ProteostaSys: a systems view of proteostasis

The transcription factor HSF-1 has long been recognised as the master regulator of the Heat Shock Response (HSR), a highly conserved transcriptional programme that is rapidly deployed to counter the threat of misfolded proteins within the cytosol/nucleus. In keeping with this role, impaired HSF-1 activity is associated with reduced tissue resilience, shortened lifespan and increased susceptibility to age-associated protein conformational disease, while enhanced HSF-1 activity is associated with greater stress resistance, increased lifespan and improved protection against protein aggregation and proteotoxicity. While HSF-1 is classically thought to promote tissue-health by increasing the levels of cytosolic/nuclear molecular chaperones and protein degradation factors, evidence is emerging that HSF-1 activity is also closely coupled to mitochondrial homeostasis. In this talk, I will present my groups recent findings that increased HSF-1 activity converges on mitochondrial homeostasis through the proteasome shuttling factor, ubiquilin-1. HSF-1 promotes ubiquilin-1 expression, which leads to increased turnover of the CDC-48 co-factor, NPL-4. This alters mitochondrial network dynamics and leads to metabolic changes that promote longevity. In addition, changes in mitochondrial homeostasis result in increased HSF-1 activity and protection of the cytosolic proteome. Our findings reveal that HSF-1 sits at the nexus between cytosolic and mitochondrial homeostasis and suggest that the interplay between HSF-1 and mitochondria is a key determinant of cell and tissue viability that could be manipulated to promote healthy ageing.

Dr John Labbadia

University College London, UK

Dr John Labbadia

University College London, UK

Dr John Labbadia is an Associate Professor within the Institute of Healthy Ageing at University College London. John has dedicated his career to understanding how cells maintain protein homeostasis, first as a PhD student working in Professor Gillian Bates’ lab at Kings College London, and then as an ALS Association Postdoctoral Fellow in Professor Rick Morimoto’s lab at Northwestern University. Following his time in the Morimoto lab, John was awarded a prestigious BBSRC David Phillips Fellowship to establish his own group at UCL, where he has been using the small nematode worm Caenorhabditis elegans, to understand how changes in the activity of stress response pathways impacts the long-term health of different tissues. His research is funded by awards from the BBSRC, Academy of Medical Sciences, Wellcome Trust, and Leverhulme Trust.

Stress granules (SGs) are ribonucleoprotein condensates that transiently form during cellular stress when protein translation is inhibited. They allow the storage of RNA-binding proteins and mRNAs, and their release for translation restoration upon stress recovery. Loss of SG dynamic properties is proposed to underlie the formation of protein aggregates in certain neurodegenerative diseases, including amyotrophic lateral sclerosis. The Hsp70 chaperone family has been implicated in limiting aberrant SG aggregation and in the disassembly of the condensates during recovery. However, a detailed understanding of how Hsp70 regulates SG dynamic properties is lacking. Here, we used C. elegans to investigate the role of constitutively expressed Hsp70 (HSP-1/Hsc70) in the dynamics of SGs in different cell types. We found that HSP-1 associates with cytosolic structures corresponding to bona fide SGs throughout the tissues of the animal. Fluorescence recovery after photobleaching of SGs in various cell types revealed that HSP-1 is highly mobile and rapidly exchanges with the cytoplasmic pool, in contrast to the core SG protein GTBP-1/G3BP1, but similar to other SG components. Reduced hsp-1 function resulted in persistent SGs in germ cells, indicating a role in SG disassembly, and altered the mobility of select SG components, suggesting a stronger effect on the more dynamic shell within the SG structure. These findings provide insights into the regulation of condensate dynamics and architecture by Hsp70, with potential implications for pathological transitions of SGs.

Dr Ambre Sala

Institute for Integrative Biology of the Cell, France

Dr Ambre Sala

Institute for Integrative Biology of the Cell, France

Ambre Sala is a CNRS researcher in the Cell Biology department of the Institute for Integrative Biology of the Cell (I2BC, Gif-sur-Yvette, France), where she heads the group “Protein homeostasis in development and aging”. Her research broadly focuses on the role of molecular chaperones in regulating physiological and pathological protein phase transitions. The team develops cell biology, genetic, and biochemistry approaches, taking advantage of the C. elegans model system to identify the cellular roles and composition of the chaperone networks that regulate these processes in various tissues and during aging. Ambre Sala previously completed her PhD thesis in 2015 at the University of Toulouse, and then worked as a postdoctoral fellow in the laboratory of Professor Richard Morimoto at Northwestern University. Ambre Sala joined the I2BC in 2023 after obtaining a tenured researcher position and a starting grant from the ATIP-Avenir program (CNRS/Inserm).

