Poster Details

Presented by Carly Pontifex, PhD candidate, University of Calgary, Canada

The Multisystem Proteinopathy (MSP) phenotype can arise from variants in several genes including HNRNPA2B1, HNRNPA1, MATR3, VCP, SQSTM1, and OPTN. Since impairments in autophagy and stress granule regulation are a common to all MSP genes, we hypothesized that two MSP mutants VCP and SQSTM1 with TIA1 modifier variants will share similarities in their autophagic and stress granule deficits. We studied stress granule dynamics, autophagic flux, stress granule loading into autophagosomes, number of autophagosomes, lysosome volume and lysosome intensity. While VCP and SQSTM1 both had aberrant stress granule dynamics and lysosomal accumulation in response to arsenite stress, they differed in autophagosome accumulation, autophagic flux, and lysosomal localization. We found that VCP has enhanced autophagic flux, while SQSTM1 had impaired autophagic flux. We also ask why TIA1 leads to a distal phenotype, with preliminary findings showing that the TIA1b isoform may be the predominant isoform contributing to pathogenesis with the TIA1b isoform stress granules colocalizing poorly to SQSTM1 while also being more expressed in distal muscle.

Presented by Carly Pontifex, PhD candidate, University of Calgary, Canada

In this review, we examine the functionally diverse AAA-ATPase, valosin-containing protein (VCP/p97), its molecular functions, the mutational landscape of VCP, and the phenotypic manifestation of VCP disease. VCP is crucial to a multitude of cellular functions including protein quality control, ERAD, autophagy, mitophagy, lysophagy, stress granule formation and clearance, DNA replication and mitosis, DNA damage response including nucleotide excision repair, ATM and ATR mediated damage response, homologous repair and non-homologous end joining. VCP variants cause multisystem proteinopathy, and pathology can arise in several tissue types such as skeletal muscle, bone, brain, motor neurons and sensory neurons and possibly cardiac muscle, with the disease course being challenging to predict.

Presented by Chad Altobelli, UCSF

VCP/p97 is a homohexameric AAA-ATPase that is ubiquitously expressed in human cells. VCP acts as a molecular motor to unfold proteins through its central pore, thereby making them available for downstream processing. There are at least 32 proteins, termed adaptors, that bind VCP directly to provide substrate specificity and control subcellular localization. One such adaptor, UBXD1, is structurally unique because it binds both the N- terminal and C-terminal domains of VCP. Biochemical assays indicate that UBXD1 inhibits the basal ATPase activity of VCP with a low nM IC50. To better understand this interaction, we determined a high resolution cyro- EM structure of full length UBXD1 in complex with VCP. The structure reveals that beyond the two predicted contacts, UBXD1 also wraps around the N-domain of VCP using a lariat-like motif and wedges itself between the D1 and D2 domains of VCP. Mutational analysis suggests that while each UBXD1 domain contributes towards VCP inhibition, most inhibition stems from the C-terminal half of the protein that includes a lariat structure anchored to the bottom of VCP. This engagement ultimately results in a shallow split- washer conformation of the hexamer and substantial separation between two protomers bridged by UBXD1. This unique complex may be important for substrate engagement or processing.

Presented by Alex Long, UCSF

VCP/p97 is a AAA+ ATPase that serves critical functions as a segregase in a diverse set of cellular processes. Familial mutations in VCP are associated with multisystem proteinopathies and neurodegeneration. VCP requires a network of more than 30 adapter proteins to modulate activity and engage protein substrates. Many disease mutants increase ATP hydrolysis and potentially alter its adapter interactions and conformational cycle, thus the development of small molecule therapeutics that restore wild type hydrolysis levels are of substantial interest. Our focus is to understand how VCP structural states and ATPase activity are modulated by disease mutations, small molecules, and adapter interactions. We have previously investigated the multi-domain adapter UBXD1, solving the structure of the UBXD1-VCP complex and identifying it as a potent inhibitor of VCP ATPase activity; however, the molecular mechanisms of this inhibition are not well understood. To dissect the contributions of adapter interactions in hydrolysis control, we are studying the small VCP-interacting protein (SVIP), another inhibitory adapter that contains a VIM-helix motif structure similar to what we identify in UBXD1. Here, I present structures of VCP-SVIP complexes and ATPase data of various SVIP mutations that advance understanding of the mechanisms of adapter-mediated VCP inhibition.

