Zn-regulated GTPase metalloprotein activator 1 modulates vertebrate zinc homeostasis.

Zinc (Zn) is an essential micronutrient and cofactor for up to 10% of proteins in living organisms. During Zn limitation, specialized enzymes called metallochaperones are predicted to allocate Zn to specific metalloproteins. This function has been putatively assigned to G3E GTPase COG0523 proteins, yet no Zn metallochaperone has been experimentally identified in any organism. Here, we functionally characterize a family of COG0523 proteins that is conserved across vertebrates. We identify Zn metalloprotease methionine aminopeptidase 1 (METAP1) as a COG0523 client, leading to the redesignation of this group of COG0523 proteins as the Zn-regulated GTPase metalloprotein activator (ZNG1) family. Using biochemical, structural, genetic, and pharmacological approaches across evolutionarily divergent models, including zebrafish and mice, we demonstrate a critical role for ZNG1 proteins in regulating cellular Zn homeostasis. Collectively, these data reveal the existence of a family of Zn metallochaperones and assign ZNG1 an important role for intracellular Zn trafficking.

RADX controls RAD51 filament dynamics to regulate replication fork stability.

The RAD51 recombinase forms nucleoprotein filaments to promote double-strand break repair, replication fork reversal, and fork stabilization. The stability of these filaments is highly regulated, as both too little and too much RAD51 activity can cause genome instability. RADX is a single-strand DNA (ssDNA) binding protein that regulates DNA replication. Here, we define its mechanism of action. We find that RADX inhibits RAD51 strand exchange and D-loop formation activities. RADX directly and selectively interacts with ATP-bound RAD51, stimulates ATP hydrolysis, and destabilizes RAD51 nucleofilaments. The RADX interaction with RAD51, in addition to its ssDNA binding capability, is required to maintain replication fork elongation rates and fork stability. Furthermore, BRCA2 can overcome the RADX-dependent RAD51 inhibition. Thus, RADX functions in opposition to BRCA2 in regulating RAD51 nucleofilament stability to ensure the right level of RAD51 function during DNA replication.

EXO5-DNA structure and BLM interactions direct DNA resection critical for ATR-dependent replication restart.

Stalled DNA replication fork restart after stress as orchestrated by ATR kinase, BLM helicase, and structure-specific nucleases enables replication, cell survival, and genome stability. Here we unveil human exonuclease V (EXO5) as an ATR-regulated DNA structure-specific nuclease and BLM partner for replication fork restart. We find that elevated EXO5 in tumors correlates with increased mutation loads and poor patient survival, suggesting that EXO5 upregulation has oncogenic potential. Structural, mechanistic, and mutational analyses of EXO5 and EXO5-DNA complexes reveal a single-stranded DNA binding channel with an adjacent ATR phosphorylation motif (T88Q89) that regulates EXO5 nuclease activity and BLM binding identified by mass spectrometric analysis. EXO5 phospho-mimetic mutant rescues the restart defect from EXO5 depletion that decreases fork progression, DNA damage repair, and cell survival. EXO5 depletion furthermore rescues survival of FANCA-deficient cells and indicates EXO5 functions epistatically with SMARCAL1 and BLM. Thus, an EXO5 axis connects ATR and BLM in directing replication fork restart.

Structure of RADX and mechanism for regulation of RAD51 nucleofilaments.

Replication fork reversal is a fundamental process required for resolution of encounters with DNA damage. A key step in the stabilization and eventual resolution of reversed forks is formation of RAD51 nucleoprotein filaments on exposed single strand DNA (ssDNA). To avoid genome instability, RAD51 filaments are tightly controlled by a variety of positive and negative regulators. RADX (RPA-related RAD51-antagonist on the X chromosome) is a recently discovered negative regulator that binds tightly to ssDNA, directly interacts with RAD51, and regulates replication fork reversal and stabilization in a context-dependent manner. Here, we present a structure-based investigation of RADX's mechanism of action. Mass photometry experiments showed that RADX forms multiple oligomeric states in a concentration-dependent manner, with a predominance of trimers in the presence of ssDNA. The structure of RADX, which has no structurally characterized orthologs, was determined ab initio by cryo-electron microscopy (cryo-EM) from maps in the 2 to 4 Å range. The structure reveals the molecular basis for RADX oligomerization and the coupled multi-valent binding of ssDNA binding. The interaction of RADX with RAD51 filaments was imaged by negative stain EM, which showed a RADX oligomer at the end of filaments. Based on these results, we propose a model in which RADX functions by capping and restricting the end of RAD51 filaments.

