Chemical and biological approaches for adapting proteostasis to ameliorate protein misfolding and aggregation diseases: progress and prognosis

Abstract

Maintaining the proteome to preserve the health of an organism in the face of developmental changes, environmental insults, infectious diseases, and rigors of aging is a formidable task. The challenge is magnified by the inheritance of mutations that render individual proteins subject to misfolding and/or aggregation. Maintenance of the proteome requires the orchestration of protein synthesis, folding, degradation, and trafficking by highly conserved/deeply integrated cellular networks. In humans, no less than 2000 genes are involved. Stress sensors detect the misfolding and aggregation of proteins in specific organelles and respond by activating stress-responsive signaling pathways. These culminate in transcriptional and posttranscriptional programs that up-regulate the homeostatic mechanisms unique to that organelle. Proteostasis is also strongly influenced by the general properties of protein folding that are intrinsic to every proteome. These include the kinetics and thermodynamics of the folding, misfolding, and aggregation of individual proteins. We examine a growing body of evidence establishing that when cellular proteostasis goes awry, it can be reestablished by deliberate chemical and biological interventions. We start with approaches that employ chemicals or biological agents to enhance the general capacity of the proteostasis network. We then introduce chemical approaches to prevent the misfolding or aggregation of specific proteins through direct binding interactions. We finish with evidence that synergy is achieved with the combination of mechanistically distinct approaches to reestablish organismal proteostasis.

Figure 1.

Energy landscape perspective on protein folding. In this view, individual positions in the X-Y-planes correspond to different protein conformations, which diminish in number as the polypeptide chain forms increasing numbers of native intrachain hydrophobic and electrostatic contacts, lowering the internal free energy as the protein approaches its native state conformational ensemble along the Z-axis. (A) A smooth folding funnel reveals the numerous pathways that a polypeptide chain can take to reach the folded conformational ensemble, reflected by the arrows moving down the folding free energy diagram. (B) A rougher folding free energy landscape also indicates that multiple parallel paths can be followed to reach the native state ensemble, however occasionally the polypeptide chain can get kinetically trapped in a folding intermediate (indicated by the red arrow).

Figure 2.

A combined energy landscape for protein folding vs. aggregation. A slice through a rough folding free energy landscape diagram (in yellow) of the type depicted in Figure 1, demonstrating that the population of a folding intermediate at high enough concentration can lead to the formation of aggregate structures (red arrow) having distinct structures and energies, some of which are more stable than the native state. The depicted intermediate could also be populated from conformational excursions from the native state ensemble. The aggregation free energy landscape (in red) is much rougher than the folding free energy diagram. (Figure adapted from Jahn and Radford [2008] and reprinted, with permission, from Elsevier © 2008.)

Figure 3.

The kinetic competition between unimolecular folding, unimolecular misfolding and concentration dependent aggregation is strongly influenced by the proteostasis network. Maximizing unimolecular folding and function by minimizing misfolding and aggregation is a main role of the protein homeostasis network. The proteostasis network is a compilation of integrated and competitive biological pathways that influence the balance between folding, trafficking and degradation, activities depicted by arrows b,d,e,f,g,h, and i. Proteostasis network pathways include ribosome-mediated protein synthesis, chaperone and enzyme mediated folding, lysosome and proteasome-mediated protein degradation, and vesicular trafficking. (Figure adapted from Balch et al. [2008] and reprinted, with permission, from the American Association for the Advancement of Science.)

Figure 4.

The mammalian heat shock response stress responsive signaling pathway matches proteostasis network capacity with demand in the cytosol. The heat shock response is turned on by Hsp90 being recruited away from the transcription factor HSF1 to deal with aggregation and/or misfolding. This allows the HSF1 transcription factor to trimerize and be phosphorylated, which initiates transcription of genes harboring the heat shock response element (HSE). Phosphatases and acetyl transferase enzymes negatively regulate the heat shock response, as does Hsp90 expression that rebinds HSF1.

Figure 5.

Reduced insulin growth factor signal enhances cytosolic proteostasis network capacity by activating the heat shock response and foxo signaling. The binding of a currently undefined ligand to the insulin/insulin-like growth factor receptor DAF-2 in C. elegans triggers insulin/insulin-like growth factor (IGF-1) signaling that negatively regulates the HSF-1 transcription factor and the FOXO transcription factor, DAF-16. Thus, reduced IGFR signaling permits increased HSF-1 and DAF-16 signaling which enhances proteostasis network capacity while also strongly influencing metabolism.

Figure 6.

Tau degradation vs. accumulation by tuning the Hsp70–Hsp40–nucleotide exchange factor pathway. Triage decisions involving the Hsp70–Hsp40 complex can be “tuned” using small molecules. By stimulating the Hsp70–Hsp40 interaction and Hsp70 ATPase activity, the stability of an Hsp70 substrate, tau, was increased. Alternatively, blocking the Hsp70–Hsp40 interaction led to ubiquitination and degradation of tau. Figure kindly provided by Jason Gestwicki.

Figure 7.

The three arms of the unfolded protein response stress-responsive signaling pathway. Proteostasis network capacity in the endoplasmic reticulum (ER) is matched to the level of newly synthesized proteins passing through the secretory pathway by the activation of intracellular signaling pathways collectively referred to as the unfolded protein response. The unfolded protein response responds to the accumulation of misfolded proteins within the lumen of the endoplasmic reticulum. Accumulation of unfolded proteins activates signaling pathways in the cytosol via the trans-membrane stress sensor proteins IRE1, ATF6, and PERK. Activation of the unfolded protein response results in translational attenuation of protein synthesis and transcriptional activation of genes regulated by the transcription factors XBP1s, ATF4, and ATF6 resulting from the three signaling arms of the unfolded protein response. (Figure adapted from Wiseman et al. [2010] and reprinted, with permission, from Elsevier © 2010.)

