Disease-causing Mutant Research Articles

Pharmacoproteomics pinpoints HSP70 interaction for correction of the most frequent Wilson disease-causing mutant of ATP7B

Pathogenic mutations in the copper transporter ATP7B have been hypothesized to affect its protein interaction landscape contributing to loss of function and, thereby, to hepatic copper toxicosis in Wilson disease. Although targeting mutant interactomes was proposed as a therapeutic strategy, druggable interactors for rescue of ATP7B mutants remain elusive. Using proteomics, we found that the frequent H1069Q substitution promotes ATP7B interaction with HSP70, thus accelerating endoplasmic reticulum (ER) degradation of the mutant protein and consequent copper accumulation in hepatic cells. This prompted us to use an HSP70 inhibitor as bait in a bioinformatics search for structurally similar Food and Drug Administration-approved drugs. Among the hits, domperidone emerged as an effective corrector that recovered trafficking and function of ATP7B-H1069Q by impairing its exposure to the HSP70 proteostatic network. Our findings suggest that HSP70-mediated degradation can be safely targeted with domperidone to rescue ER-retained ATP7B mutants and, hence, to counter the onset of Wilson disease.

Evolution of the human mitochondrial ABCB7 [2Fe–2S](GS)4 cluster exporter and the molecular mechanism of an E433K disease-causing mutation

Iron-sulfur cluster proteins play key roles in a multitude of cellular processes. Iron-sulfur cofactors are assembled primarily in mitochondria and are then exported to the cytosol by use of an ABCB7 transporter. It has been shown that the yeast mitochondrial transporter Atm1 can export glutathione-coordinated iron-sulfur clusters, [2Fe–2S](SG)4, providing a source of cluster units for cytosolic iron-sulfur cluster assembly systems. This pathway is consistent with the endosymbiotic model of mitochondrial evolution where homologous bacterial heavy metal transporters, utilizing metal glutathione adducts, were adapted for use in eukaryotic mitochondria. Herein, the basis for endosymbiotic evolution of the human cluster export protein (ABCB7) is developed through a BLAST analysis of transporters from ancient proteobacteria. In addition, a functional comparison of native human protein, versus a disease-causing mutant, demonstrates a key role for residue E433 in promoting cluster transport. Dysfunction in mitochondrial export of Fe–S clusters is a likely cause of the disease condition X-linked sideroblastic anemia.

GDF6-CD99 Signaling Regulates Src and Ewing Sarcoma Growth

SUMMARYWe report here that the autocrine signaling mediated by growth and differentiation factor 6 (GDF6), a member of the bone morphogenetic protein (BMP) family of cytokines, maintains Ewing sarcoma growth by preventing Src hyperactivation. Surprisingly, Ewing sarcoma depends on the prodomain, not the BMP domain, of GDF6. We demonstrate that the GDF6 prodomain is a ligand for CD99, a transmembrane protein that has been widely used as a marker of Ewing sarcoma. The binding of the GDF6 prodomain to the CD99 extracellular domain results in recruitment of CSK (C-terminal Src kinase) to the YQKKK motif in the intracellular domain of CD99, inhibiting Src activity. GDF6 silencing causes hyperactivation of Src and p21-dependent growth arrest. We demonstrate that two GDF6 prodomain mutants linked to Klippel-Feil syndrome are hyperactive in CD99-Src signaling. These results reveal a cytokine signaling pathway that regulates the CSK-Src axis and cancer cell proliferation and suggest the gain-of-function activity for disease-causing GDF6 mutants.

Functional analysis of thyroid peroxidase gene mutations resulting in congenital hypothyroidism.