Application of a quantitative framework to improve the accuracy of a bacterial infection model.

Laboratory models are critical to basic and translational microbiology research. Models serve multiple purposes, from providing tractable systems to study cell biology to allowing the investigation of inaccessible clinical and environmental ecosystems. Although there is a recognized need for improved model systems, there is a gap in rational approaches to accomplish this goal. We recently developed a framework for assessing the accuracy of microbial models by quantifying how closely each gene is expressed in the natural environment and in various models. The accuracy of the model is defined as the percentage of genes that are similarly expressed in the natural environment and the model. Here, we leverage this framework to develop and validate two generalizable approaches for improving model accuracy, and as proof of concept, we apply these approaches to improve models of Pseudomonas aeruginosa infecting the cystic fibrosis (CF) lung. First, we identify two models, an in vitro synthetic CF sputum medium model (SCFM2) and an epithelial cell model, that accurately recapitulate different gene sets. By combining these models, we developed the epithelial cell-SCFM2 model which improves the accuracy of over 500 genes. Second, to improve the accuracy of specific genes, we mined publicly available transcriptome data, which identified zinc limitation as a cue present in the CF lung and absent in SCFM2. Induction of zinc limitation in SCFM2 resulted in accurate expression of 90% of P. aeruginosa genes. These approaches provide generalizable, quantitative frameworks for microbiological model improvement that can be applied to any system of interest.

Molecular basis and functional consequences of the interaction between the base excision repair DNA glycosylase NEIL1 and RPA.

NEIL1 is a DNA glycosylase that recognizes and initiates base excision repair of oxidized bases. The ubiquitous ssDNA binding scaffolding protein, replication protein A (RPA), modulates NEIL1 activity in a manner that depends on DNA structure. Interaction between NEIL1 and RPA has been reported, but the molecular basis of this interaction has yet to be investigated. Using a combination of NMR spectroscopy and isothermal titration calorimetry (ITC), we show that NEIL1 interacts with RPA through two contact points. An interaction with the RPA32C protein recruitment domain was mapped to a motif in the common interaction domain (CID) of NEIL1 and a dissociation constant (Kd) of 200 nM was measured. A substantially weaker secondary interaction with the tandem RPA70AB ssDNA binding domains was also mapped to the CID. Together these two contact points reveal NEIL1 has a high overall affinity (Kd ∼ 20 nM) for RPA. A homology model of the complex of RPA32C with the NEIL1 RPA binding motif in the CID was generated and used to design a set of mutations in NEIL1 to disrupt the interaction, which was confirmed by ITC. The mutant NEIL1 remains catalytically active against a thymine glycol lesion in duplex DNA in vitro. Testing the functional effect of disrupting the NEIL1-RPA interaction in vivo using a Fluorescence Multiplex-Host Cell Reactivation (FM-HCR) reporter assay revealed an unexpected role for NEIL1 in nucleotide excision repair. These findings are discussed in the context of the role of NEIL1 in replication-associated repair.

RADX Promotes Genome Stability and Modulates Chemosensitivity by Regulating RAD51 at Replication Forks.

RAD51 promotes homology-directed repair (HDR), replication fork reversal, and stalled fork protection. Defects in these functions cause genomic instability and tumorigenesis but also generate hypersensitivity to cancer therapeutics. Here we describe the identification of RADX as an RPA-like, single-strand DNA binding protein. RADX is recruited to replication forks, where it prevents fork collapse by regulating RAD51. When RADX is inactivated, excessive RAD51 activity slows replication elongation and causes double-strand breaks. In cancer cells lacking BRCA2, RADX deletion restores fork protection without restoring HDR. Furthermore, RADX inactivation confers chemotherapy and PARP inhibitor resistance to cancer cells with reduced BRCA2/RAD51 pathway function. By antagonizing RAD51 at forks, RADX allows cells to maintain a high capacity for HDR while ensuring that replication functions of RAD51 are properly regulated. Thus, RADX is essential to achieve the proper balance of RAD51 activity to maintain genome stability.

Two interaction surfaces between XPA and RPA organize the preincision complex in nucleotide excision repair.