The progression of neurodegenerative diseases such as Alzheimer’s disease (AD) or Parkinson’s disease (PD), is linked to the spreading of protein aggregation across interconnected brain regions. Fibrillar seeds are transferred between cells, inducing misfolding and aggregation of its native monomeric counterpart. A critical rate-limiting step in this process is the rupture of endolysosomal vesicles, which releases transferred aggregates into the cytosol, where they promote further aggregation of endogenous proteins. While the pathways underlying endolysosomal damage and repair have been increasingly studied, many aspects remain unclear. We conducted a genome-wide RNAi screen in a novel C. elegans model of tau spreading and identified sphingolipid metabolism genes as critical for endolysosomal integrity. Using C. elegans and cell culture models, we investigated how disruption of sphingolipid homeostasis affects endolysosomal membrane integrity and contributes to tau aggregation and toxicity. Fluorescence recovery after photobleaching and C-Laurdan dye imaging revealed that silencing sphingolipid metabolism genes decreases endolysosomal vesicle membrane fluidity, increasing endolysosomal rupture. The accumulation of aggregated tau in endolysosomal vesicles further aggravated endomembrane rigidification and damage and promoted seeded tau aggregation, potentially by facilitating the escape of tau seeds from the endolysosomal system. Supplementation with unsaturated fatty acids improved membrane fluidity, suppressing endolysosomal rupture and seeded tau aggregation in cell models, and alleviating tau-associated neurotoxicity in C. elegans. Conversely, supplementation with the saturated fatty acid palmitic acid worsened these effects. These findings provide insights into how impaired sphingolipid homeostasis drives tau pathology, suggesting membrane fluidity restoration as a potential therapeutic strategy for AD.

Professor Carmen Nussbaum

Ludwig Maximilians University Munich, Germany

Professor Carmen Nussbaum

Ludwig Maximilians University Munich, Germany

Carmen Nussbaum studied Biology and pursued her doctoral research at the Technical University of Munich, earning her PhD in December 2008. She then joined Northwestern University in Evanston, IL, USA, as a postdoctoral fellow. From 2014 to 2022, she led a research group at Heidelberg University and the German Cancer Research Center. Since 2022, she holds a W2 Professorship in the Department of Neuroanatomy at Ludwig-Maximilians-University of Munich. Her research utilises C. elegans and cell culture models to study the molecular mechanisms underlying neurodegenerative diseases. She focused on the dual role of the Hsp70 disaggregation machinery in the prion-like propagation of α-synuclein and tau, demonstrating that while chaperone-mediated disaggregation solubilises protein aggregates, it also generates small, seeding-competent toxic species. More recently, her work has focused on the role of endolysosomal integrity in prion-like aggregate propagation.

Since the 1950s, humans have produced 8 billion tons of plastic. This plastic, the vast majority of which is not recycled, is in the form of plastic containers, synthetic fibers used in clothing, paints, adhesives, etc. These plastics break down with use and enter our food and water as micro and nano-sized particles. Plastic is also added to personal care products as microspheres that do not need to break down to enter waterways. Moreover, tire wear particles account for 28% of microplastics that enter the environment. A recent retrospective study revealed that microplastic accumulation in the human brain is increasing at an alarming rate. This has been found to contribute to neuroinflammation and to promote the progression of Alzheimer’s Disease symptoms. We asked whether microplastic from a synthetic soccer turf (micro-rubber) causes protein damage/misfolding, overwhelms the proteostasis network, and contributes to proteostasis collapse. To address this, we utilised C. elegans sensors of protein folding, and found that exposure to micro-rubber particles accelerated the formation of large visible protein aggregates in body wall muscle cells. Interestingly, animals that were exposed to micro-rubber were impaired in their ability to launch a heat shock response. Taken together, the data suggests that micro-rubber particles contribute to a decline in proteostasis, which may be relevant to neurodegenerative disease progression.

Dr Elise A Kikis

University of the South (Sewanee), UK

Dr Elise A Kikis

University of the South (Sewanee), UK

Elise A Kikis earned a BA in Biology from Cornell University and a PhD in Plant Biology from the University of California, Berkeley. She currently serves the University of the South (Sewanee) as Professor and Chair of the biology department and as co-director of the Office of Undergraduate Research and Scholarship. She joined the Sewanee faculty in 2012 as an assistant professor with the goal of inspiring the next generation of scientists to think critically and address pressing scientific questions. Prior to beginning her faculty position, she conducted post-doctoral research under the supervision of Richard Morimoto at Northwestern University. There, she solidified her interest in the study of protein folding and the regulation of proteostasis. She has published numerous research articles, many with Sewanee undergraduate student co-authors, in internationally recognised journals and has successfully competed for extramural funding from the National Institutes of Health, the Kennedy’s Disease Association, and the Appalachian College Association. She enjoys fostering teamwork and collaboration in her laboratory and training undergraduates as they begin their careers.