Thyroid peroxidase (TPO) is essential for thyroid hormone biosynthesis. TPO mutations might lead to congenital hypothyroidism. In the present study, we analysed the function of a compound heterozygous TPO mutation in a Chinese family. We studied a 23-year-old Chinese girl with a history of growth retardation and severe constipation from the age of 3months, who was diagnosed as having congenital hypothyroidism. Genomic DNA was extracted from peripheral blood samples obtained from the patient's family members. The genomic DNA was sequenced to detect mutations in a panel of genes associated with congenital hypothyroidism. Bioinformatic analysis and structural modelling predicted the potential disease-causing potential mutant genes and the microstructure of the mutant protein, respectively. Western blotting and ELISA were used to measure protein expression, and guaiacol oxidation assay measured the TPO activity of the mutant protein. We identified a compound heterozygous mutation (c.C1993T, c.T2473C) in the TPO gene. Bioinformatic analysis predicted that the TPO mutations were potentially disease causing. Structural modelling predicted damage to the microstructure of the mutant TPO protein. Western blotting and ELISA showed reduced protein levels of the mutant TPO protein compared with that of the wild-type protein. The mutant TPO protein showed weaker activity compared with that of the wild-type protein. A novel compound heterozygous mutation of TPO gene was identified in a Chinese family. This mutation might alter the extracellular microstructure of TPO, and decrease its expression and the activity, resulting in congenital hypothyroidism.

A Genetic Screen for Human Genes Suppressing FUS Induced Toxicity in Yeast.

FUS is a nucleic acid binding protein that, when mutated, cause a subset of familial amyotrophic lateral sclerosis (ALS). Expression of FUS in yeast recapitulates several pathological features of the disease-causing mutant proteins, including nuclear to cytoplasmic translocation, formation of cytoplasmic inclusions, and cytotoxicity. Genetic screens using the yeast model of FUS have identified yeast genes and their corresponding human homologs suppressing FUS induced toxicity in yeast, neurons and animal models. To expand the search for human suppressor genes of FUS induced toxicity, we carried out a genome-scale genetic screen using a newly constructed library containing 13570 human genes cloned in an inducible yeast-expression vector. Through multiple rounds of verification, we found 37 human genes that, when overexpressed, suppress FUS induced toxicity in yeast. Human genes with DNA or RNA binding functions are overrepresented among the identified suppressor genes, supporting that perturbations of RNA metabolism is a key underlying mechanism of FUS toxicity.

Structural And Computational Perspectives of Selectively Targeting Mutant Proteins.

Diseases are often caused by mutant proteins. Many drugs have limited effectiveness and/or toxic side effects because of a failure to selectively target the disease-causing mutant variant, rather than the functional wild type protein. Otherwise, the drugs may even target different proteins with similar structural features. Designing drugs that successfully target mutant proteins selectively represents a major challenge. Decades of cancer research have led to an abundance of potential therapeutic targets, often touted to be "master regulators". For many of these proteins, there are no FDA-approved drugs available; for others, off-target effects result in dose-limiting toxicity. Cancer-related proteins are an excellent medium to carry the story of mutant-specific targeting, as the disease is both initiated and sustained by mutant proteins; furthermore, current chemotherapies generally fail at adequate selective distinction. This review discusses some of the challenges associated with selective targeting from a structural biology perspective, as well as some of the developments in algorithm approach and computational workflow that can be applied to address those issues. One of the most widely researched proteins in cancer biology is p53, a tumor suppressor. Here, p53 is discussed as a specific example of a challenging target, with contemporary drugs and methodologies used as examples of burgeoning successes. The oncogene KRAS, which has been described as "undruggable", is another extensively investigated protein in cancer biology. This review also examines KRAS to exemplify progress made towards selective targeting of diseasecausing mutant proteins. Finally, possible future directions relevant to the topic are discussed.

The pathogenicity, structural and functional exploration of human HMGB1 single nucleotide polymorphisms using in silico study

