Biochemical, Cell Biological, and Genetic Assays to Analyze Amyloid and Prion Aggregation in Yeast

Protein aggregates are associated with a variety of debilitating human diseases, but they can have functional roles as well. Both pathological and nonpathological protein aggregates display tremendous diversity, with substantial differences in aggregate size, morphology, and structure. Among the different aggregation types, amyloids are particularly remarkable, because of their high degree of order and their ability to form self-perpetuating conformational states. Amyloids form the structural basis for a group of proteins called prions, which have the ability to generate new phenotypes by a simple switch in protein conformation that does not involve changes in the sequence of the DNA. Although protein aggregates are notoriously difficult to study, recent technological developments and, in particular, the use of yeast prions as model systems, have been very instrumental in understanding fundamental aspects of aggregation. Here, we provide a range of biochemical, cell biological and yeast genetic methods that are currently used in our laboratory to study protein aggregation and the formation of amyloids and prions

Granulostasis: Protein Quality Control of RNP Granules

Ribonucleoprotein (RNP) granules transport, store, or degrade messenger RNAs, thereby indirectly regulating protein synthesis. Normally, RNP granules are highly dynamic compartments. However, because of aging or severe environmental stress, RNP granules, in particular stress granules (SGs), convert into solid, aggregate-like inclusions. There is increasing evidence that such RNA-protein inclusions are associated with several age-related neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS), fronto-temporal dementia (FTD) and Alzheimer's disease (AD). Thus, understanding what triggers the conversion of RNP granules into aggregates and identifying the cellular players that control RNP granules will be critical to develop treatments for these diseases. In this review article, we discuss recent insight into RNP and SG formation. More specifically, we examine the evidence for liquid-liquid phase separation (LLPS) as an organizing principle of RNP granules and the role of aggregation-prone RNA-binding proteins (RBPs) in this process. We further discuss recent findings that liquid-like SGs can sequester misfolded proteins, which promote an aberrant conversion of liquid SGs into solid aggregates. Importantly, very recent studies show that a specific protein quality control (PQC) process prevents the accumulation of misfolding-prone proteins in SGs and, by doing so, maintains the dynamic state of SGs. This quality control process has been referred to as granulostasis and it relies on the specific action of the HSPB8-BAG3-HSP70 complex. Additional players such as p97/valosin containing protein (VCP) and other molecular chaperones (e.g., HSPB1) participate, directly or indirectly, in granulostasis, and ensure the timely elimination of defective ribosomal products and other misfolded proteins from SGs. Finally, we discuss recent findings that, in the stress recovery phase, SGs are preferentially disassembled with the assistance of chaperones, and we discuss evidence for a back-up system that targets aberrant SGs to the aggresome for autophagy-mediated clearance. Altogether the findings discussed here provide evidence for an intricate network of interactions between RNP granules and various components of the PQC machinery. Molecular chaperones in particular are emerging as key players that control the composition and dynamics of RNP granules, which may be important to protect against age-related diseases

Cooperation of a ubiquitin domain protein and an E3 ubiquitin ligase during chaperone/proteasome coupling

Background: Molecular chaperones recognize nonnative proteins and orchestrate cellular folding processes in conjunction with regulatory cofactors. However, not every attempt to fold a protein is successful, and misfolded proteins can be directed to the cellular degradation machinery for destruction. Molecular mechanisms underlying the cooperation of molecular chaperones with the degradation machinery remain largely enigmatic so far. Results: By characterizing the chaperone cofactors BAG-1 and CHIP, we gained insight into the cooperation of the molecular chaperones Hsc70 and Hsp70 with the ubiquitin/proteasome system, a major system for protein degradation in eukaryotic cells. The cofactor CHIP acts as a ubiquitin ligase in the ubiquitination of chaperone substrates such as the raf-1 protein kinase and the glucocorticoid hormone receptor. During targeting of signaling molecules to the proteasome, CHIP may cooperate with BAG-1, a ubiquitin domain protein previously shown to act as a coupling factor between Hsc/Hsp70 and the proteasome. BAG-1 directly interacts with CHIP; it accepts substrates from Hsc/Hsp70 and presents associated proteins to the CHIP ubiquitin conjugation machinery. Consequently, BAG-1 promotes CHIP-induced degradation of the glucocorticoid hormone receptor in vivo. Conclusions: The ubiquitin domain protein BAG-1 and the CHIP ubiquitin ligase can cooperate to shift the activity of the Hsc/Hsp70 chaperone system from protein folding to degradation. The chaperone cofactors thus act as key regulators to influence protein quality control

Mutualism, class composition, and the reshaping of worker organisation in platform work and the gig economy

This article contributes an understanding of mutualism as a foundational element in emergent worker collectivism. We challenge mainstream institutionalist accounts in industrial relations, especially from the Global North, that downplay processes of bottom-up regeneration of working-class organisation. We discuss compositional accounts of class formation and examine previous understandings of mutualism, then apply our conceptual framework to evidence from international literature and our own research on platform work in Italy and the UK. Three important themes emerge in understanding worker self-organisation: the demographics of the workforce, including migration backgrounds and social ties beyond the workplace; the existence of social relations in the ethnic/political/local community; and the relevance of free spaces of resource sharing and recomposition in the absence of a fixed place of work. We conclude that an understanding of mutualism can help to grasp emergent solidarities among new groups of workers within and beyond both platform work and trade unions