*YOUNG INVESTIGATOR AWARD WINNER*

Presented by Eileen Lynch PhD, from the Weihl lab, department of Neurology, Washington University

Valosin-containing protein (VCP) is critical for the maintenance of proteostasis, with roles in endocytosis, autophagy, the proteasome, and many other cellular processes. Mutations in VCP can lead to a spectrum of diseases including frontotemporal dementia (FTD), amyotrophic lateral sclerosis (ALS), and inclusion body myopathy (IBM). Cytoplasmic aggregates of the normally nuclear-localized protein TDP-43 are a characteristic feature of pathology in both the brains of patients with FTD/ALS and the muscles of patients with IBM. TDP-43 is an RNA-binding protein that in certain disease conditions behaves in a prion-like manner, forming insoluble aggregates and inducing other TDP-43 monomers to misfold and aggregate, a process referred to as seeding. Previous work from our lab has demonstrated that VCP inhibition or knockdown facilitates TDP-43 seeding in primary mouse cortical neurons. More recently we have also developed a seeding assay that will be used to determine differences in TDP-43 seeding in WT versus VCP patient iPSC-derived motor neurons. The same WT and VCP patient iPSC lines were differentiated into skeletal myocytes. At baseline, VCP patient myocytes have increased insoluble TDP-43 which further increases with arsenite-induced stress. Additionally, mouse C2C12 differentiated myotubes stressed with arsenite and then treated with a VCP inhibitor during recovery had delayed clearance of insoluble TDP-43. To study TDP-43 aggregation in skeletal muscle in vivo, we developed a transgenic mouse line with doxycycline-inducible cytoplasmic mislocalized human TDP-43 specific to skeletal muscle (HSA-hTDP-43ΔNLS). These mice form abundant sarcoplasmic TDP-43 insoluble aggregates when the transgene expression is activated. When the transgene is turned off, insoluble TDP-43 is cleared from muscle but TDP-43 aggregate seeding persists. When the HSA-hTDP-43ΔNLS mice were crossed with VCPR155H/WT mice, the resulting mice have increased persistence of insoluble TDP-43 but decreased seed generation during recovery. We hypothesize that regular VCP function is involved in the clearance of insoluble TDP-43 aggregates but this process may generate more seeding-competent intermediates initially. Together, these studies support a role for VCP in the seeding and clearance of TDP-43 aggregates in both neurons and muscle, suggesting similar mechanisms between the tissue types that could be targeted therapeutically.

This poster will be presented at the YIA oral session on February 24

Presented by Deirdre Mack, PhD candidate, University of Utah

Cells must maintain a balance between generating, folding, transporting, and degrading proteins in order to maintain proper protein homeostasis, or proteostasis. A central player in the maintenance of mammalian proteostasis is VCP (also known as p97 or Cdc48), a AAA+ ATPase (ATPase associated with diverse cellular activities) that leverages the power of ATP hydrolysis to pull ubiquitinated substrates from a variety of organelles and unfold them before proteasomal degradation. Mutations in VCP can lead to diseases associated with dysregulation of proteostasis; thus, while it is known that VCP is critical to cellular health, much about its mechanism remains unknown. In general, VCP must bind, translocate, and release the unfolded substrate. Each of these steps is dependent on VCP’s interactions with multiple binding partners, yet how these interactions are coordinated has not been fully characterized. Although it is well established that VCP complexes bind and process ubiquitinated substrates, it has also been shown that VCP cannot release these substrates without the help of another binding partner: Otu1. Otu1 is a deubiquitinase (DUB) that interacts with VCP and trims ubiquitin moieties on substrates to allow for efficient unfolding and release. Yet, how this trimming occurs during substrate unfolding remains an open question. To elucidate how VCP is regulated by Otu1 and how Otu1 interacts with polyubiquinated substrate, I aim to determine a high-resolution structure of the VCP-Otu1 complex with polyubiquitinated substrate via cryo-EM and an unfolding assay. In order to capture VCP bound to Otu1 while in the process of deubiquitinating substrate, I have reconstituted active complexes in vitro using purified VCP hexamers, the Otu1 DUB in its wild-type or catalytically inactive form (Otu1C120S), the substrate recruiting heterodimer Ufd1/Npl4, and a fluorescent polyubiquitinated substrate. To determine how Otu1 influences the rate of unfolding activity of VCP I use the same components (Otu1, VCP, UN, and substrate) in a fluorescence based unfolding activity assay. This work will elucidate how polyubiquitinated substrates are deubiquitinated, will construct a more complete understanding of how VCP processes its substrates, and has implications for the development of therapeutics for degenerative diseases associated with failure in protein quality control.