The xeroderma pigmentosum protein A (XPA) and replication protein A (RPA) proteins fulfill essential roles in the assembly of the preincision complex in the nucleotide excision repair (NER) pathway. We have previously characterized the two interaction sites, one between the XPA N-terminal (XPA-N) disordered domain and the RPA32 C-terminal domain (RPA32C), and the other with the XPA DNA binding domain (DBD) and the RPA70AB DBDs. Here, we show that XPA mutations that inhibit the physical interaction in either site reduce NER activity in biochemical and cellular systems. Combining mutations in the two sites leads to an additive inhibition of NER, implying that they fulfill distinct roles. Our data suggest a model in which the interaction between XPA-N and RPA32C is important for the initial association of XPA with NER complexes, while the interaction between XPA DBD and RPA70AB is needed for structural organization of the complex to license the dual incision reaction. Integrative structural models of complexes of XPA and RPA bound to single-stranded/double-stranded DNA (ss/dsDNA) junction substrates that mimic the NER bubble reveal key features of the architecture of XPA and RPA in the preincision complex. Most critical among these is that the shape of the NER bubble is far from colinear as depicted in current models, but rather the two strands of unwound DNA must assume a U-shape with the two ss/dsDNA junctions localized in close proximity. Our data suggest that the interaction between XPA and RPA70 is key for the organization of the NER preincision complex.

Comparative analysis of the physical properties of murine and human S100A7: Insight into why zinc piracy is mediated by human but not murine S100A7.

S100 proteins are a subfamily of EF-hand calcium-binding proteins found primarily in vertebrate animals. They are distinguished by binding of transition metals and functioning in both the intracellular and extracellular milieu. S100A7 functions in the protection of the skin and mucous membranes and is a biomarker in inflammatory skin disease. A recent study of Neisseria gonorrhoeae infection revealed that human but not murine S100A7 could be used to evade host nutritional immunity. To understand the molecular basis for this difference, we carried out a comparative analysis of the physical and structural properties of human and murine S100A7. The X-ray crystal structure of Ca2+-loaded mouse S100A7 (mS100A7) was determined to 1.69 Å resolution, and Ca2+-induced conformational changes were assessed by NMR. Unlike human S100A7 (hS100A7), which exhibits conformational changes in response to binding of Ca2+, no significant changes in mS100A7 were detected. Dynamic light scattering, circular dichroism, and a competition chelator assay were used to compare the Zn2+ affinity and the effects of ion binding on mS100A7 versus hS100A7. Alignment of their sequences revealed a substantial difference in the C-terminal region, which is an important mediator of protein-protein interactions, suggesting a rationale for the specificity of N. gonorrhoeae for hS100A7. These data, along with more detailed analysis of S100A7 sequence conservation across different species, support the proposal that, although hS100A7 is highly conserved in many mammals, the murine protein is a distinct ortholog. Our results highlight the potential limitations of using mouse models for studying bacterial infections in humans.

Promotion of BRCA2-Dependent Homologous Recombination by DSS1 via RPA Targeting and DNA Mimicry.

The tumor suppressor BRCA2 is thought to facilitate the handoff of ssDNA from replication protein A (RPA) to the RAD51 recombinase during DNA break and replication fork repair by homologous recombination. However, we find that RPA-RAD51 exchange requires the BRCA2 partner DSS1. Biochemical, structural, and in vivo analyses reveal that DSS1 allows the BRCA2-DSS1 complex to physically and functionally interact with RPA. Mechanistically, DSS1 acts as a DNA mimic to attenuate the affinity of RPA for ssDNA. A mutation in the solvent-exposed acidic domain of DSS1 compromises the efficacy of RPA-RAD51 exchange. Thus, by targeting RPA and mimicking DNA, DSS1 functions with BRCA2 in a two-component homologous recombination mediator complex in genome maintenance and tumor suppression. Our findings may provide a paradigm for understanding the roles of DSS1 in other biological processes.

Modification of the 4Fe-4S Cluster Charge Transport Pathway Alters RNA Synthesis by Yeast DNA Primase.

