HSP70-binding motifs function as protein quality control degrons

Abildgaard, Amanda B; Voutsinos, Vasileios; Petersen, S.; Larsen, Fia B; Kampmeyer, Caroline; Johansson, Kristoffer E.; Stein, Amelie; Ravid, Tommer; Andréasson, Claes; Jensen, Michael K.; Lindorff‐Larsen, Kresten; Hartmann‐Petersen, Rasmus

doi:10.1007/s00018-022-04679-3

Cited by 16 publications

(19 citation statements)

References 82 publications

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“…Along with the 132 nt oligos three 126 nt long control oligos were made as well, each consisting of a 66 nt long oligo flanked by the same complementary Gibson overlaps. The three control oligos used were based on the APPY degron (- RLLL), which is 22 aa long sequence and two variants that are known to mildly (-RAAA) or strongly (-DAAA) stabilize the APPY degron 55 . The 132 nt long oligos library, and the three control oligos, were ordered from IDT as three separate libraries in a way that excludes the presence of Odds, Evens and CT oligos of the same protein in the same library tube, thus producing three libraries referred to as Odds (complexity = 93), Evens (complexity = 91) and CT (complexity = 10).…”

Section: Methodsmentioning

confidence: 99%

“…The reigning hypothesis explaining the degradation of destabilized or misfolded proteins, states that these proteins, through local or global unfolding events, transiently expose PQC degradation signals (degrons). Degrons are recognized by E3 ubiquitin-protein ligases and/or molecular chaperones such as HSP70 [52][53][54][55] , which in turn direct the protein for proteasomal degradation.…”

Section: Inherent Pqc Degrons In Aspa Map To Buried Regions That Are ...mentioning

confidence: 99%

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Deep mutational scanning reveals a tight correlation between protein degradation and toxicity of thousands of non-native aspartoacylase protein variants

Grønbæk-Thygesen,

Voutsinos,

Johansson

et al. 2023

Preprint

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When the structural stability of a protein is compromised, the protein may form non-native interactions with other cell proteins and thus becomes a hazard to the cell. To mitigate this danger, destabilized proteins are targeted by the cellular protein quality control (PQC) network, which either corrects the folding defect or targets the protein for degradation. However, the details of how the protein folding and degradation systems collaborate to combat potentially toxic non-native proteins are unknown. To address this issue, we performed systematic studies on destabilized variants of the cytosolic aspartoacylase, ASPA, where loss-of-function variants are linked to Canavan’s disease, an autosomal recessive and lethal neurological disorder, characterized by the spongy degeneration of the white matter in the brain. Using Variant Abundance by Massively Parallel sequencing (VAMP-seq), we determined the abundance of 6152 out of the 6260 (∼98%) possible single-site missense and nonsense ASPA variants in cultured human cells. The majority of the low abundance ASPA variants are degraded through the ubiquitin-proteasome system (UPS) and become toxic upon prolonged expression. Variant cellular abundance data correlates with predicted thermodynamic stability, evolutionary conservation, and separates most known disease-linked variants from benign variants. Systematic mapping of degradation signals (degrons) shows that inherent primary degrons in ASPA are located in buried regions, and reveals that the wild-type ASPA C-terminal region functions as a degron. Collectively, our data can be used to interpret Canavan’s disease variants and also offer mechanistic insight into how ASPA missense variants are targeted by the PQC system. These are essential steps towards future implementation of precision medicine for Canavan’s disease.

