The Proteasome Activators Blm10/PA200 Enhance the Proteasomal Degradation of N-Terminal Huntingtin.

The Blm10/PA200 family of proteasome activators modulates the peptidase activity of the core particle (20S CP). They participate in opening the 20S CP gate, thus facilitating the degradation of unstructured proteins such as tau and Dnm1 in a ubiquitin- and ATP-independent manner. Furthermore, PA200 also participates in the degradation of acetylated histones. In our study, we use a combination of yeast and human cell systems to investigate the role of Blm10/PA200 in the degradation of N-terminal Huntingtin fragments (N-Htt). We demonstrate that the human PA200 binds to N-Htt. The loss of Blm10 in yeast or PA200 in human cells results in increased mutant N-Htt aggregate formation and elevated cellular toxicity. Furthermore, Blm10 in vitro accelerates the proteasomal degradation of soluble N-Htt. Collectively, our data suggest N-Htt as a new substrate for Blm10/PA200-proteasomes and point to new approaches in Huntington\u27s disease (HD) research

Elevated Proteasome Capacity Extends Replicative Lifespan in Saccharomyces cerevisiae

Aging is characterized by the accumulation of damaged cellular macromolecules caused by declining repair and elimination pathways. An integral component employed by cells to counter toxic protein aggregates is the conserved ubiquitin/proteasome system (UPS). Previous studies have described an age-dependent decline of proteasomal function and increased longevity correlates with sustained proteasome capacity in centenarians and in naked mole rats, a long-lived rodent. Proof for a direct impact of enhanced proteasome function on longevity, however, is still lacking. To determine the importance of proteasome function in yeast aging, we established a method to modulate UPS capacity by manipulating levels of the UPS–related transcription factor Rpn4. While cells lacking RPN4 exhibit a decreased non-adaptable proteasome pool, loss of UBR2, an ubiquitin ligase that regulates Rpn4 turnover, results in elevated Rpn4 levels, which upregulates UPS components. Increased UPS capacity significantly enhances replicative lifespan (RLS) and resistance to proteotoxic stress, while reduced UPS capacity has opposing consequences. Despite tight transcriptional co-regulation of the UPS and oxidative detoxification systems, the impact of proteasome capacity on lifespan is independent of the latter, since elimination of Yap1, a key regulator of the oxidative stress response, does not affect lifespan extension of cells with higher proteasome capacity. Moreover, since elevated proteasome capacity results in improved clearance of toxic huntingtin fragments in a yeast model for neurodegenerative diseases, we speculate that the observed lifespan extension originates from prolonged elimination of damaged proteins in old mother cells. Epistasis analyses indicate that proteasome-mediated modulation of lifespan is at least partially distinct from dietary restriction, Tor1, and Sir2. These findings demonstrate that UPS capacity determines yeast RLS by a mechanism that is distinct from known longevity pathways and raise the possibility that interventions to promote enhanced proteasome function will have beneficial effects on longevity and age-related disease in humans