DNA synthesis during replication begins with the generation of an ∼10-nucleotide primer by DNA primase. Primase contains a redox-active 4Fe-4S cluster in the C-terminal domain of the p58 subunit (p58C). The redox state of this 4Fe-4S cluster can be modulated via the transport of charge through the protein and the DNA substrate (redox switching); changes in the redox state of the cluster alter the ability of p58C to associate with its substrate. The efficiency of redox switching in p58C can be altered by mutating tyrosine residues that bridge the 4Fe-4S cluster and the nucleic acid binding site. Here, we report the effects of mutating bridging tyrosines to phenylalanines in yeast p58C. High-resolution crystal structures show that these mutations, even with six tyrosines simultaneously mutated, do not perturb the three-dimensional structure of the protein. In contrast, measurements of the electrochemical properties on DNA-modified electrodes of p58C containing multiple tyrosine to phenylalanine mutations reveal deficiencies in their ability to engage in DNA charge transport. Significantly, this loss of electrochemical activity correlates with decreased primase activity. While single-site mutants showed modest decreases in activity compared to that of the wild-type primase, the protein containing six mutations exhibited a 10-fold or greater decrease. Thus, many possible tyrosine-mediated pathways for charge transport in yeast p58C exist, but inhibiting these pathways together diminishes the ability of yeast primase to generate primers. These results support a model in which redox switching is essential for primase activity.

Neutrophil-Associated Responses to Vibrio cholerae Infection in a Natural Host Model.

Vibrio cholerae, the cause of human cholera, is an aquatic bacterium found in association with a variety of animals in the environment, including many teleost fish species. V. cholerae infection induces a proinflammatory response followed by a noninflammatory convalescent phase. Neutrophils are integral to this early immune response. However, the relationship between the neutrophil-associated protein calprotectin and V. cholerae has not been investigated, nor have the effects of limiting transition metals on V. cholerae growth. Zebrafish are useful as a natural V. cholerae model as the entire infectious cycle can be recapitulated in the presence of an intact intestinal microbiome and mature immune responses. Here, we demonstrate that zebrafish produce a significant neutrophil, interleukin 8 (IL-8), and calprotectin response following V. cholerae infection. Bacterial growth was completely inhibited by purified calprotectin protein or the chemical chelator N,N,N',N'-tetrakis(2-pyridinylmethyl)-1,2-ethanediamine (TPEN), but growth was recovered by the addition of the transition metals zinc and manganese. The expression of downstream calprotectin targets was also significantly increased in the zebrafish. These findings illuminate the role of host calprotectin in combating V. cholerae infection. Inhibition of V. cholerae growth through metal limitation may provide new approaches in the development of anti-V. cholerae therapeutics. This study also establishes a major role for calprotectin in combating infectious diseases in zebrafish.

Adherence Enables Neisseria gonorrhoeae to Overcome Zinc Limitation Imposed by Nutritional Immunity Proteins.

Neisseria gonorrhoeae (Gc) must overcome the limitation of metals such as zinc to colonize mucosal surfaces in its obligate human host. While the zinc-binding nutritional immunity proteins calprotectin (S100A8/A9) and psoriasin (S100A7) are abundant in human cervicovaginal lavage fluid, Gc possesses TonB-dependent transporters TdfH and TdfJ that bind and extract zinc from the human version of these proteins, respectively. Here we investigated the contribution of zinc acquisition to Gc infection of epithelial cells of the female genital tract. We found that TdfH and TdfJ were dispensable for survival of strain FA1090 Gc that was associated with Ect1 human immortalized epithelial cells, when zinc was limited by calprotectin and psoriasin. In contrast, suspension-grown bacteria declined in viability under the same conditions. Exposure to murine calprotectin, which Gc cannot use as a zinc source, similarly reduced survival of suspension-grown Gc, but not Ect1-associated Gc. We ruled out epithelial cells as a contributor to the enhanced growth of cell-associated Gc under zinc limitation. Instead, we found that attachment to glass was sufficient to enhance bacterial growth when zinc was sequestered. We compared the transcriptional profiles of WT Gc adherent to glass coverslips or in suspension, when zinc was sequestered with murine calprotectin or provided in excess, from which we identified open reading frames that were increased by zinc sequestration in adherent Gc. One of these, ZnuA, was necessary but not sufficient for survival of Gc under zinc-limiting conditions. These results show that adherence protects Gc from zinc-dependent growth restriction by host nutritional immunity proteins.

Mechanism of action of nucleotide excision repair machinery.

Nucleotide excision repair (NER) is a versatile DNA repair pathway essential for the removal of a broad spectrum of structurally diverse DNA lesions arising from a variety of sources, including UV irradiation and environmental toxins. Although the core factors and basic stages involved in NER have been identified, the mechanisms of the NER machinery are not well understood. This review summarizes our current understanding of the mechanisms and order of assembly in the core global genome (GG-NER) pathway.

