The accumulation of misfolded proteins is associated with numerous degenerative conditions, cancers and genetic diseases. These pathological imbalances in protein homeostasis (termed proteostasis), result from the improper triage and disposal of damaged and defective proteins from the cell. The ubiquitin-proteasome system is a key pathway for the molecular control of misfolded cytosolic proteins, co-opting a cascade of ubiquitin ligases to direct terminally damaged proteins to the proteasome via modification with chains of the small protein, ubiquitin. Despite the evidence for ubiquitination in this critical pathway, the precise complement of ubiquitin ligases and deubiquitinases that modulate this process remains under investigation. Whilst chaperones act as the first line of defence against protein misfolding, the ubiquitination machinery has a pivotal role in targeting terminally defunct cytosolic proteins for destruction. Recent work points to a complex assemblage of chaperones, ubiquitination machinery and subcellular quarantine as components of the cellular arsenal against proteinopathies. In this review, we examine the contribution of these pathways and cellular compartments to the maintenance of the cytosolic proteome. Here we will particularly focus on the ubiquitin code and the critical enzymes which regulate misfolded proteins in the cytosol, the molecular point of origin for many neurodegenerative and genetic diseases.

The efficacy of superoxide dismutase-1 (SOD1) folding impacts neuronal loss in motor system neurodegenerative diseases. Mutations can prevent SOD1 post-translational processing leading to misfolding and cytoplasmic aggregation in familial amyotrophic lateral sclerosis (ALS). Evidence of immature, wild-type SOD1 misfolding has also been observed in sporadic ALS, non-SOD1 familial ALS and Parkinson's disease. The copper chaperone for SOD1 (hCCS) is a dedicated and specific chaperone that assists SOD1 folding and maturation to produce the active enzyme. Misfolded or misfolding prone SOD1 also interacts with heat shock proteins and macrophage migration inhibitory factor to aid folding, refolding or degradation. Recognition of specific SOD1 structures by the molecular chaperone network and timely dissociation of SOD1-chaperone complexes are, therefore, important steps in SOD1 processing. Harnessing these interactions for therapeutic benefit is actively pursued as is the modulation of SOD1 behaviour with pharmacological and peptide chaperones. This review highlights the structural and mechanistic aspects of a selection of SOD1-chaperone interactions together with their impact on disease models.

Phosphorylation by protein kinases is a fundamental mechanism of signal transduction. Many kinase families contain one or several members that, although evolutionarily conserved, lack the residues required for catalytic activity. Studies combining structural, biochemical, and functional approaches revealed that these pseudokinases have crucial roles in vivo and may even represent attractive targets for pharmacological intervention. Pseudokinases mediate signal transduction by a diversity of mechanisms, including allosteric regulation of their active counterparts, assembly of signaling hubs, or modulation of protein localization. One such pseudokinase, named Tra1 in yeast and transformation/transcription domain-associated protein (TRRAP) in mammals, is the only member lacking all catalytic residues within the phosphatidylinositol 3-kinase related kinase (PIKK) family of kinases. PIKKs are related to the PI3K family of lipid kinases, but function as Serine/Threonine protein kinases and have pivotal roles in diverse processes such as DNA damage sensing and repair, metabolic control of cell growth, nonsense-mediated decay, or transcription initiation. Tra1/TRRAP is the largest subunit of two distinct transcriptional co-activator complexes, SAGA and NuA4/TIP60, which it recruits to promoters upon transcription factor binding. Here, we review our current knowledge on the Tra1/TRRAP pseudokinase, focusing on its role as a scaffold for SAGA and NuA4/TIP60 complex assembly and recruitment to chromatin. We further discuss its evolutionary history within the PIKK family and highlight recent findings that reveal the importance of molecular chaperones in pseudokinase folding, function, and conservation.

FKBP51 and FKBP52 are two iconic members of the family of peptidyl-prolyl-( cis/trans )-isomerases (EC: 5.2.1.8), which comprises proteins that catalyze the cis/trans isomerization of peptidyl-prolyl peptide bonds in unfolded and partially folded polypeptide chains and native state proteins. Originally, both proteins have been studied as molecular chaperones belonging to the steroid receptor heterocomplex, where they were first discovered. In addition to their expected role in receptor folding and chaperoning, FKBP51 and FKBP52 are also involved in many biological processes, such as signal transduction, transcriptional regulation, protein transport, cancer development, and cell differentiation, just to mention a few examples. Recent studies have revealed that both proteins are subject of post-translational modifications such as phosphorylation, SUMOlyation, and acetylation. In this work, we summarize recent advances in the study of these immunophilins portraying them as scaffolding proteins capable to organize protein heterocomplexes, describing some of their antagonistic properties in the physiology of the cell, and the putative regulation of their properties by those post-translational modifications.

Twenty-five years ago, we obtained our first mass spectra of molecular chaperones in complex with protein ligands and entered a new field of gas-phase structural biology. It is perhaps now time to pause and reflect, and to ask how many of our initial structure predictions and models derived from mass spectrometry (MS) datasets were correct. With recent advances in structure determination, many of the most challenging complexes that we studied over the years have become tractable by other structural biology approaches enabling such comparisons to be made. Moreover, in the light of powerful new electron microscopy methods, what role is there now for MS? In considering these questions, I will give my personal view on progress and problems as well as my predictions for future directions.