Mark S. Hipp

Molecular Chaperone Functions in Protein Folding and Proteostasis

The biological functions of proteins are governed by their three-dimensional fold. Protein folding, maintenance of proteome integrity, and protein homeostasis (proteostasis) critically depend on a complex network of molecular chaperones. Disruption of proteostasis is implicated in aging and the pathogenesis of numerous degenerative diseases. In the cytosol, different classes of molecular chaperones cooperate in evolutionarily conserved folding pathways. Nascent polypeptides interact cotranslationally with a first set of chaperones, including trigger factor and the Hsp70 system, which prevent premature (mis)folding. Folding occurs upon controlled release of newly synthesized proteins from these factors or after transfer to downstream chaperones such as the chaperonins. Chaperonins are large, cylindrical complexes that provide a central compartment for a single protein chain to fold unimpaired by aggregation. This review focuses on recent advances in understanding the mechanisms of chaperone action in promoting and regulating protein folding and on the pathological consequences of protein misfolding and aggregation.

Proteostasis impairment in protein-misfolding and -aggregation diseases

Cells possess an extensive network of components to safeguard proteome integrity and maintain protein homeostasis (proteostasis). When this proteostasis network (PN) declines in performance, as may be the case during aging, newly synthesized proteins are no longer able to fold efficiently and metastable proteins lose their functionally active conformations, particularly under conditions of cell stress. Apart from loss-of-function effects, a critical consequence of PN deficiency is the accumulation of cytotoxic protein aggregates, which are also associated with many age-dependent neurodegenerative diseases and other medical disorders. Here we discuss recent evidence that the chronic production of aberrantly folded and aggregated proteins in these diseases is harmful by overtaxing PN capacity, setting in motion a vicious cycle of increasing proteome imbalance that eventually leads to PN collapse and cell death.

PolyQ Proteins Interfere with Nuclear Degradation of Cytosolic Proteins by Sequestering the Sis1p Chaperone

Dysfunction of protein quality control contributes to the cellular pathology of polyglutamine (polyQ) expansion diseases and other neurodegenerative disorders associated with aggregate deposition. Here we analyzed how polyQ aggregation interferes with the clearance of misfolded proteins by the ubiquitin-proteasome system (UPS). We show in a yeast model that polyQ-expanded proteins inhibit the UPS-mediated degradation of misfolded cytosolic carboxypeptidase Y(∗) fused to green fluorescent protein (GFP) (CG(∗)) without blocking ubiquitylation or proteasome function. Quantitative proteomic analysis reveals that the polyQ aggregates sequester the low-abundant and essential Hsp40 chaperone Sis1p. Overexpression of Sis1p restores CG(∗) degradation. Surprisingly, we find that Sis1p, and its homolog DnaJB1 in mammalian cells, mediates the delivery of misfolded proteins into the nucleus for proteasomal degradation. Sis1p shuttles between cytosol and nucleus, and its cellular level limits the capacity of this quality control pathway. Upon depletion of Sis1p by polyQ aggregation, misfolded proteins are barred from entering the nucleus and form cytoplasmic inclusions.

Cytoplasmic protein aggregates interfere with nucleocytoplasmic transport of protein and RNA

Location, location, location Aggregates of certain disease-associated proteins are involved in neurodegeneration. Woerner et al. now show that the exact location of these aggregates in the cell may be the key to their pathology (see the Perspective by Da Cruz and Cleveland). An artificial aggregate-prone protein caused problems when expressed in the cytoplasm but not when expressed in the nucleus. Cytoplasmic aggregates interfered with nucleocytoplasmic import and export. Perhaps if we can shunt pathological aggregates to the nucleus in the future, we will be able to ameliorate some forms of degenerative disease. Science, this issue p. 173; see also p. 125 Protein aggregates in the cytoplasm soak up accessory factors needed for transport of other proteins and RNA across the nuclear envelope. [Also see Perspective by Da Cruz and Cleveland] Amyloid-like protein aggregation is associated with neurodegeneration and other pathologies. The nature of the toxic aggregate species and their mechanism of action remain elusive. Here, we analyzed the compartment specificity of aggregate toxicity using artificial β-sheet proteins, as well as fragments of mutant huntingtin and TAR DNA binding protein–43 (TDP-43). Aggregation in the cytoplasm interfered with nucleocytoplasmic protein and RNA transport. In contrast, the same proteins did not inhibit transport when forming inclusions in the nucleus at or around the nucleolus. Protein aggregation in the cytoplasm, but not the nucleus, caused the sequestration and mislocalization of proteins containing disordered and low-complexity sequences, including multiple factors of the nuclear import and export machinery. Thus, impairment of nucleocytoplasmic transport may contribute to the cellular pathology of various aggregate deposition diseases.

FAT10, a ubiquitin-independent signal for proteasomal degradation

ABSTRACT FAT10 is a small ubiquitin-like modifier that is encoded in the major histocompatibility complex and is synergistically inducible by tumor necrosis factor alpha and gamma interferon. It is composed of two ubiquitin-like domains and possesses a free C-terminal diglycine motif that is required for the formation of FAT10 conjugates. Here we show that unconjugated FAT10 and a FAT10 conjugate were rapidly degraded by the proteasome at a similar rate. Fusion of FAT10 to the N terminus of very long-lived proteins enhanced their degradation rate as potently as fusion with ubiquitin did. FAT10-green fluorescent protein fusion proteins were not cleaved but entirely degraded, suggesting that FAT10-specific deconjugating enzymes were not present in the analyzed cell lines. Interestingly, the prevention of ubiquitylation of FAT10 by mutation of all lysines or by expression in ubiquitylation-deficient cells did not affect FAT10 degradation. Thus, conjugation with FAT10 is an alternative and ubiquitin-independent targeting mechanism for degradation by the proteasome, which, in contrast to polyubiquitylation, is cytokine inducible and irreversible.