A key interaction with RPA orients XPA in NER complexes.

The XPA protein functions together with the single-stranded DNA (ssDNA) binding protein RPA as the central scaffold to ensure proper positioning of repair factors in multi-protein nucleotide excision repair (NER) machinery. We previously determined the structure of a short motif in the disordered XPA N-terminus bound to the RPA32C domain. However, a second contact between the XPA DNA-binding domain (XPA DBD) and the RPA70AB tandem ssDNA-binding domains, which is likely to influence the orientation of XPA and RPA on the damaged DNA substrate, remains poorly characterized. NMR was used to map the binding interfaces of XPA DBD and RPA70AB. Combining NMR and X-ray scattering data with comprehensive docking and refinement revealed how XPA DBD and RPA70AB orient on model NER DNA substrates. The structural model enabled design of XPA mutations that inhibit the interaction with RPA70AB. These mutations decreased activity in cell-based NER assays, demonstrating the functional importance of XPA DBD-RPA70AB interaction. Our results inform ongoing controversy about where XPA is bound within the NER bubble, provide structural insights into the molecular basis for malfunction of disease-associated XPA missense mutations, and contribute to understanding of the structure and mechanical action of the NER machinery.

The Zinc Transporter ZnuABC Is Critical for the Virulence of Chromobacterium violaceum and Contributes to Diverse Zinc-Dependent Physiological Processes.

Chromobacterium violaceum is a ubiquitous environmental bacterium that causes sporadic life-threatening infections in humans. How C. violaceum acquires zinc to colonize environmental and host niches is unknown. In this work, we demonstrated that C. violaceum employs the zinc uptake system ZnuABC to overcome zinc limitation in the host, ensuring the zinc supply for several physiological demands. Our data indicated that the C. violaceum ZnuABC transporter is encoded in a zur-CV_RS15045-CV_RS15040-znuCBA operon. This operon was repressed by the zinc uptake regulator Zur and derepressed in the presence of the host protein calprotectin (CP) and the synthetic metal chelator EDTA. A ΔznuCBA mutant strain showed impaired growth under these zinc-chelated conditions. Moreover, the deletion of znuCBA provoked reductions in violacein production, swimming motility, biofilm formation, and bacterial competition. Remarkably, the ΔznuCBA mutant strain was highly attenuated for virulence in an in vivo mouse infection model and showed low capacities to colonize the liver, grow in the presence of CP, and resist neutrophil killing. Overall, our findings demonstrate that ZnuABC is essential for C. violaceum virulence, contributing to subversion of zinc-based host nutritional immunity.

A fragment-based approach to discovery of Receptor for Advanced Glycation End products inhibitors.

The Receptor for Advanced Glycation End products (RAGE) is a pattern recognition receptor that signals for inflammation via the NF-κB pathway. RAGE has been pursued as a potential target to suppress symptoms of diabetes and is of interest in a number of other diseases associated with chronic inflammation, such as inflammatory bowel disease and bronchopulmonary dysplasia. Screening and optimization have previously produced small molecules that inhibit the activity of RAGE in cell-based assays, but efforts to develop a therapeutically viable direct-binding RAGE inhibitor have yet to be successful. Here, we show that a fragment-based approach can be applied to discover fundamentally new types of RAGE inhibitors that specifically target the ligand-binding surface. A series of systematic assays of structural stability, solubility, and crystallization were performed to select constructs of the RAGE ligand-binding domain and optimize conditions for NMR-based screening and co-crystallization of RAGE with hit fragments. An NMR-based screen of a highly curated ~14 000-member fragment library produced 21 fragment leads. Of these, three were selected for elaboration based on structure-activity relationships generated through cycles of structural analysis by X-ray crystallography, structure-guided design principles, and synthetic chemistry. These results, combined with crystal structures of the first linked fragment compounds, demonstrate the applicability of the fragment-based approach to the discovery of RAGE inhibitors.

The novel interaction between Neisseria gonorrhoeae TdfJ and human S100A7 allows gonococci to subvert host zinc restriction.