Mass spectrometric analysis of signal dependent protein modifications

C/EBPα und C/EBPβ sind Transkriptionsfaktoren, die die Zellproliferation und Zelldifferenzierung in vielen Geweben regulieren. C/EBPα und C/EBPβ spielen auch eine onkogene Rolle in akuter myeloischer Leukämie und anaplastisch-großzelligen Lymphomen (ALCL). In dieser Studie wird C/EBPα auf neue posttranslationale Modifikationen, wie z. B. Arginin-Methylierungen und Citrullinierungen, sowie Lysin-Methylierungen und Acetylierungen, mit massenspektrometrischen Mitteln untersucht. Es werden eine modifizierbare C/EBPα-Arginin-Citrullinierungsposition und die C/EBPα-Lysinsumoylierung eingehend auf ihren Einfluss auf C/EBPα-Proteininteraktionsnetzwerk überprüft. Außerdem wurde im Verlauf dieser Studie ein Hochdurchsatz-Screening-Verfahren entwickelt, das wir Protein Interaktions-Screening auf einer Peptid Matrix (PrISMa) nennen. Dieses Verfahren dient der Aufklärung des modifikationsabhängigen Proteininteraktionsnetzwerkes von C/EBPβ. PrISMa basiert auf einer Peptidmembran, auf der C/EBPβ-Peptide synthetisiert sind. Viele dieser Peptide enthalten methylierte Arginine und Lysine, acetylierte Lysine, citrullinierte Arginine und phosphorylierte Serine, Tyrosine und Threonine. Mittels PrISMa konnten Interaktionen von C/EBPβ mit Histonacetyltransferasen, dem Mediatorkomplex, Proteinen des nukleären Transports und RNA bindenden und spleißenden Proteinen verifiziert und kartiert werden. Des Weiteren konnte mit Hilfe von PrISMa eine große Anzahl von publizierten C/EBPβ Interaktionspartner spezifischen C/EBPβ-Sequenzen zugeordnet werden. C/EBPβ wird in ALCL in hohem Maße exprimiert und ist für die Zellproliferation dieser Krebsart wichtig. In dieser Studie wird das Proteininteraktionsnetzwerk C/EBPβ in einer ALCL Zelllinie aufgeklärt, um tiefere Einsichten über die Funktion von C/EBPβ als Onkogen zu erlangen.C/EBPα and C/EBPβ regulate cell proliferation and differentiation in multiple cell types. Moreover, C/EBPα and C/EBPβ are known to play oncogenic roles in acute myeloid leukemia and anaplastic large cell lymphomas (ALCLs). In this study C/EBPα is screened for novel posttranslational modifications (PTMs), such as arginine methylation and citrullination, as well as lysine methylation and acetylation by using mass spectrometry. A in this survey identified C/EBPα site of arginine citrullination and the C/EBPα lysine sumoylation are scrutinized for their impact on C/EBPα protein interaction network. A new high-throughput method named Protein Interaction Screen on peptide Matrices (PrISMa) is introduced in this study. This method was developed to determine the C/EBPβ protein interaction network in a PTM dependent manner. The PrISMa survey is based on a peptide membrane spotted with C/EBPβ peptides. Many of these C/EBPβ peptide sequences contain amino acid sequences comprising arginine and lysine methylation, lysine acetylation, arginine citrullination and serine, tyrosine and threonine phosphorylation. By means of PrISMa the C/EBPβ interplay with histone acetyltransferases, the mediator complex, proteins of the nucleoplasmic transport and RNA processing proteins is verified and specified by mapping these interactions to C/EBPβ amino acid sequences. Furthermore, PrISMa provides a map of C/EBPβ protein interaction patterns for a great number of the up to date published C/EBPβ protein interaction partners. C/EBPβ is highly expressed in ALCL cell lines and is essential for the cell proliferation of this type of cancer. In this study the C/EBPβ protein-protein interaction pattern in an ALCL cell line is unraveled providing valuable insight into the protein interaction network of C/EBPβ as an oncogene

PRISMA: Protein Interaction Screen on Peptide Matrix Reveals Interaction Footprints and Modifications- Dependent Interactome of Intrinsically Disordered C/EBPβ

Summary: CCAAT enhancer-binding protein beta (C/EBPβ) is a pioneer transcription factor that specifies cell differentiation. C/EBPβ is intrinsically unstructured, a molecular feature common to many proteins involved in signal processing and epigenetics. The structure of C/EBPβ differs depending on alternative translation initiation and multiple post-translational modifications (PTM). Mutation of distinct PTM sites in C/EBPβ alters protein interactions and cell differentiation, suggesting that a C/EBPβ PTM indexing code determines epigenetic outcomes. Herein, we systematically explored the interactome of C/EBPβ using an array technique based on spot-synthesized C/EBPβ-derived linear tiling peptides with and without PTM, combined with mass spectrometric proteomic analysis of protein interactions. We identified interaction footprints of ∼1,300 proteins in nuclear extracts, many with chromatin modifying, chromatin remodeling, and RNA processing functions. The results suggest that C/EBPβ acts as a multi-tasking molecular switchboard, integrating signal-dependent modifications and structural plasticity to orchestrate interactions with numerous protein complexes directing cell fate and function. : Proteomics; Systems Biology; Transcriptomics Subject Areas: Proteomics, Systems Biology, Transcriptomic

Modernisierungstheorien: Anregungspotenziale für die Frauen- und Geschlechterforschung

Oechsle M, Geissler B. Modernisierungstheorien: Anregungspotenziale für die Frauen- und Geschlechterforschung. In: Becker R, Kortendiek B, eds. Handbuch Frauen- und Geschlechterforschung. Geschlecht & Gesellschaft. Vol 35. 2nd ed. Wiesbaden: VS Verlag für Sozialwissenschaften; 2008: 203-211