The human HMGB1 gene mutations have a major impact on several immune-related diseases and cancer. The detrimental effect of non-synonymous mutations of HMGB1 has not been investigated yet, hence the present study aims to examine single nucleotide polymorphisms and their implications on the structure-function of human HMGB1. The multifaceted HMGB1 protein acts as pleiotropic cytokine and regulates essential genes for coordinated cellular functions. The mutational effect on HMGB1 was analyzed by sequence-based homology methods, supervised learning methods, and structure-based methods. The study identified 58 non-synonymous mutations in human HMGB1, out of which only 2 mutations; R10T (rs61742222) and F103C (rs61733675) were classified as the SNPs with highest deleterious and disease-causing mutants. The effect of these mutations in structure of HMGB1 was scrutinized and the R10T mutant found to have a distinct structural behaviour in the B-box domain. In addition, R10T mutant predicted that it affects the MoRF function of HMGB1 and it could disrupt the DNA binding or/and protein partner interaction activity by HMGB1. F103C mutation takes place at the TLR binding and cytokine inducing region of HMGB1, hence it could affect the protein binding activity which involves in many cellular signaling. The study identified potent mutations R10T (a cancer-causing somatic mutation) and F103C (a novel mutation) and these mutations either directly or indirectly hinder DNA binding activity and TLR and cytokine binding of HMGB1. These findings will help in understanding the molecular basis of these promising mutations and functional role of human HMGB1 in cancer and immunological diseases.AbbreviationsAGERAdvanced glycosylation end product-specific receptorCXCLChemokine (C-X-C motif) liganddbSNPThe single nucleotide polymorphism databaseHMGB1High mobility group box 1LINCSLINear Constraint SolverMDSMolecular dynamics simulationMoRFMolecular recognition featuresNPTNumber of particle, Pressure and TemperatureNVTNumber of particle, Volume and TemperaturensSNPNon-synonymous SNPPBCPartial boundary conditionPCAPrincipal component analysisPMEPartial mesh EwaldRMSDRoot mean square deviationRMSFRoot mean square fluctuationSNPSingle nucleotide polymorphismSPCSingle-point chargeTLRToll-like receptorUTRUn-translated RegionCommunicated by Ramaswamy H. Sarma

Brain-penetrant PQR620 mTOR and PQR530 PI3K/mTOR inhibitor reduce huntingtin levels in cell models of HD

One of the pathological hallmarks of Huntington disease (HD) is accumulation of the disease-causing mutant huntingtin (mHTT), which leads to the disruption of a variety of cellular functions, ultimately resulting in cell death. Induction of autophagy, for example by the inhibition of mechanistic target of rapamycin (mTOR) signaling, has been shown to reduce HTT levels and aggregates. While rapalogs like rapamycin allosterically inhibit the mTOR complex 1 (TORC1), ATP-competitive mTOR inhibitors suppress activities of TORC1 and TORC2 and have been shown to be more efficient in inducing autophagy and reducing protein levels and aggregates than rapalogs. The ability to cross the blood-brain barrier of first generation catalytic mTOR inhibitors has so far been limited, and therefore sufficient target coverage in the brain could not be reached. Two novel, brain penetrant compounds – the mTORC1/2 inhibitor PQR620, and the dual pan-phosphoinositide 3-kinase (PI3K) and mTORC1/2 kinase inhibitor PQR530 - were evaluated by assessing their potential to induce autophagy and reducing mHTT levels. For this purpose, expression levels of autophagic markers and well-defined mTOR targets were analyzed in STHdh cells and HEK293T cells and in mouse brains. Both compounds potently inhibited mTOR signaling in cell models as well as in mouse brain. As proof of principle, reduction of aggregates and levels of soluble mHTT were demonstrated upon treatment with both compounds. Originally developed for cancer treatment, these second generation mTORC1/2 and PI3K/mTOR inhibitors show brain penetrance and efficacy in cell models of HD, making them candidate molecules for further investigations in HD.

Pleiotropic requirements for human TDP-43 in the regulation of cell and organelle homeostasis.

TDP-43 is an RNA-binding protein that forms cytoplasmic aggregates in multiple neurodegenerative diseases. Although the loss of normal TDP-43 functions likely contributes to disease pathogenesis, the cell biological consequences of human TDP-43 depletion are not well understood. We, therefore, generated human TDP-43 knockout (KO) cells and subjected them to parallel cell biological and transcriptomic analyses. These efforts yielded three important discoveries. First, complete loss of TDP-43 resulted in widespread morphological defects related to multiple organelles, including Golgi, endosomes, lysosomes, mitochondria, and the nuclear envelope. Second, we identified a new role for TDP-43 in controlling mRNA splicing of Nup188 (nuclear pore protein). Third, analysis of multiple amyotrophic lateral sclerosis causing TDP-43 mutations revealed a broad ability to support splicing of TDP-43 target genes. However, as some TDP-43 disease-causing mutants failed to fully support the regulation of specific target transcripts, our results raise the possibility of mutation-specific loss-of-function contributions to disease pathology.