Presented by Dale Martin, PhD, University of Waterloo, Canada

Protein mislocalization is one of the first steps in neurodegeneration, leading to proteostasis deficiencies and cell death, typically linked to protein aggregation. Thus, defining the mechanisms that regulate subcellular protein localization is critical for understanding cell biology and disease progression, as well as for developing effective therapeutics targeted to the earliest stages in disease pathogenesis. The pleiotropic effects combined with the disconnect between genotype and phenotype suggest modifiers may play a role in Multisystem proteinopathy (MSP). We have identified palmitoylation of VCP and one of its co-factors, small VCP-interacting protein (SVIP), as a novel disease modifier in MSP. Palmitoylation involves the reversible addition of the fatty acid palmitate to cysteine residues and promotes membrane binding, protein-protein interactions, and protein stability. Much like phosphorylation, reversibility is highly dynamic making palmitoylation an emerging drug target. Many disease-causing VCP mutations exist and targeting each mutation separately is difficult and costly. Consequently, identifying a common pathway or target affected by more than one VCP mutation, like palmitoylation, could provide a common therapeutic strategy for VCP diseases and, potentially, other neuronal, muscle, and bone diseases. We recently showed that dynamic palmitoylation acts as a novel regulator of VCP and VCP co-factors in MSP and amyotrophic lateral sclerosis. We have identified two novel pathways that specifically alter VCP localization and regulate mutant VCP toxicity: (i) VCP binding to and palmitoylation by palmitoylating enzymes (ii) in conjunction with fatty acylation of VCP co-factor SVIP. We aim to map the inter-relationships of VCP, SVIP, and their palmitoylating enzymes to understand their complex biological role in VCP cell biology and elucidate VCP dysregulation in MSP disease pathogenesis.

Presented by Carly Pontifex, PhD candidate, University of Calgary, Canada

Multisystem Proteinopathy (MSP) genes can be divided into two types, the RNA-binding splicing regulators that are sequestered into stress granules and ubiquitin-binding autophagy regulators. According to Wheeler et al. 2022, RNA-binding splicing regulators that cause MSP such as HNRNPA1 and HNRNPA2B1 are upregulated during myogenic differentiation. We hypothesize that there is a common mechanism that functionally unites these genes and contributes to the pathogenesis of MSP. We used single nuclear RNA sequencing from frozen muscle biopsies derived from two control patients and four myopathy patients, including a sporadic inclusion body myositis patient and a SQSTM1 MSP patient. When Ingenuity Pathway Analysis was used to analyze the top markers of pre-committed myoblast clusters, results suggested impairments in myogenic differentiation. Patient-derived mt-VCP and mt-SQSTM1 fibroblasts were converted to myoblasts using lentiviral MYOD1 transduction followed by staining with late-stage differentiation marker desmin. VCP and SQSTM1 mutants exhibit impaired myogenic differentiation compared to controls. These results suggest that the pathogenesis of inclusion body myopathy in MSP may be driven not only by accelerated atrophy of myofibers, but also by impaired maturation of myonuclei.

Presented by Maxwell Tucker, graduate student at UCSF.

VCP/p97 is a AAA+ segregase that unfolds and disassembles ubiquitinated macromolecular targets via hydrolysis-driven substrate translocation through a central pore. Mutations in VCP are associated with several diseases, including frontotemporal dementia and ALS. One hallmark of these disease states is the disruption of lysosomal regulation. Intriguingly, VCP and several cofactors have been directly linked to lysosomal regulation through the lysophagy pathway, in which damaged lysosomes are degraded through autophagy. While the necessity of VCP complexes in lysosomal regulation is well established, the precise functional role remains unclear. In this work, we are using in situ cryo-electron tomography (cryo-ET) to study the localization of VCP within the ultrastructure of damaged lysosomes. This technique allows for nanometer-resolution within the native context of a cell. We have developed a pipeline for initiating lysosome damage and targeting VCP-associated damaged lysosomes using correlative fluorescence microscopy and FIB-milling. These samples are used for high resolution cryo-ET collection and 3D structural characterization of VCP-like densities in proximity to damaged membranes. Placing VCP in the cellular ultrastructure of a damaged lysosome will shed new light on its function in this critical pathway.