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Section: Methodsmentioning

confidence: 99%

Section: Inherent Pqc Degrons In Aspa Map To Buried Regions That Are ...mentioning

confidence: 99%

Deep mutational scanning reveals a tight correlation between protein degradation and toxicity of thousands of non-native aspartoacylase protein variants

Grønbæk-Thygesen,

Voutsinos,

Johansson

et al. 2023

Preprint

Self Cite

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“…For persistently misfolded proteins, release from Hsp70 is mediated by dedicated armadillo and BAG-type NEFs that carry specialized substrate release domains that compete the substrate off the chaperone substrate binding site ( Rauch et al, 2016 ; Gowda et al, 2018 ; Rosam et al, 2018 ). This orchestrated release enables other factors such as ubiquitin E3 ligases to get access to the hydrophobic peptides that constitute the Hsp70 binding site and also function as degrons ( Rauch et al, 2016 ; Gowda et al, 2018 ; Rosam et al, 2018 ; Abildgaard et al, 2023 ). For example, the metazoan E3 ligase CHIP associates with Hsp70 and will eventually ubiquitinate persistently misfolded proteins that undergo repeated Hsp70 interaction cycles ( Murata et al, 2001 ).…”

Section: Disaggregation Is a Decision Point For Refolding And Degrada...mentioning

confidence: 99%

Reversible protein assemblies in the proteostasis network in health and disease

Kohler

Andréasson

2023

Front. Mol. Biosci.

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While proteins populating their native conformations constitute the functional entities of cells, protein aggregates are traditionally associated with cellular dysfunction, stress and disease. During recent years, it has become clear that large aggregate-like protein condensates formed via liquid-liquid phase separation age into more solid aggregate-like particles that harbor misfolded proteins and are decorated by protein quality control factors. The constituent proteins of the condensates/aggregates are disentangled by protein disaggregation systems mainly based on Hsp70 and AAA ATPase Hsp100 chaperones prior to their handover to refolding and degradation systems. Here, we discuss the functional roles that condensate formation/aggregation and disaggregation play in protein quality control to maintain proteostasis and why it matters for understanding health and disease.

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“…Recently, methods have been developed to control the degradation of selected proteins to study their function, − design synthetic circuits, , regulate gene expression, , and degrade disease-causing and other nondesired proteins. , To facilitate degradation, different destabilizing domains (or “degrons”) have been developed that guide selected proteins to the degradation machinery. These rely on the interaction with cellular E3 ligases, − 26S proteasome, , chaperones, − or through currently unknown mechanisms.…”

Section: Introductionmentioning

confidence: 99%

Subunits of an E3 Ligase Complex as Degrons for Efficient Degradation of Cytosolic, Nuclear, and Membrane Proteins

Verbič,

Lebar,

Praznik

et al. 2024

ACS Synth. Biol.

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Protein degradation is a highly regulated cellular process crucial to enable the high dynamic range of the response to external and internal stimuli and to balance protein biosynthesis to maintain cell homeostasis. Within mammalian cells, hundreds of E3 ubiquitin ligases target specific protein substrates and could be repurposed for synthetic biology. Here, we present a systematic analysis of the four protein subunits of the multiprotein E3 ligase complex as scaffolds for the designed degrons. While all of them were functional, the fusion of a fragment of Skp1 with the target protein enabled the most effective degradation. Combination with heterodimerizing peptides, protease substrate sites, and chemically inducible dimerizers enabled the regulation of protein degradation. While the investigated subunits of E3 ligases showed variable degradation efficiency of the membrane and cytosolic and nuclear proteins, the bipartite SSD (SOCSbox-Skp1(ΔC111)) degron enabled fast degradation of protein targets in all tested cellular compartments, including the nucleus and plasma membrane, in different cell lines and could be chemically regulated. These subunits could be employed for research as well as for diverse applications, as demonstrated in the regulation of Cas9 and chimeric antigen receptor proteins.

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HSP70-binding motifs function as protein quality control degrons

Cited by 16 publications

References 82 publications

Deep mutational scanning reveals a tight correlation between protein degradation and toxicity of thousands of non-native aspartoacylase protein variants

Deep mutational scanning reveals a tight correlation between protein degradation and toxicity of thousands of non-native aspartoacylase protein variants

Reversible protein assemblies in the proteostasis network in health and disease

Subunits of an E3 Ligase Complex as Degrons for Efficient Degradation of Cytosolic, Nuclear, and Membrane Proteins

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