Ubiquitin accumulation in autophagy-deficient mice is dependent on the Nrf2-mediated stress response pathway: a potential role for protein aggregation in autophagic substrate selection

Inactivation of the essential autophagy gene Atg5 results in selective accumulation of aggregation-prone proteins independently of substrate ubiquitination.

Indirect inhibition of 26S proteasome activity in a cellular model of Huntington’s disease

Rather than directly impairing 26S proteasomes, misfolded huntingtin may disrupt cellular proteostasis and lead to competition for limited 26S proteasome capacity.

Proteasome inhibition leads to NF-κb-independent IL-8 transactivation in human endothelial cells through induction of AP-1

IL‐8 is an important mediator of leukocyte trafficking and activation, participating in tumor angiogenesis, inflammatory processes and coronary atherosclerosis. Under flow conditions IL‐8, in conjunction with MCP‐1, triggers the firm adhesion of monocytes to the vascular endothelium. While previous studies have suggested the requirement of NF‐κB for IL‐8 secretion by endothelial cells, we investigated the possibility of IL‐8 transactivation under conditions of NF‐κB suppression. Inhibition of the proteasome by MG‐132 or lactacystin completely blocked TNF‐α‐induced IκBα degradation as well as NF‐κB activity in human arterial endothelial cells. Surprisingly, basal secretion of IL‐8 protein was eight‐ to tenfold induced by proteasome inhibitors, while MCP‐1 expression was, as expected, completely down‐regulated. IL‐8 was up‐regulated at the transcriptional level, and promoter studies proved a more than ninefold induction of transcription factor AP‐1 activity to be the cause of increased IL‐8 transcription. Mutation of the AP‐1 binding site in an IL‐8 promoter construct completely abrogated this effect, while mutation of the NF‐κB motif did not influence IL‐8 transactivation by proteasome inhibitors. With DNA binding assays we found a seven‐ to eightfold induction of phosphorylated c‐Jun and hence JNK kinase activity under MG‐132 treatment. Induction of JNK kinase appeared independent of the cell type, even in tumor cell lines not responding to proteasome inhibitors. Since neither inactivation of p53 in wild‐type p53 cells nor reintroduction of functional p53 into p53–/– cells affected MG‐132‐inducible IL‐8 secretion, a direct influence of p53 on IL‐8 regulation could be excluded. These results show that proteasome inhibitors can not only lead to functional AP‐1 induction by enhanced c‐Jun phosphorylation, but also transactivate the IL‐8 gene in human endothelial cells despite complete suppression of NF‐κB activity.

The UBA domains of NUB1L are required for binding but not for accelerated degradation of the ubiquitin-like modifier FAT10.

Proteins selected for degradation are labeled with multiple molecules of ubiquitin and are subsequently cleaved by the 26 S proteasome. A family of proteins containing at least one ubiquitin-associated (UBA) domain and one ubiquitin-like (UBL) domain have been shown to act as soluble ubiquitin receptors of the 26 S proteasome and introduce a new level of specificity into the degradation system. They bind ubiquitylated proteins via their UBA domains and the 26 S proteasome via their UBL domain and facilitate the contact between substrate and protease. NEDD8 ultimate buster-1 long (NUB1L) belongs to this class of proteins and contains one UBL and three UBA domains. We recently reported that NUB1L interacts with the ubiquitin-like modifier FAT10 and accelerates its degradation and that of its conjugates. Here we show that a deletion mutant of NUB1L lacking the UBL domain is still able to bind FAT10 but not the proteasome and no longer accelerates FAT10 degradation. A version of NUB1L lacking all three UBA domains, on the other hand, looses the ability to bind FAT10 but is still able to interact with the proteasome and accelerates the degradation of FAT10. The degradation of a FAT10 mutant containing only the C-terminal UBL domain is also still accelerated by NUB1L, even though the two proteins do not interact. In addition, we show that FAT10 and either one of its UBL domains alone can interact directly with the 26 S proteasome. We propose that NUB1L not only acts as a linker between the 26 S proteasome and ubiquitin-like proteins, but also as a facilitator of proteasomal degradation.

Proteotoxic stress and ageing triggers the loss of redox homeostasis across cellular compartments

The cellular proteostasis network integrates the protein folding and clearance machineries in multiple sub‐cellular compartments of the eukaryotic cell. The endoplasmic reticulum (ER) is the site of synthesis and folding of membrane and secretory proteins. A distinctive feature of the ER is its tightly controlled redox homeostasis necessary for the formation of inter‐ and intra‐molecular disulphide bonds. Employing genetically encoded in vivo sensors reporting on the redox state in an organelle‐specific manner, we show in the nematode Caenorhabditis elegans that the redox state of the ER is subject to profound changes during worm lifetime. In young animals, the ER is oxidizing and this shifts towards reducing conditions during ageing, whereas in the cytosol the redox state becomes more oxidizing with age. Likewise, the redox state in the cytosol and the ER change in an opposing manner in response to proteotoxic challenges in C. elegans and in HeLa cells revealing conservation of redox homeostasis. Moreover, we show that organelle redox homeostasis is regulated across tissues within C. elegans providing a new measure for organismal fitness.