Presented by Johannes Van Den Boom, PhD, University of Duisburg-Essen, De

The AAA+ ATPase p97 is a major protein unfolding machine with hundreds of clients in diverse cellular pathways that are critical for cell homeostasis, proliferation and signaling. Client proteins are recruited to p97 by at least two types of alternative adapters. The Ufd1-Npl4 adapter along with accessory adapters targets ubiquitylated clients in the majority of pathways and uses ubiquitin as a universal unfolding tag. In contrast, the family of SEP-domain adapters such as p37 can target clients directly to p97 in a ubiquitin-independent manner. We now present a cryo-EM structure of p97 and p37 in the act of disassembling a protein phosphatase-1 (PP1) complex that together with biochemical evidence suggests a hold-and-extract mechanism for PP1 complex disassembly. PP1 is held in an inactive complex with its binding partners SDS22 and inhibitor-3 (I3) during PP1 biogenesis and formation of active PP1 holoenzymes with substrate specifiers requires disassembly of the SDS22-PP1-I3 complex by p97. During the disassembly reaction, SDS22 with PP1 is firmly locked directly onto one of the N-domains of p97. The p37 adapter is located underneath and bridges two N-domains with multivalent interactions to the PP1 complex suggesting that p37 helps position PP1-SDS22 complex. I3 is inserted into the central pore of p97 in active staircase conformation while I3 is still attached to PP1 indicating that I3 is stripped off PP1 by threading I3 through the pore. Our data uncover how p97 releases PP1 from its inhibitory partners for activation and demonstrate a remarkable plasticity in substrate threading by p97.

Presented by Yihong Ye, PhD, Senior Investigator, NIH, NIDDK, Laboratory of Molecular Biology.

In eukaryotic cells, a protein translation surveillance system named ribosome-associated Quality Control (RQC) degrades partially translated products, resolving translation stress caused by prolonged ribosome stalling. Central players of this process include ribosome-associated ubiquitin ligases, which collaborate with the AAA ATPase p97 to target ubiquitinated RQC substrates for proteasomal degradation. While this pathway has been well characterized for ribosomes stalled on mRNAs encoding soluble proteins, little is known about how cells handle ribosomes stalled while making a membrane or secretory proteins at the endoplasmic reticulum (ER), a key protein biogenesis hub of eukaryotic cells. Using two distinct mammalian ER RQC reporters, we performed genome-wide CRISPR knockout screens, which identified players involved in ER RQC processes. Our study delineates two parallel quality control systems: One shuttles a defective RQC substrate from the ER to the lysosome in a p97-independent mechanism, while the other targets an ER RQC substrate to the proteasome via the p97-UFD1-NPL4 complex. Interestingly, both processes require post-translational modification of the large ribosomal subunit RPL26 with a small ubiquitin-like protein named UFM1. While both RQC substrates are released into the ER lumen for transporting to the Golgi, an ER RQC substrate translated from a mRNA with no stop codon is retrieved back to the ER, from where it is retrotranslocated into the cytosol by p97 and degraded by the proteasome. Our study has revealed an unexpected role for p97 in maintaining ER homeostasis by safeguarding protein translocation at the ER membrane.

Speaker at session#1, Structural VCP &Biochemistry, February 23.