Protein disulfide formation contributes to protein folding and function, as evidenced by numerous oxidoreductases that maintain disulfide status within various cell compartments.  Many of these oxidoreductases, including thioredoxins (Trxs) and protein disulfide isomerases (PDIs), arose from a common evolutionary precursor and employ transient thiol-disulfide exchange mechanisms when interacting with their redox partners.  Because disulfide bonds that form between Trx superfamily members and their redox partners are often short-lived, we developed a chemical cross-linking strategy that irreversibly captures these proteins when interacting with potential redox partners.  Using the thiol-reactive cross-linker divinyl sulfone (DVSF), Trx2, a cytosolic Trx in baker’s yeast, undergoes cross-linking with its most prominent redox partners via modification of its active site cysteines. Likewise, upon DVSF treatment, PDIs that promote oxidative protein folding in the ER form cross-linked complexes with known redox partners, including many chaperones and protein clearance factors. More recently, we have turned our attention to less studied redox networks, seeking to identify the interaction partners of disulfide maintenance systems in mitochondria.  Particularly, we have characterised the putative redox partners of yeast Trx3, a disulfide reductase that resides in the mitochondrial matrix. We have found that Trx3 interacts with numerous metabolic enzymes, including members of the branched chain amino acid biosynthesis pathway, as well as proteins in the proteostasis network.  Our current work suggests disulfide bond homeostasis and flux through specific metabolic pathways may be linked in previously undescribed ways.

Professor James West

The College of Wooster, USA

Professor James West

The College of Wooster, USA

James West received his PhD in Biochemistry from Vanderbilt University in 2005, working in Larry Marnett’s laboratory on apoptotic cell death mechanisms and cellular responses to protein-damaging lipid peroxidation byproducts. Subsequently, he performed postdoctoral research in the Department of Molecular Biosciences at Northwestern University in the laboratory of Rick Morimoto, studying small molecule activators of the heat shock response and parallel stress responses. He began a faculty position at The College of Wooster in 2008, where his research program has focused on interrogating protein disulfide formation and reduction networks, primarily in a baker’s yeast model system.  

There is no cure or effective treatment for neurodegenerative protein conformational diseases (PCDs), such as Alzheimer’s, Parkinson’s, and Huntington’s. All these ailments are characterised by toxic protein aggregation, resulting in progressive neuronal damage and cell death. Emerging evidence reveals that bacteria play a significant role in the pathogenesis of neurodegenerative diseases; however, the microbial identity and the underlying mechanisms remain elusive. In a screen of over 220 unique bacterial species from the Human Microbiome Project, we found proteotoxic and proteoprotective bacteria. We demonstrated that employing bacteriophage-mediated strategies to eliminate detrimental bacteria associated with proteotoxicity alleviates disease pathogenicity. Furthermore, among the beneficial microbes, a single bacterial strain, Prevotella corporis, provides protection against misfolding and aggregation. We employed the Caenorhabditis elegans and Drosophila melanogaster models expressing polyglutamine and Aβ₄₂ to demonstrate that colonisation of the gut suppresses aggregation in both models. Further analysis revealed that P. corporis activates the expression of heat shock genes and can rapidly disassemble existing protein aggregates. While P. corporis did not have any effect on lifespan, it enhanced the thermotolerance and disaggregation activity of the host, suggesting general improvement in proteostasis. Collectively, we reveal how bacteria affect host proteostasis and influence disease pathology, opening new opportunities to manage neurodegenerative diseases by targeting the gut.

Dr Daniel Czyż

University of Florida, USA

Dr Daniel Czyż

University of Florida, USA

Dr Daniel Czyż (chysh) is an Associate Professor in the Department of Microbiology and Cell Science at the University of Florida. He received his BS in Biochemistry from the University of Illinois at Chicago and a PhD from Northwestern University, where he studied the regulation of the heat shock response using Caenorhabditis elegans. During his postdoc in the Department of Microbiology at the University of Chicago, he pioneered drug-repurposing and host-targeting approaches against intracellular bacterial infections. Dr Czyż has been a faculty at the University of Florida since 2018. His group studies microbial pathogenesis in neurodegenerative diseases employing C. elegans and tissue culture models. Recently, the Czyż Group has characterised over 200 different bacteria for their role in host proteostasis, revealing proteotoxic and proteoprotective species that modulate disease pathogenesis by influencing host stress responses. Antibiotics are the major contributor to resistance and gut dysbiosis, which is implicated in the pathogenesis of neurodegenerative diseases. The Czyż Group utilises non-traditional approaches, including bacteriophages, to target proteotoxic and antibiotic-resistant bacteria and restore gut eubiosis, potentially alleviating disease progression. Dr Czyż received numerous distinctions for teaching and research, including the UF Exemplary Online Award for Student Engagement, IFAS/CALS Innovative Teaching Award, NACTA Educator Award, Richard Jones Outstanding New Faculty Research Award, and much more. He served as the Vice-Chair (2020-2021) and Chair (2021-2022) of the Advisory Council at the National Institute of Antimicrobial Resistance Research and Education (NIAMRRE) and hosted the 2023 NIAMRRE Annual Conference at UF.