CMT disease severity correlates with mutation-induced open conformation of histidyl-tRNA synthetase, not aminoacylation loss, in patient cells

Aminoacyl-transfer RNA (tRNA) synthetases (aaRSs) are the largest protein family causatively linked to neurodegenerative Charcot-Marie-Tooth (CMT) disease. Dominant mutations cause the disease, and studies of CMT disease-causing mutant glycyl-tRNA synthetase (GlyRS) and tyrosyl-tRNA synthetase (TyrRS) showed their mutations create neomorphic structures consistent with a gain-of-function mechanism. In contrast, based on a haploid yeast model, loss of aminoacylation function was reported for CMT disease mutants in histidyl-tRNA synthetase (HisRS). However, neither that nor prior work of any CMT disease-causing aaRS investigated the aminoacylation status of tRNAs in the cellular milieu of actual patients. Using an assay that interrogated aminoacylation levels in patient cells, we investigated a HisRS-linked CMT disease family with the most severe disease phenotype. Strikingly, no difference in charged tRNA levels between normal and diseased family members was found. In confirmation, recombinant versions of 4 other HisRS CMT disease-causing mutants showed no correlation between activity loss in vitro and severity of phenotype in vivo. Indeed, a mutation having the most detrimental impact on activity was associated with a mild disease phenotype. In further work, using 3 independent biophysical analyses, structural opening (relaxation) of mutant HisRSs at the dimer interface best correlated with disease severity. In fact, the HisRS mutation in the severely afflicted patient family caused the largest degree of structural relaxation. These data suggest that HisRS-linked CMT disease arises from open conformation-induced mechanisms distinct from loss of aminoacylation.

RNA Secondary Structure-Based Design of Antisense Peptide Nucleic Acids for Modulating Disease-Associated Aberrant Tau Pre-mRNA Alternative Splicing.

Alternative splicing of tau pre-mRNA is regulated by a 5′ splice site (5′ss) hairpin present at the exon 10–intron 10 junction. Single mutations within the hairpin sequence alter hairpin structural stability and/or the binding of splicing factors, resulting in disease-causing aberrant splicing of exon 10. The hairpin structure contains about seven stably formed base pairs and thus may be suitable for targeting through antisense strands. Here, we used antisense peptide nucleic acids (asPNAs) to probe and target the tau pre-mRNA exon 10 5′ss hairpin structure through strand invasion. We characterized by electrophoretic mobility shift assay the binding of the designed asPNAs to model tau splice site hairpins. The relatively short (10–15 mer) asPNAs showed nanomolar binding to wild-type hairpins as well as a disease-causing mutant hairpin C+19G, albeit with reduced binding strength. Thus, the structural stabilizing effect of C+19G mutation could be revealed by asPNA binding. In addition, our cell culture minigene splicing assay data revealed that application of an asPNA targeting the 3′ arm of the hairpin resulted in an increased exon 10 inclusion level for the disease-associated mutant C+19G, probably by exposing the 5′ss as well as inhibiting the binding of protein factors to the intronic spicing silencer. On the contrary, the application of asPNAs targeting the 5′ arm of the hairpin caused an increased exon 10 exclusion for a disease-associated mutant C+14U, mainly by blocking the 5′ss. PNAs could enter cells through conjugation with amino sugar neamine or by cotransfection with minigene plasmids using a commercially available transfection reagent.

