Inotersen Treatment for Patients with Hereditary Transthyretin Amyloidosis

ATPases associated with diverse cellular activities (AAA+ proteins) are macromolecular machines that convert the chemical energy contained in ATP molecules into powerful mechanical forces to remodel a vast array of cellular substrates, including protein aggregates, macromolecular complexes and polymers. AAA+ proteins have key functionalities encompassing unfolding and disassembly of such substrates in different subcellular localizations and, hence, power a plethora of fundamental cellular processes, including protein quality control, cytoskeleton remodelling and membrane dynamics. Over the past 35 years, many of the key elements required for AAA+ activity have been identified through genetic, biochemical and structural analyses. However, how ATP powers substrate remodelling and whether a shared mechanism underlies the functional diversity of the AAA+ superfamily were uncertain. Advances in cryo-electron microscopy have enabled high-resolution structure determination of AAA+ proteins trapped in the act of processing substrates, revealing a conserved core mechanism of action. It has also become apparent that this common mechanistic principle is structurally adjusted to carry out a diverse array of biological functions. Here, we review how substrate-bound structures of AAA+ proteins have expanded our understanding of ATP-driven protein remodelling.

The molecular principles governing the activity and functional diversity of AAA+ proteins

ClpXP is an ATP-dependent protease in which the ClpX AAA+ motor binds, unfolds, and translocates specific protein substrates into the degradation chamber of ClpP. We present cryo-EM studies of the E. coli enzyme that show how asymmetric hexameric rings of ClpX bind symmetric heptameric rings of ClpP and interact with protein substrates. Subunits in the ClpX hexamer assume a spiral conformation and interact with two-residue segments of substrate in the axial channel, as observed for other AAA+ proteases and protein-remodeling machines. Strictly sequential models of ATP hydrolysis and a power stroke that moves two residues of the substrate per translocation step have been inferred from these structural features for other AAA+ unfoldases, but biochemical and single-molecule biophysical studies indicate that ClpXP operates by a probabilistic mechanism in which five to eight residues are translocated for each ATP hydrolyzed. We propose structure-based models that could account for the functional results.

Structures of the ATP-fueled ClpXP proteolytic machine bound to protein substrate.

The ClpXP degradation machine consists of a hexameric AAA+ unfoldase (ClpX) and a pair of heptameric serine protease rings (ClpP) that unfold, translocate, and subsequently degrade client proteins. ClpXP is an important target for drug development against infectious diseases. Although structures are available for isolated ClpX and ClpP rings, it remains unknown how symmetry mismatched ClpX and ClpP work in tandem for processive substrate translocation into the ClpP proteolytic chamber. Here, we present cryo-EM structures of the substrate-bound ClpXP complex from Neisseria meningitidis at 2.3 to 3.3 A resolution. The structures allow development of a model in which the sequential hydrolysis of ATP is coupled to motions of ClpX loops that lead to directional substrate translocation and ClpX rotation relative to ClpP. Our data add to the growing body of evidence that AAA+ molecular machines generate translocating forces by a common mechanism.

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A processive rotary mechanism couples substrate unfolding and proteolysis in the ClpXP degradation machinery.

Adapting the proteostasis capacity to sustain brain healthspan

Hand-over-hand translocation is emerging as the conserved mechanism by which ATP hydrolysis drives substrate translocation within the classical clade of AAA+ proteins. However, the operating principles of the distantly related HCLR clade, which includes the important quality control protease Lon, remains poorly defined. We determined a cryo-electron microscopy structure of Y. pestis Lon trapped in the act of processing substrate. This structure revealed that sequential ATP hydrolysis and hand-over-hand substrate translocation are conserved in this AAA+ protease. However, Lon processes substrates through a distinct molecular mechanism involving structural features unique to the HCLR clade. Our findings define a previously unobserved translocation mechanism that is likely conserved across HCLR proteins and reveal how fundamentally distinct structural configurations of distantly-related AAA+ enzymes can power hand-over-hand substrate translocation.

https://biorxiv.org/content/biorxiv/early/2019/04/24/617159.full-text.pdf

Distinct Structural Features of the Lon Protease Drive Conserved Hand-over-Hand Substrate Translocation

Systemic amyloidosis represents a class of disorders in which misfolded proteins are secreted by effector organs and deposited as proteotoxic aggregates at downstream target tissues. Despite being well-described clinically, the contribution of effector organs such as the liver to the pathogenesis of these diseases is poorly understood. Here, we utilize a patient-specific induced pluripotent stem cell (iPSC)-based model of hereditary transthyretin (TTR) amyloid disease (ATTR amyloidosis) in order to define the contributions of hepatic cells to the distal proteotoxicity of secreted TTR. To this end, we employ a gene correction strategy to generate isogenic, ATTR amyloidosis patient-specific iPSCs expressing either amyloidogenic or wild-type TTR. We further utilize this gene editing strategy in combination with single cell RNAseq to identify multiple hepatic proteostasis factors, including many components of adaptive unfolded protein response (UPR) signaling pathways, whose expression correlates with the production of destabilized TTR variants in iPSC-derived hepatic cells. We further demonstrate that enhancing ER proteostasis within ATTR amyloidosis iPSC-derived hepatic lineages via stress-independent activation of aforementioned adaptive UPR signaling preferentially reduces the secretion of destabilized amyloidogenic TTR. Together, these results suggest the potential of the liver to chaperone-at-a-distance and impact pathogenesis at downstream target cells in the context of systemic amyloid disease, and further highlight the promise of UPR modulating therapeutics for the treatment of TTR-mediated and other amyloid diseases.

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Amyloidogenic Proteins Drive Hepatic Proteostasis Remodeling in an Induced Pluripotent Stem Cell Model of Systemic Amyloid Disease

Alteration to endoplasmic reticulum (ER) proteostasis is observed on a variety of neurodegenerative diseases associated with abnormal protein aggregation. Activation of the unfolded protein response (UPR) enables an adaptive reaction to recover ER proteostasis and cell function. The UPR is initiated by specialized stress sensors that engage gene expression programs through the concerted action of the transcription factors ATF4, ATF6f, and XBP1s. Although UPR signaling is generally studied as unique linear signaling branches, correlative evidence suggests that ATF6f and XBP1s may physically interact to regulate a subset of UPR-target genes. Here, we designed an ATF6f-XBP1s fusion protein termed UPRplus that behaves as a heterodimer in terms of its selective transcriptional activity. Cell-based studies demonstrated that UPRplus has stronger an effect in reducing the abnormal aggregation of mutant huntingtin and alpha-synuclein when compared to XBP1s or ATF6 alone. We developed a gene transfer approach to deliver UPRplus into the brain using adeno-associated viruses (AAVs) and demonstrated potent neuroprotection in vivo in preclinical models of Parkinson’s and Huntington’s disease. These results support the concept where directing UPR-mediated gene expression toward specific adaptive programs may serve as a possible strategy to optimize the beneficial effects of the pathway in different disease conditions.

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R.L. Wiseman

Papers

Distinct Structural Features of the Lon Protease Drive Conserved Hand-over-Hand Substrate Translocation

Amyloidogenic Proteins Drive Hepatic Proteostasis Remodeling in an Induced Pluripotent Stem Cell Model of Systemic Amyloid Disease

Enforced dimerization between XBP1s and ATF6f enhances the protective effects of the unfolded protein response (UPR) in models of neurodegeneration