Figure 8.

Small molecule activation of the unfolded protein response improves the folding, trafficking, and function of folding compromised secreted proteins. Small molecule activation of one or more arms of the unfolded protein response stress responsive signaling pathway (Fig. 7) in patient-derived fibroblasts partially restores mutant enzyme folding, trafficking and lysosomal activity in the case of mutated, misfolding-prone enzymes associated with distinct lysosomal storage diseases. Chaperones and folding enzymes, increased in concentration in response to activation of the unfolded protein response, bind to folding intermediates and transition states of proteins undergoing folding, resculpting the folding free energy diagrams of misfolding-prone enzymes so as to maximize the population of the folded ensemble, while minimizing misfolding and aggregation–increasing the concentration of properly folded mutant enzyme that can traffic to the acidic environment of the lysosome, the environment in which these enzymes were evolved to function. (Figure adapted from Mu et al. [2008] and reprinted, with permission, from Elsevier © 2008.)

Figure 9.

Characterization of proteostasis network components used by the client protein cftr using immunoisolation followed by mass spectrometry. The cystic fibrosis transmembrane conductance regulator (CFTR) interactome (panels A and B) was characterized by immunoisolating both wild type and mutant (ΔF508) CFTR followed by characterization of the interacting proteins by MudPIT mass spectrometry. A ΔF508 CFTR folding intermediate in the cytosol appears to be sequestered by the Hsp90 chaperone–Aha1 cochaperone complex leading to endoplasmic reticulum-associated degradation and poor secretion (Panel C: note lack of C band reflecting CFTR on plasma membrane). Reducing the concentration of Aha1 enhances the folding of ΔF508 CFTR by altering the proteostasis network in such a fashion that it can now more efficiently fold ΔF508 CFTR. Figure kindly provided by William E. Balch.

Figure 10.

Prolonging an emergent property of stress responsive signaling. Guanabenz prolongs eIF2α-mediated translational attenuation associated with activation of the PERK arm of the unfolded protein response, enhancing proteostasis by decreasing the protein load on the proteostasis network and increasing the folding enzyme and chaperone–cochaperone stoichiometry relative to that of the client proteins. Guanabenz inhibits the GADD34-mediated negative feedback loop by direct binding to GADD34, the regulatory subunit of the phosphatase, preventing its association with protein phosphatase 1 (PP1), the catalytic subunit of the phosphatase. Importantly, guanabenz does not inhibit the constitutive eIF2α phosphatase CReP-PP1 heterodimer, thus translational attenuation ultimately ceases, just more slowly.

Figure 11.

Endoplasmic reticulum chaperones appear to be regulated by calcium binding—altering ER calcium levels to regulate chaperone function. Endoplasmic reticulum chaperones including calnexin, calreticulin, and Bip (Hsp70) have Ca²⁺ binding sites that appear to be important for regulating the function of these critical chaperones. (Figure kindly provided by Derrick Ong.)

Figure 12.

Pharmacologic chaperone strategy to enhance the folding trafficking and function of misfolding-prone proteins. Pharmacologic chaperone binding stabilizes the folded state of mutant proteins, increasing the population of the folded mutant enzyme ensemble in the endoplasmic reticulum that can engage the trafficking receptor and be trafficked to the lysosome, increasing the mutant enzyme concentration in the lysosome. Mutant enzyme inhibition in the lysosome can be minimized by creating pharmacologic chaperones that bind with much higher affinity at pH 7 than they do at pH 5—the operating pH of the lysosome.

Figure 13.

Kinetic stabilizer strategy to prevent the misfolding and aggregation of transthyretin into amyloid resulting in the degradation of postmitotic tissue. Rate-limiting transthyretin (TTR) tetramer dissociation proceeding through the dimer shown, monomer misfolding and thermodynamically favorable misassembly into a spectrum of aggregate types, including amyloid fibrils, is genetically and pathologically linked to several degenerative diseases that selectively compromise postmitotic tissue, including the heart and the nervous system. Occupancy of one of the thyroxine binding sites at the weaker dimer–dimer interface with a small molecule kinetic stabilizer (shown in black and red CPK view) is sufficient to make the tetramer dissociation barrier insurmountable under physiological conditions, precluding amyloidogenesis, while still allowing tetrameric TTR to function.

Figure 14.

Mechanistically distinct manipulators of the proteostasis network can result in additive or synergistic rescue of proteostasis. Coapplication of a small molecule unfolded protein response activator and a pharmacologic chaperone affords a synergistic rescue of mutant lysosomal enzyme proteostasis. A small molecule unfolded protein response activator resculpts the folding free energy diagram of a mutant enzyme and “pushes” more misfolding-prone mutant enzyme toward the native conformational ensemble, while the pharmacologic chaperone binds to the native state of a mutant lysosomal enzyme, stabilizing it and therefore increasing the concentration of the mutant enzyme•pharmacologic chaperone complex, effectively “pulling” more of the mutant protein toward the native state. Treatment of patient-derived cells with a pharmacologic chaperone and an unfolded protein response activator leads to more folding, trafficking and mutant enzyme function than the sum of the individual treatments.