Molecular basis of the scalp-ear-nipple syndrome unraveled by the characterization of disease-causing KCTD1 mutants

The scalp-ear-nipple (SEN) syndrome is an autosomal-dominant disorder characterized by cutis aplasia of the scalp and malformations of breast, external ears, digits, and nails. Genetic analyses have shown that the disease is caused by missense mutations of the KCTD1 protein, although the functional/structural basis of SEN insurgence is hitherto unknown. With the aim of unravelling the molecular basis of the SEN syndrome associated with KCTD1 mutations we here expressed and characterized several disease causing mutants. A preliminary dissection of the protein provides insights into the role that individual domains play in KCTD1 stability. The characterization of SEN-causing mutants indicates that, although the mutation sites are located in distant regions of the BTB domain or of the pre-BTB region, all of them are unable to interact with the transcription factor AP-2α, a well-known KCTD1 biological partner. Notably, all mutations, including the one located in the pre-BTB region, produce a significant destabilization of the protein. The structural role of the pre-BTB region in KCTD1 and other proteins of the family is corroborated by its sequence conservation in orthologs and paralogs. Interestingly, SEN-causing mutations also favor the tendency of KCTD1 to adopt structural states that are characterized by the ability to bind the β-amyloid fluorescent dye thioflavin T. The formation of aggregation-prone species may have important implications for the disease etiology. Collectively, these findings provide an intriguing picture of the functional and structural alterations induced by KCTD1 mutations that ultimately lead to disease.

Protein folding state-dependent sorting at the Golgi apparatus.

In eukaryotic cells, organelle-specific protein quality control (PQC) is critical for maintaining cellular homeostasis. Despite the Golgi apparatus being the major protein processing and sorting site within the secretory pathway, how it contributes to PQC has remained largely unknown. Using different chemical biology-based protein unfolding systems, we reveal the segregation of unfolded proteins from folded proteins in the Golgi. Quality control (QC) substrates are subsequently exported in distinct carriers, which likely contain unfolded proteins as well as highly oligomerized cargo that mimic protein aggregates. At an additional sorting step, oligomerized proteins are committed to lysosomal degradation, while unfolded proteins localize to the endoplasmic reticulum (ER) and associate with chaperones. These results highlight the existence of checkpoints at which QC substrates are selected for Golgi export and lysosomal degradation. Our data also suggest that the steady-state ER localization of misfolded proteins, observed for several disease-causing mutants, may have different origins.

OR05-2 Targeted Inhibition of Glutamate Dehydrogenase by Alpha-Tocopherol: A Potential Novel Treatment for Hyperinsulinism Hyperammonemia Syndrome

Congenital hyperinsulinism is a rare genetic disorder that causes severe hypoglycemia and can lead to permanent brain damage if inadequately controlled. Gain of function mutations in glutamate dehydrogenase (GDH) result in hyperinsulinism hyperammonemia (HI/HA) syndrome, the second most common cause of congenital hyperinsulinism. Current therapies inhibit insulin secretion but do not target GDH overactivity directly and thus fail to treat the spectrum of clinical manifestations observed, which in addition to hyperinsulinism and hyperammonemia, include seizures and developmental delays. The neurological symptoms are profound and occur independent of the hypoglycemia. We investigated the efficacy of alpha-tocopherol as a targeted GDH inhibitor in vitro and in vivo models of HI/HA syndrome. Human embryonic kidney (HEK293T) cells were transduced with lentivirus to overexpress either wild-type GDH or H454Y GDH, a well-characterized disease-causing mutant. GDH enzyme kinetics were determined spectrophotometrically in cell homogenates perfused with escalating concentrations of alpha-tocopherol on a background of 10mM glutamine. To evaluate the inhibitory effect of alpha-tocopherol on GDH in vivo, wild-type (WT, n=10) and H454Y GDH transgenic (Tg, n=13) adult mice were treated with oral alpha-tocopherol (75 μg/g body weight/dose) or vehicle twice daily for 3 doses and then fasted for 6 hours. Nadir fasting plasma glucoses were measured as a clinical correlate of GDH activity and compared between alpha-tocopherol and vehicle-treated wild-type and transgenic mice, respectively. Alpha-tocopherol effectively inhibited activity of wild-type and H454Y mutant GDH in HEK293T cells with 50% inhibitory concentration (IC50) of 4.1 and 3.1 μM, respectively. In vivo, nadir fasting plasma glucose was significantly higher in alpha-tocopherol-treated versus vehicle-treated WT and Tg mice (WT: mean nadir plasma glucose 94.3 ± 9.6 mg/dl v. 80.1 ± 9.6 mg/dl, p=0.003; Tg: mean nadir plasma glucose 71.9± 11.3 mg/dl v. 57.2 ± 12.6 mg/dl, p=0.002). Alpha-tocopherol effectively inhibits GDH in vitro and ameliorates fasting hypoglycemia in vivo in a mouse model of HI/HA syndrome. Based upon these findings, alpha-tocopherol is a promising potential treatment for HI/HA syndrome. Our next step will be to study the safety and efficacy of oral alpha-tocopherol supplementation in human subjects with HI/HA syndrome.