Neisseria gonorrhoeae causes the sexually-transmitted infection gonorrhea, a global disease that is difficult to treat and for which there is no vaccine. This pathogen employs an arsenal of conserved outer membrane proteins called TonB-dependent transporters (TdTs) that allow the gonococcus to overcome nutritional immunity, the host strategy of sequestering essential nutrients away from invading bacteria to handicap infectious ability. N. gonorrhoeae produces eight known TdTs, of which four are utilized for acquisition of iron or iron chelates from host-derived proteins or xenosiderophores produced by other bacteria. Of the remaining TdTs, two of them, TdfH and TdfJ, facilitate zinc uptake. TdfH was recently shown to bind Calprotectin, a member of the S100 protein family, and subsequently extract its zinc, which is then internalized by N. gonorrhoeae. Like Calprotectin, other S100s are also capable of binding transition metals such as zinc and copper, and thus have demonstrated growth suppression of numerous other pathogens via metal sequestration. Considering the functional and structural similarities of the TdTs and of the S100s, as well as the upregulation in response to Zn limitation shown by TdfH and TdfJ, we sought to evaluate whether other S100s have the ability to support gonococcal growth by means of zinc acquisition and to frame this growth in the context of the TdTs. We found that both S100A7 and S10012 are utilized by N. gonorrhoeae as a zinc source in a mechanism that depends on the zinc transport system ZnuABC. Moreover, TdfJ binds directly to S100A7, from which it internalizes zinc. This interaction is restricted to the human version of S100A7, and zinc presence in S100A7 is required to fully support gonococcal growth. These studies highlight how gonococci co-opt human nutritional immunity, by presenting a novel interaction between TdfJ and human S100A7 for overcoming host zinc restriction.

Yeast require redox switching in DNA primase.

Eukaryotic DNA primases contain a [4Fe4S] cluster in the C-terminal domain of the p58 subunit (p58C) that affects substrate affinity but is not required for catalysis. We show that, in yeast primase, the cluster serves as a DNA-mediated redox switch governing DNA binding, just as in human primase. Despite a different structural arrangement of tyrosines to facilitate electron transfer between the DNA substrate and [4Fe4S] cluster, in yeast, mutation of tyrosines Y395 and Y397 alters the same electron transfer chemistry and redox switch. Mutation of conserved tyrosine 395 diminishes the extent of p58C participation in normal redox-switching reactions, whereas mutation of conserved tyrosine 397 causes oxidative cluster degradation to the [3Fe4S]+ species during p58C redox signaling. Switching between oxidized and reduced states in the presence of the Y397 mutations thus puts primase [4Fe4S] cluster integrity and function at risk. Consistent with these observations, we find that yeast tolerate mutations to Y395 in p58C, but the single-residue mutation Y397L in p58C is lethal. Our data thus show that a constellation of tyrosines for protein-DNA electron transfer mediates the redox switch in eukaryotic primases and is required for primase function in vivo.

Disrupted structure and aberrant function of CHIP mediates the loss of motor and cognitive function in preclinical models of SCAR16.

CHIP (carboxyl terminus of heat shock 70-interacting protein) has long been recognized as an active member of the cellular protein quality control system given the ability of CHIP to function as both a co-chaperone and ubiquitin ligase. We discovered a genetic disease, now known as spinocerebellar autosomal recessive 16 (SCAR16), resulting from a coding mutation that caused a loss of CHIP ubiquitin ligase function. The initial mutation describing SCAR16 was a missense mutation in the ubiquitin ligase domain of CHIP (p.T246M). Using multiple biophysical and cellular approaches, we demonstrated that T246M mutation results in structural disorganization and misfolding of the CHIP U-box domain, promoting oligomerization, and increased proteasome-dependent turnover. CHIP-T246M has no ligase activity, but maintains interactions with chaperones and chaperone-related functions. To establish preclinical models of SCAR16, we engineered T246M at the endogenous locus in both mice and rats. Animals homozygous for T246M had both cognitive and motor cerebellar dysfunction distinct from those observed in the CHIP null animal model, as well as deficits in learning and memory, reflective of the cognitive deficits reported in SCAR16 patients. We conclude that the T246M mutation is not equivalent to the total loss of CHIP, supporting the concept that disease-causing CHIP mutations have different biophysical and functional repercussions on CHIP function that may directly correlate to the spectrum of clinical phenotypes observed in SCAR16 patients. Our findings both further expand our basic understanding of CHIP biology and provide meaningful mechanistic insight underlying the molecular drivers of SCAR16 disease pathology, which may be used to inform the development of novel therapeutics for this devastating disease.