abstract [en]

    Extracellular amino acids induce the yeast SPS sensor to endoproteolytically cleave transcription factors Stp1 and Stp2 in a process termed receptor-activated proteolysis (RAP). Ssy5, the activating endoprotease, is synthesized with a large N-terminal prodomain and a C-terminal chymotrypsin-like catalytic (Cat) domain. During biogenesis, Ssy5 cleaves itself and the prodomain and Cat domain remain associated, forming an inactive primed protease. Here we show that the prodomain is a potent inhibitor of Cat domain activity and that its inactivation is a requisite for RAP. Accordingly, amino acid-induced signals trigger proteasome-dependent degradation of the prodomain. A mutation that stabilizes the prodomain prevents Stp1 processing, whereas destabilizing mutations lead to constitutive RAP-independent Stp1 processing. We fused a conditional degron to the prodomain to synthetically reprogram the amino acid-responsive SPS signaling pathway, placing it under temperature control. Our results define a regulatory mechanism that is novel for eukaryotic proteases functioning within cells

  • 20. Pfirrmann, Thorsten
    et al.
    Lokapally, Ashwin
    Andréasson, Claes
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Ljungdahl, Per O
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Hollemann, Thomas
    SOMA: A Single Oligonucleotide Mutagenesis and Cloning Approach2013In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, no 6, article id e64870Article in journal (Refereed)
    Abstract [en]

    Modern biology research requires simple techniques for efficient and restriction site-independent modification of genetic material. Classical cloning and mutagenesis strategies are limited by their dependency on restriction sites and the use of complementary primer pairs. Here, we describe the Single Oligonucleotide Mutagenesis and Cloning Approach (SOMA) that is independent of restriction sites and only requires a single mutagenic oligonucleotide to modify a plasmid. We demonstrate the broad application spectrum of SOMA with three examples. First, we present a novel plasmid that in a standardized and rapid fashion can be used as a template for SOMA to generate GFP-reporters. We successfully use such a reporter to assess the in vivo knock-down quality of morpholinos in Xenopus laevis embryos. In a second example, we show how to use a SOMA-based protocol for restriction-site independent cloning to generate chimeric proteins by domain swapping between the two human hRMD5a and hRMD5b isoforms. Last, we show that SOMA simplifies the generation of randomized single-site mutagenized gene libraries. As an example we random-mutagenize a single codon affecting the catalytic activity of the yeast Ssy5 endoprotease and identify a spectrum of tolerated and non-tolerated substitutions. Thus, SOMA represents a highly efficient alternative to classical cloning and mutagenesis strategies.

  • 21.
    Suhm, Tamara
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Habernig, Lukas
    Rzepka, Magdalena
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Kaimal, Jayasankar Mohanakrishnan
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Andréasson, Claes
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Büttner, Sabrina
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute. University of Graz, Austria.
    Ott, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    A novel system to monitor mitochondrial translation in yeast2018In: Microbial Cell, ISSN 2311-2638, Vol. 5, no 3, p. 158-164Article in journal (Refereed)
    Abstract [en]

    The mitochondrial genome is responsible for the production of a handful of polypeptides that are core subunits of the membrane-bound oxidative phosphorylation system. Until now the mechanistic studies of mitochondrial protein synthesis inside cells have been conducted with inhibition of cytoplasmic protein synthesis to reduce the background of nuclear gene expression with the undesired consequence of major disturbances of cellular signaling cascades. Here we have generated a system that allows direct monitoring of mitochondrial translation in unperturbed cells. A recoded gene for superfolder GFP was inserted into the yeast (Saccharomyces cerevisiae) mitochondrial genome and enabled the detection of translation through fluorescence microscopy and flow cytometry in functional mitochondria. This novel tool allows the investigation of the function and regulation of mitochondrial translation during stress signaling, aging and mitochondrial biogenesis.

  • 22.
    Suhm, Tamara
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Kaimal, Jayasankar Mohanakrishnan
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Dawitz, Hannah
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Peselj, Carlotta
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Masser, Anna E.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Hanzén, Sarah
    Ambrožič, Matevž
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Smialowska, Agata
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Björck, Markus L.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Brzezinski, Peter
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Nyström, Thomas
    Büttner, Sabrina
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute. University of Graz, Austria.
    Andréasson, Claes
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Ott, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Mitochondrial Translation Efficiency Controls Cytoplasmic Protein Homeostasis2018In: Cell Metabolism, ISSN 1550-4131, E-ISSN 1932-7420, Vol. 27, no 6, p. 1309-1322Article in journal (Refereed)
    Abstract [en]

    Cellular proteostasis ismaintained via the coordinated synthesis, maintenance, and breakdown of proteins in the cytosol and organelles. While biogenesis of the mitochondrial membrane complexes that execute oxidative phosphorylation depends on cytoplasmic translation, it is unknown how translation within mitochondria impacts cytoplasmic proteostasis and nuclear gene expression. Here we have analyzed the effects of mutations in the highly conserved accuracy center of the yeast mitoribosome. Decreased accuracy of mitochondrial translation shortened chronological lifespan, impaired management of cytosolic protein aggregates, and elicited a general transcriptional stress response. In striking contrast, increased accuracy extended lifespan, improved cytosolic aggregate clearance, and suppressed a normally stress-induced, Msn2/4-dependent interor-ganellar proteostasis transcription program (IPTP) that regulates genes important for mitochondrial proteostasis. Collectively, the data demonstrate that cytosolic protein homeostasis and nuclear stress signaling are controlled by mitochondrial translation efficiency in an inter-connected organelle quality control network that determines cellular lifespan.

1 - 22 of 22
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