Gene Editing–Mediated Disruption of Epidermolytic Ichthyosis–Associated KRT10 Alleles Restores Filament Stability in Keratinocytes

Epidermolytic ichthyosis is a skin fragility disorder caused by dominant-negative mutations in KRT1 or KRT10. No definitive restorative therapies exist that target these genetic faults. Gene editing can be used to efficiently introduce frameshift mutations to inactivate mutant genes. This can be applied to counter the effect of dominantly inherited diseases such as epidermolytic ichthyosis. In this study, we used transcription activator-like effector nuclease technology, to disrupt disease-causing mutant KRT10 alleles in an exvivo cellular approach, with the intent of developing a therapy for patients with epidermolytic ichthyosis. A transcription activator-like effector nuclease was designed to specifically target a region of KRT10, upstream of a premature termination codon known to induce a genetic knockout. This proved highly efficient at gene disruption in a patient-derived keratinocyte cell line. In addition, analysis for off-target effects indicated no promiscuous gene editing-mediated disruption. Reversion of the keratin intermediate filament fragility phenotype associated with epidermolytic ichthyosis was observed by the immunofluorescence analysis of correctly gene-edited single-cell clones. This was in concurrence with immunofluorescence and ultrastructure analysis of murine xenograft models. The efficiency of this approach was subsequently confirmed in primary patient keratinocytes. Our data demonstrate the feasibility of an exvivo gene-editing therapy for more than 95.6% of dominant KRT10 mutations.

Measurement of Myofilament-Localized Calcium Dynamics in Adult Cardiomyocytes and the Effect of Hypertrophic Cardiomyopathy Mutations.

Subcellular Ca2+ indicators have yet to be developed for the myofilament where disease mutation or small molecules may alter contractility through myofilament Ca2+ sensitivity. Here, we develop and characterize genetically encoded Ca2+ indicators restricted to the myofilament to directly visualize Ca2+ changes in the sarcomere. To produce and validate myofilament-restricted Ca2+ imaging probes in an adenoviral transduction adult cardiomyocyte model using drugs that alter myofilament function (MYK-461, omecamtiv mecarbil, and levosimendan) or following cotransduction of 2 established hypertrophic cardiomyopathy disease-causing mutants (cTnT [Troponin T] R92Q and cTnI [Troponin I] R145G) that alter myofilament Ca2+ handling. When expressed in adult ventricular cardiomyocytes RGECO-TnT (Troponin T)/TnI (Troponin I) sensors localize correctly to the sarcomere without contractile impairment. Both sensors report cyclical changes in fluorescence in paced cardiomyocytes with reduced Ca2+ on and increased Ca2+ off rates compared with unconjugated RGECO. RGECO-TnT/TnI revealed changes to localized Ca2+ handling conferred by MYK-461 and levosimendan, including an increase in Ca2+ binding rates with both levosimendan and MYK-461 not detected by an unrestricted protein sensor. Coadenoviral transduction of RGECO-TnT/TnI with hypertrophic cardiomyopathy causing thin filament mutants showed that the mutations increase myofilament [Ca2+] in systole, lengthen time to peak systolic [Ca2+], and delay [Ca2+] release. This contrasts with the effect of the same mutations on cytoplasmic Ca2+, when measured using unrestricted RGECO where changes to peak systolic Ca2+ are inconsistent between the 2 mutations. These data contrast with previous findings using chemical dyes that show no alteration of [Ca2+] transient amplitude or time to peak Ca2+. RGECO-TnT/TnI are functionally equivalent. They visualize Ca2+ within the myofilament and reveal unrecognized aspects of small molecule and disease-associated mutations in living cells.

