Protein homeostasis as a therapeutic target for diseases of protein conformation

Abstract

Protein misfolding and aggregation are widely implicated in an increasing number of human diseases providing for new therapeutic opportunities targeting protein homeostasis (proteostasis). The cellular response to proteotoxicity is highly regulated by stress signaling pathways, molecular chaperones, transport and clearance machineries that function as a proteostasis network (PN) to protect the stability and functional properties of the proteome. Consequently, the PN is essential at the cellular and organismal level for development and lifespan. However, when challenged during aging, stress, and disease, the folding and clearance machineries can become compromised leading to both gain-of-function and loss-of-function proteinopathies. Here, we assess the role of small molecules that activate the heat shock response, the unfolded protein response, and clearance mechanisms to increase PN capacity and protect cellular proteostasis against proteotoxicity. We propose that this strategy to enhance cell stress pathways and chaperone activity establishes a cytoprotective state against misfolding and/or aggregation and represents a promising therapeutic avenue to prevent the cellular damage associated with the variety of protein conformational diseases.

Fig. 1

Pathways and modifiers influencing proteostasis. Genetic, epigenetic, physiological and environmental stressors affect proteostasis and cause the accumulation of misfunctional proteins. Small molecule modulators of the activities of the proteostasis network pathways (small molecule proteostasis regulators) facilitate chaperone-mediate refolding and/or induce the degradation of misfolded and damaged proteins therefore rebalancing cellular proteostasis. In parenthesis are indicated some of the genes responsible for proteostasis maintenance.

Fig. 2

The heat shock response (HSR) pathway. The mammalian HSR is governed by the transcription factor HSF-1. In absence of stress, chaperones maintain HSF-1 as an inert monomer. With the appearance of misfolded proteins in the cytoplasm, chaperones are titrated away from HSF-1 (1) to deal with misfolding and aggregation. This allows HSF-1 to trimerize (2), bind to its consensus sequence (3) and be post-translational modified (4) therefore inducing the transcription of chaperones and heat shock proteins (hsps) (5). HSF-1 activation pathway is negatively regulated by the newly expressed chaperones and hsps, which rebind HSF-1 (6), and by acetyl transferases (HATs) (7). SIRT1 positively regulates HSF1 DNA-binding activity (7).

Fig. 3

The unfolded protein response (UPR) pathway and its chemical modulators. Accumulation of misfolded proteins in the ER lumen activate the three UPR signal transducers ATF6, PERK and IRE1. This results in the production of transcription factors that migrate into the nucleus and activate UPR target genes that cause attenuation of protein synthesis and increase both the ER folding-capacity and ER-associated degradation.

Fig. 4

ER-associated degradation (ERAD) and its chemical modulators. Proteins entering the endoplasmic reticulum (ER) are immediately recognized by BiP and often modified by the addition of a GlcNAc₂-Man₉-Glc₃ glycan. Glucosidases I and II sequentially remove two terminal glucoses (G) from the glycan and generate monoglucosylated substrates that are recognized by calnexin and calreticulin (calreticulin is a soluble protein and is not shown), which facilitate substrate folding. Once the substrate is released from the calnexin–calreticulin cycle, glucosidase II trims the last glucose. Proteins that have adopted their native conformation are demannosylated by mannosidases I and II and exit the ER. If proteins have not been folded properly, they re-enter the calnexin–calreticulin cycle. Such proteins are reglucosylated by UDP-glucose:glycoprotein glucosyltransferase (UGGT), which promotes re-entry into the folding cycle. Terminally misfolded proteins are processed by mannosidase I and then targeted for ERAD with the participation, through an undetermined mechanism, of the ER degradation-enhancing -mannosidase-like lectins (EDEM). Retrotranslocation of the misfolding-prone substrate to the cytoplasm is mediated by p97 complex. The small molecule ERAD inhibitors kifunesine and Eeyarestatin I block different steps of the ERAD pathways.

Fig. 5

Autophagy, the ubiquitin proteasome system (UPS) and their chemical modulators. Both mTOR-dependent and -independent pathways induce autophagy, the main degradation pathway for aggregate-prone proteins. One of the major pathways that regulate mTOR in mammalian cells is the PI3K–Akt pathway, which is triggered by the binding of insulin growth factors to its receptor (IR). An mTOR-independent mechanism instead involves G protein-coupled receptors, which regulate intracellular inositol and inositol 1,4,5-trisphosphate (IP₃) levels. Inositol and IP₃ are negative regulators of autophagy. Induction of autophagy involves the formation of a phagophore, a double-membrane structure that sequesters aggregated proteins, thus creating an autophagosome. Autophagosomes fuse with lysosome to form autolysosomes in which lysosomal hydrolases degrade their content. Chaperone-mediated autophagy (CMA) instead targets cytosolic proteins to the lysosome surface where they bind to the transmembrane protein lysosome-associated membrane protein type 2A (LAMP-2A). LAMP-2A mediates the protein translocation across the lysosomal membrane for degradation. Unfolded and misfolded proteins that cannot be re-folded correctly by molecular chaperones are polyubiquitinated and then targeted for degradation by the proteasome. SV, sodium valproate; CA, carbamazepine.

Fig. 6

Modulation of ER calcium levels by small molecules alters mutant protein-chaperone interaction and restores proteostasis. The activity of ER-resident chaperones are regulated by calcium. Increased ER calcium concentration is beneficial for those misfolded-prone proteins (such as those involved in different lysosomal storage diseases), which require chaperone-assisted folding (left panel). On the contrary, a reduction in ER calcium levels allows ΔF508CFTR to escape from chaperone-mediated proteasomal degradation (right panel). The question mark indicates that the original findings on thapsigargin and curcumin were not reproducible.

Fig. 7

The Hsp70 chaperone cycle and chemical modulators of its activity. (1) Binding of ATP to the nucleotide binding domain (NBD) of Hsp70 causes the lid to be in an open state, which has a weak affinity for peptide substrates. When a peptide binds to the Hsp70 substrate binding domain (SBD) (2), ATP is hydrolyzed and this event leads to a conformational change that causes the lid closure (3) and increases the affinity of Hsp70 for the substrate. J-domain containing co-chaperones, such as Hsp40, increase the ATPase activity of Hsp70. Replacement of ADP with ATP is required for the release of the folded substrate (4). Small molecules alter Hsp70 functional activity by interfering with different regions of the chaperone. DSG, 15-deoxyspergualin; SGL, 3′-sulfogalactolipid; MB, methylene blue; MY, myricetin.