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All lifeforms have to sense changes in their environment and adapt to possibly detrimental conditions. On a cellular level, the highly elaborate proteostasis network (PN) consisting of housekeeping and stress-induced proteins, confers this tolerance against stress and maintains cellular protein homoestasis. This is essential for survival, as an accumulation of stress-induced protein aggregation will eventually affect the functionality of crucial cellular components and ultimately lead to cell death. The guardians of this balance are the molecular chaperones and their activity-regulating co-haperones. They are engaged in all aspects of protein biogenesis, maintenance and degradation, especially during stress.
The heat shock proteins (HSPs) are the major chaperones in mammals and encompass constitutive and stress-induced isoforms. Among them, the HSP70 and the HSP90 family are the most abundant HSPs and their activity is involved in a great variety of homoestasis and stress-induced tasks.
As part of the protein triage the E3 ligase CHIP (C-terminal HSC70-interacting protein) is an essential activity regulating co-chaperone of HSP70 and HSP90 which provides a link between chaperone mediated protein-folding and various degradation pathways. Due to its decisive function, CHIP is involved in a wide array of cellular processes, especially in clearing misfolded HSP70 client proteins that are prone to aggregate. As a consequence, CHIP was reported to confer protection against many aggregation-induced pathologies of the neuronal system. Additionally, CHIP has been identified as a critical factor in various types of cancer and is implied to affect the development and the longevity of mammals.
Despite the significant progress in the understanding of CHIP’s structure and function, many aspects surrounding its chaperone dependency and its substrate recognition remain unclear. Moreover, due to the variety of substrates in diverse cellular pathways, there are yet many connections to elucidate between CHIP and components of the cellular proteostasis network.
The work of this thesis was focused on the role of CHIP in acute stress response and the corresponding status of chaperone association. Moreover, it was investigated if CHIP, as the connecting ligase of folding and degradation systems, might also provide a link between the PN and the reorganisation of the cellular architecture upon stress exposure.
This has become of increasing interest as recent reports highlight the importance of spatial sequestration in protein quality control.
To this end, subcellular distribution of CHIP was analysed by live-cell microscopy during heat stress. It became obvious that during the heat-induced challenge of the chaperone system, CHIP migrated to new cellular sites. Further experiments suggested that the observed migration to the plasma membrane is a chaperone-independent process and in vitro reconstitution of membrane association confirmed the competitive nature of membranes and chaperones for CHIP binding. A detailed in vivo and in vitro analysis of the newly observed membrane association of CHIP revealed a distinct lipid specificity and a novel direct association with lipids. Binding experiments with recombinantly purified deletion mutants of CHIP identified the TPR domain and a positive patch in the coiled-coil domain as main determinants for the lipid association. Through biochemical and biophysical approaches, the structural integrity and functionality of CHIP upon membrane binding was confirmed and further characterised.
Moreover, mass spectrometry analysis provided a high confidence identification of chaperone-free interactors of CHIP at the plasma membrane and other membranous compartments.
In accordance with the lipid specificity, the Golgi apparatus was one of these sites. Only chaperone-free CHIP had a significant effect on the morphology of the organelle, again confirming the competitive role of chaperones and lipids. With respect to the physiological consequences of the changed localisation of CHIP, preliminary results indicated increased cell death when the ligase localises to cellular membranes. The results lead to the conclusion that CHIP acts as an initiator of early stress adaptation and as a sensor for the severity and strength of the stress reaction.
Cells respond to protein misfolding and aggregation in the cytosol by adjusting gene transcription and a number of post-transcriptional processes. In parallel to functional reactions, cellular structure changes as well; however, the mechanisms underlying the early adaptation of cellular compartments to cytosolic protein misfolding are less clear. Here we show that the mammalian ubiquitin ligase C-terminal Hsp70-interacting protein (CHIP), if freed from chaperones during acute stress, can dock on cellular membranes thus performing a proteostasis sensor function. We reconstituted this process in vitro and found that mainly phosphatidic acid and phosphatidylinositol-4-phosphate enhance association of chaperone-free CHIP with liposomes. HSP70 and membranes compete for mutually exclusive binding to the tetratricopeptide repeat domain of CHIP. At new cellular locations, access to compartment-specific substrates would enable CHIP to participate in the reorganization of the respective organelles, as exemplified by the fragmentation of the Golgi apparatus (effector function).
In bioengineering, scaffold proteins have been increasingly used to recruit molecules to parts of a cell, or to enhance the efficacy of biosynthetic or signalling pathways. For example, scaffolds can be used to make weak or non-immunogenic small molecules immunogenic by attaching them to the scaffold, in this role called carrier. Here, we present the dodecin from Mycobacterium tuberculosis (mtDod) as a new scaffold protein. MtDod is a homododecameric complex of spherical shape, high stability and robust assembly, which allows the attachment of cargo at its surface. We show that mtDod, either directly loaded with cargo or equipped with domains for non-covalent and covalent loading of cargo, can be produced recombinantly in high quantity and quality in Escherichia coli. Fusions of mtDod with proteins of up to four times the size of mtDod, e.g. with monomeric superfolder green fluorescent protein creating a 437 kDa large dodecamer, were successfully purified, showing mtDod’s ability to function as recruitment hub. Further, mtDod equipped with SYNZIP and SpyCatcher domains for post-translational recruitment of cargo was prepared of which the mtDod/SpyCatcher system proved to be particularly useful. In a case study, we finally show that mtDod-peptide fusions allow producing antibodies against human heat shock proteins and the C-terminus of heat shock cognate 70 interacting protein (CHIP).