New CRISPR/Cas9 mouse model confirms association of phosphorylation of ACTN4 at serine 159 with proteinuria and focal segmental glomerulosclerosis

Mutations in α‐actinin‐4 (ACTN4)—an important cytoskeleton protein that binds actin—cause a form of kidney injury in humans called familial focal segmental glomerulosclerosis (FSGS). FSGS is characterized by proteinuria and eventual progression to renal failure. All of the known disease‐causing mutations result in increased ACTN4 binding affinity to actin. We previously showed that this increased actin binding was associated with biomechanical changes that left podocytes vulnerable to mechanical stress and eventual detachment. We also previously detected with mass spectrometry that ACTN4 is phosphorylated at the serine 159 site (S159) in cultured human podocytes. Moreover, phosphomimetic S159D ACTN4 protein (which mimics the effect of phosphorylation at S159) confers similar increased actin binding affinity as disease‐causing mutant ACTN4. In the current study, we hypothesized that the changes in binding affinity associated with phosphorylation of S159 may also render the podocyte vulnerable to mechanical stress and lead to eventual proteinuria and FSGS in vivo. Using CRISPR/Cas9 technology, we developed a new phosphomimetic mouse model Actn4 S160D (analogous to S159D mutation in humans). We first isolated primary podocytes from wild‐type (WT) and homozygous Actn4S160D/S160D mice. Similar to podocytes carrying disease‐causing mutant Actn4, homozygous Actn4S160D/S160D podocytes demonstrated more aligned actin filaments and increased number of focal adhesions (quantified using vinculin staining) in comparison with WT podocytes. We subjected both WT and homozygous Actn4S160D/S160D mice to subtotal nephrectomy to induce glomerular hyperfiltration and increase glomerular mechanical stress upon podocytes. We found that in comparison with WT, homozygous Actn4S160D/S/160D mice demonstrated significantly increased albuminuria at 2, 3, and 7 weeks after nephrectomy. Additionally, kidney sections were used for PAS staining and examined by a renal pathologist to assess for glomerulosclerosis. Whereas only 1% of glomeruli were found to be sclerosed in WT, 12% were sclerosed in homozygous Actn4S160D/S/160D mice (p<0.001). Our findings confirm that increased phosphorylation at S159 is pathologic, in showing that a phosphomimetic mouse model develops albuminuria and FSGS. Moreover, we show that phosphomimetic podocytes demonstrate changes in actin alignment and focal adhesion, markers of podocyte dysfunction that are shared by disease‐causing mutant ACTN4 podocytes. Altogether, these findings add to the building evidence suggesting that abnormal phosphorylation at S159 leads to changes on a protein, cellular, and whole animal level that are similar to mutant ACTN4‐mediated disease. This evidence provides new insights into the importance of a post‐translational modification affecting the podocyte cytoskeleton.Support or Funding InformationK01DK114329 and R37DK059588This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Interaction of NaV1.2 IQ Motif with Disease-Causing Mutants of Calmodulin