Presented by Isabelle Rouiller, PhD, the University of Melbourne, Australia

Single-particle cryo-electron microscopy (cryo-EM) has proven effective in determining the structure of VCP in different conformations including those linked to mutants associated with multisystem proteinopathy development, as well as its interactions with substrates and co-factors [1]. However, most published cryo-EM maps exhibit heterogeneous resolution, leading to ambiguities and unresolved segments within the VCP, VCP/co-factors and VCP/substrate protein complexes. This artifact stems from the average of many images of the complexes needed to retrieve the signal from noisy cryo-EM data, resulting in reduced definition and, at times, elimination of density from flexible regions of the protein. Flexible regions of VCP, such as the indispensable N-domains [2], play pivotal roles in co-factor and substrate recruitment, and their dynamic properties are influenced by disease-causing mutations [3,4]. Regrettably, these regions of critical functional importance often suffer from diminished resolution in cryo-EM studies. Our recently developed image analysis method relies on a 3D-to-2D flexible fitting strategy, enabling the modification of an atomic structure through Molecular Dynamics (MD) simulations while utilizing the cryo-EM images as constraints [5]. This innovative approach facilitated a comprehensive exploration of the conformational dynamics within VCP as captured in the cryo-EM data. Specifically, it enabled the characterization of the entire spectrum of conformations adopted by VCP's N-domains [6] and enabled the detection of novel particle conformations within our cryo-EM dataset. Leveraging this methodology, we conducted a comparative analysis between the dynamic properties of wild-type VCP and VCP bearing the R155P mutation. Additionally, our findings were corroborated through Cross-Linking mass spectrometry (XL-MS), Hydrogen Deuterium Exchange mass spectrometry (HDX-MS) and NMR spectroscopy. Collectively, our methodology enables the analysis of VCP’s diverse conformational landscape embraced by VCP, providing crucial insights for drug discovery. A comprehensive structural characterization of VCP's conformations will aid in identifying novel active sites, designing higher-affinity ligands, and targeting disease-associated conformations or modulating protein function. [1] Valimehr et al. AAA ATPase multifunctional protein, VCP/p97; an important therapeutic target. Biomolecules. 2023 Apr 24;13(5):737. doi: 10.3390/biom13050737. [2] Rouiller, I., et al., Conformational changes of the multifunction p97 AAA ATPase during its ATPase cycle. Nat Struct Biol, 2002. 9(12): p. 950-7. [3] Mountassif et al., Cryo-EM of the pathogenic VCP variant R155P reveals long-range conformational changes in the D2 ATPase ring. Biochem Biophys Res Commun, 2015. [4] Halawani et al., Hereditary inclusion body myopathy-linked p97/VCP mutations in the NH2 domain and the D1 ring modulate p97/VCP ATPase activity and D2 ring conformation. Mol Cell Biol, 2009. 29(16): p. 4484-94. [5] Vuillemot et al., MDSPACE: Extracting Continuous Conformational Landscapes from Cryo-EM Single Particle Datasets Using 3D-to-2D Flexible Fitting based on Molecular Dynamics Simulation. J Mol Biol, 2023: p. 167951. [6] Valimehr et al., Analysis of the conformational landscape of the N-domains of the AAA ATPase: disentangling continuous conformational variability of partially symmetrical complexes. Manuscript under review.

Presented by Mengcheng Wu, PhD Student, Caltech

p97 is an abundant protein that participates in multiple cellular functions, including Endoplasmic-reticulum-associated protein degradation(ERAD), DNA damage response, and autophagy. Binding with the correct cofactor(s) is crucial for p97's involvement in these pathways. Numerous p97 mutations can cause the neurodegenerative disease multisystem proteinopathy type 1(MSP1), characterized by symptoms such as muscle and/or bone weakness. Although it is suspected some cellular functions are impaired due to incorrect cofactor binding, the specific pathogenesis mechanism at the cellular level remains unclear. To address this gap, we developed three p97 mutant cell lines(R155H, K164Q, F267del) on HEK293 cells to express p97 mutants while knocking down endogenous p97. Our primary objectives are to analyze the p97 interactome in the three mutant cell lines using liquid chromatography–mass spectrometry(LC-MS). This analysis aims to determine (1) if there is a common pathogenesis mechanism and (2) to identify the specific mechanism(s) potentially causing the neurodegenerative disease MSP-1.

Presented by Wenyan Li, PhD, University of Utah

The linear chains of amino acids are synthesized on ribosomes, then it must fold into unique three-dimensional structures to obtain their biological functions. The product of truncated polypeptides by stalling of ribosomes during protein synthesis must be eliminated. Ribosome Quality control Complex (RQC) sense the 60S subunit obstructing with the nascent chain then mediates nascent chain degradation to maintain cellular protein homeostasis. In this work, we describe the cryo–electron microscopy structure of the whole RQC complex. Through analyzing the structure, we elucidate the mechanism of how p97/Cdc48 functions in this process.