Fusion of Protein Aggregates Facilitates Asymmetric Damage Segregation

Asymmetric segregation of damaged proteins at cell division generates a cell that retains damage and a clean cell that supports population survival. In cells that divide asymmetrically, such as Saccharomyces cerevisiae, segregation of damaged proteins is achieved by retention and active transport. We have previously shown that in the symmetrically dividing Schizosaccharomyces pombe there is a transition between symmetric and asymmetric segregation of damaged proteins. Yet how this transition and generation of damage-free cells are achieved remained unknown. Here, by combining in vivo imaging of Hsp104-associated aggregates, a form of damage, with mathematical modeling, we find that fusion of protein aggregates facilitates asymmetric segregation. Our model predicts that, after stress, the increased number of aggregates fuse into a single large unit, which is inherited asymmetrically by one daughter cell, whereas the other one is born clean. We experimentally confirmed that fusion increases segregation asymmetry, for a range of stresses, and identified Hsp16 as a fusion factor. Our work shows that fusion of protein aggregates promotes the formation of damage-free cells. Fusion of cellular factors may represent a general mechanism for their asymmetric segregation at division

Identification of Triterpenes and β-sitosterol in the Bark of Plane Tree Extracts

Plane tree is planted as ornamental tree in urban areas. This tree naturally sheds its bark during the spring; however, the shed bark is commonly regarded as a waste material without any significant application.On the other hand, the bark of plane tree may be an important source of industrially relevant compounds, most notably betulinic acid. In our study a Supercritical Fluid Ultra Performance Convergence Chromatography (UPC2) system coupled with Evaporative Light Scattering Detector (ELSD), along with conventional HPLC, GC-MS and NMR were successfully utilized to analyze triterpenes in the extracts from the bark of plane tree. We show that not only betulinic acid, but other important triterpenes: betulin, betulinic aldehyde and β-sitosterol are also present in the extract of the plane tree bark. Among these the main compound is betulinic acid, with up to an order of magnitude larger concentration than the other constituents. The applied extraction method has a significant role on the concentration of the different compounds in the extracts. Most notably, neat scCO2 is not suitable to extract the polar betulinic acid, however betulin and betulinic aldehyde can be extracted selectively

On the structure of phase transition maps for three or more coexisting phases

This paper is partly based on a lecture delivered by the author at the ERC workshop "Geometric Partial Differential Equations" held in Pisa in September 2012. What is presented here is an expanded version of that lecture.Comment: 23 pages, 6 figure

The social protection of workers in the platform economy

This study investigates the social protection of workers in the platform economy at the request of the European Parliament’s Employment and Social Affairs Committee. The report reviews literature and previous research on the platform economy with the aims of defining it and developing a typology for understanding its nature. It discusses the growth and drivers of the platform economy, as well as benefits and challenges for workers, reporting findings from 50 interviews conducted with expert stakeholders in eight European countries and from an original survey of 1,200 platform workers. It dissects the different normative layers that need to be considered when looking at the challenges of social protection of platform workers from a legal perspective. Finally, the report draws conclusions and makes recommendations concerning arrangements for the provision of social protection for workers in this growing sector of the economyEste estudio investiga la protección social de los trabajadores en la economía de plataforma a petición de la Comisión de Empleo y Asuntos Sociales del Parlamento Europeo. El informe revisa la literatura y las investigaciones previas sobre la economía de plataforma con el objetivo de definirla y desarrollar una tipología para comprender su naturaleza. Discute el crecimiento y los motores de la economía planificada, así como los beneficios y los desafíos para los trabajadores, y presenta los resultados de 50 entrevistas realizadas a expertos de ocho países europeos y de una encuesta original de 1.200 trabajadores de la plataforma. Disecciona los diferentes estratos normativos que deben considerarse cuando se analizan los retos de la protección social de los trabajadores de las plataformas desde una perspectiva jurídica. Por último, el informe extrae conclusiones y formula recomendaciones relativas a las disposiciones para la provisión de protección social para los trabajadores de este creciente sector de la economía

Adaptable P body physical states differentially regulate bicoid mRNA storage during early Drosophila development.

Ribonucleoprotein condensates can exhibit diverse physical states in vitro and in vivo. Despite considerable progress, the relevance of condensate physical states for in vivo biological function remains limited. Here, we investigated the physical properties of processing bodies (P bodies) and their impact on mRNA storage in mature Drosophila oocytes. We show that the conserved DEAD-box RNA helicase Me31B forms viscous P body condensates, which adopt an arrested physical state. We demonstrate that structurally distinct proteins and protein-protein interactions, together with RNA, regulate the physical properties of P bodies. Using live imaging and in situ hybridization, we show that the arrested state and integrity of P bodies support the storage of bicoid (bcd) mRNA and that egg activation modulates P body properties, leading to the release of bcd for translation in the early embryo. Together, this work provides an example of how physical states of condensates regulate cellular function in development