Calmodulin (CaM) binds constitutively to an IQ motif (residues 1901-1927, NaV1.2IQp) in the cytosolic C-terminal tail of the a-subunit of the neuronal voltage-gated sodium channel NaV1.2. Solution structures of CaM C-domain (CaMC) bound to NaV1.2IQp showed calcium binding reverses the orientation of (Ca2+)2-CaMC (2M5E.pdb) relative to apo CaMC (2KXW.pdb). Our structure of full-length apo human CaM bound to human NaV1.2IQp showed that only CaMc interacts with NaV1.2IQp. Thermodynamic and NMR analysis of the CaM-NaV1.2IQp complex showed no evidence for CaMN-NaV1.2IQp interactions, and indicated nearly identical affinity for calcium binding to the 4 EF-hand sites of CaM. Residues in NaV1.2IQp had different chemical environments when bound to apo and (Ca2+)4-CaM, consistent with the calcium-trigged change observed in the (Ca2+)2-CaMC-NaV1.2IQp interface (2M5E.pdb). Apo human CaM carrying cardiomyopathy-causing mutations [N53I(N54I), D95V(D96V), A102V(A103V), E104A(E105A), D129G(D130G), and F141L(F142L)] all bound tightly to NaV1.2IQp. However, mutations in CaMC inhibited Ca2+-mediated conformational changes of CaM bound to NaV1.2IQp. This suggests that high cytosolic Ca2+ levels would not be sufficient to trigger reversal of the orientation of these CaMC mutants. The effects of other recently described disease-causing CaM mutations are being investigated. NIH R01 GM57001 (MAS), NIH T32 NS045549 (RM), UI CCOM FUTURE in Biomedicine (AMK)

Cooperative subunit dynamics modulate p97 function

p97 is an essential hexameric AAA+ ATPase involved in a wide range of cellular processes. Mutations in the enzyme are implicated in the etiology of an autosomal dominant neurological disease in which patients are heterozygous with respect to p97 alleles, containing one copy each of WT and disease-causing mutant genes, so that, in vivo, p97 molecules can be heterogeneous in subunit composition. Studies of p97 have, however, focused on homohexameric constructs, where protomers are either entirely WT or contain a disease-causing mutation, showing that for WT p97, the N-terminal domain (NTD) of each subunit can exist in either a down (ADP) or up (ATP) conformation. NMR studies establish that, in the ADP-bound state, the up/down NTD equilibrium shifts progressively toward the up conformation as a function of disease mutant severity. To understand NTD functional dynamics in biologically relevant p97 heterohexamers comprising both WT and disease-causing mutant subunits, we performed a methyl-transverse relaxation optimized spectroscopy (TROSY) NMR study on a series of constructs in which only one of the protomer types is NMR-labeled. Our results show positive cooperativity of NTD up/down equilibria between neighboring protomers, allowing us to define interprotomer pathways that mediate the allosteric communication between subunits. Notably, the perturbed up/down NTD equilibrium in mutant subunits is partially restored by neighboring WT protomers, as is the two-pronged binding of the UBXD1 adaptor that is affected in disease. This work highlights the plasticity of p97 and how subtle perturbations to its free-energy landscape lead to significant changes in NTD conformation and adaptor binding.

Secretion and Uptake of \u03b1-Synuclein Via Extracellular Vesicles in Cultured Cells

In Parkinson’s disease and other Lewy body disorders, the propagation of pathology has been accredited to the spreading of extracellular α-synuclein (α-syn). Although the pathogenic mechanisms are not fully understood, cell-to-cell transfer of α-syn via exosomes and other extracellular vesicles (EVs) has been reported. Here, we investigated whether altered molecular properties of α-syn can influence the distribution and secretion of α-syn in human neuroblastoma cells. Different α-syn variants, including α-syn:hemi-Venus and disease-causing mutants, were overexpressed and EVs were isolated from the conditioned medium. Of the secreted α-syn, 0.1–2% was associated with vesicles. The major part of EV α-syn was attached to the outer membrane of vesicles, whereas a smaller fraction was found in their lumen. For α-syn expressed with N-terminal hemi-Venus, the relative levels associated with EVs were higher than for WT α-syn. Moreover, such EV-associated α-syn:hemi-Venus species were internalized in recipient cells to a higher degree than the corresponding free-floating forms. Among the disease-causing mutants, A53T α-syn displayed an increased association with EVs. Taken together, our data suggest that α-syn species with presumably lost physiological functions or altered aggregation properties may shift the cellular processing towards vesicular secretion. Our findings thus lend further support to the tenet that EVs can mediate spreading of harmful α-syn species and thereby contribute to the pathology in α-synucleinopathies.