Abstract Biallelic variants in PYROXD1 are associated with a life limiting muscle and connective tissue disorder characterised by generalised muscle weakness, breathing and feeding difficulties, distal laxity, hypernasal speech, blue sclera and osteopenia. PYROXD1 encodes an oxidoreductase implicated in mitochondrial function and tRNA ligase activity. Herein we present the first comprehensive suite of mouse models designed to elucidate PYROXD1 redox functions and the underlying pathogenetic basis for PYROXD1 disorders. A LacZ reporter strain reveals ubiquitous expression of Pyroxd1, particularly in the developing head, eye, heart and skin. Complete knockout (KO) of Pyroxd1 resulted in embryonic lethality between ED4.5–E9.5, suggesting Pyroxd1 performs a unique function during embryogenesis that cannot be substituted by other redox enzymes. Skeletal muscle tissue-specific KO, or stage-specific tamoxifen-induced KO during gestation or post-weaning, were viable but not sustainable models. Tamoxifen-inducible KO cell lines derived from these models provide valuable tools for dissecting Pyroxd1 function. Homozygous mice harbouring the recurrent human variant NM_024854.5:c.464A > G;p.(N155S), termed Pyroxd1 N155S, phenocopied a severe PYROXD1 disorder, presenting from ~ 10 weeks of age with a progressive myopathy, myofibrillar disorganisation, decreased contractile strength, muscle hypotrophy and osteopenia. Conversely, homozygous Pyroxd1 N155G mice, created incidentally during CRISPR editing, were phenotypically normal and provide an important benign control. Proteomic analyses reveal distinct molecular signatures between acute Pyroxd1 KO and Pyroxd1 N155S models, highlighting differential effects of complete loss-of-function (KO) compared to partial enzymatic activity (N155S). Pyroxd1 activity is critical for numerous cell essential processes, including protein biosynthesis and turnover, tRNA ligase complex activity and the unfolded protein response, mitochondrial respiratory chain function (especially complex 1), with an implicated broader role in the exon-ligation step of pre-mRNA splicing. Together, these murine Pyroxd1 models establish an enabling platform for gaining mechanistic insight and guiding the path of future therapeutic development for PYROXD1 disorders.

Synaptotagmin-1 (syt1) functions as the Ca2+-dependent sensor that triggers the rapid and synchronous release of neurotransmitters from neurotransmitter-containing vesicles during neuronal exocytosis. The syt1 protein has two homologous tandem C2 domains that interact with phospholipids in a Ca2+-dependent manner. Despite the crucial role of syt1 in exocytosis, the precise interactions between Ca2+, syt1, and phospholipids are not fully understood. In a study involving recessive lethal mutations in the syt1 gene, a specific mutation named AD3 was generated in Drosophila syt1, resulting in a significant reduction in Ca2+-dependent exocytosis. Further investigation revealed that the AD3 mutation was a missense mutation located in a conserved consensus sequence within the C2B domain of Drosophila syt1. However, the biophysical impact of the AD3 mutation had not been analyzed. Our study uses x-ray crystallography, isothermal titration calorimetry, thermodynamic analysis, and molecular dynamics simulation to show that the primary defect caused by the AD3 mutation in the syt1 protein is reduced thermodynamic stability. This instability alters the population of Ca2+-receptive states, leading to two major consequences: decreased affinity for calcium ions and compromised stabilization of the domain normally enhanced by Ca2+. We conclude that this conserved residue acts as a structural constraint, delimiting the movement of loop 3 within the pocket and ultimately influencing the affinity of the calcium ion binding with the C2 domain.

The mouse epididymis is a long tubule connecting the testis to the vas deferens. Its primary functions are to mature spermatozoa into motile and fertile cells and to protect them from pathogens that ascend the male tract. We previously demonstrated that a functional extracellular amyloid matrix surrounds spermatozoa in the epididymal lumen and has host defense functions, properties not unlike that of an extracellular biofilm that encloses and protects a bacterial community. Here we show the epididymal amyloid matrix also structurally resembles a biofilm by containing eDNA, eRNA, and mucin-like polysaccharides. Further these structural components exhibit comparable behaviors and perform functions such as their counterparts in bacterial biofilms. Our studies suggest that nature has used the ancient building blocks of bacterial biofilms to form an analogous structure that nurtures and protects the mammalian male germline.

Corneal blindness affects greater than 5 million individuals, with over 180,000 corneal transplantations (CTs) performed annually. Inhigh-risk CTs, almost all grafts are rejected within 10 years. Herein, adeno-associated virus (AAV) ex vivo gene therapy was investigated to establish immune tolerance in the corneal allograft to prevent high-risk CT rejection. Our previous work has demonstrated that HLA-G contributes to ocular immune privilege by inhibiting both immune cells and neovascularization; however, homodimerization is a rate-limiting step for optimal HLA-G function. Therefore, a chimeric protein termed single chain immunomodulator (sclM), was engineered to mimic the native activity of the secreted HLA-G dimer complex and eliminate the need for homodimerization. In a murine corneal burn model, AAV8-sclM significantly reduced corneal vascularization and fibrosis. Next, ex vivo AAV8-scIM gene delivery to corneal allografts was evaluated in a high-risk CT rejection rabbit model. All sclM treated corneas were well tolerated and transparent after 42 days while 83% of vehicle treated corneas were rejected. Histologically, AAV-scIM treated corneas were devoid of immune cell infiltration, vascularization, with minimal fibrosis at the host-graft interface. The data collectively demonstrate that sclM gene therapy prevents corneal neovascularization, reduces trauma-induced corneal fibrosis, and prevents allogeneic CT rejection in a high-risk large animal model.

Mutations in the DYSF gene, encoding the protein dysferlin, lead to several forms of muscular dystrophy. In healthy skeletal muscle, dysferlin concentrates in the transverse tubules and is involved in repairing the sarcolemma and stabilizing Ca2+ signaling after membrane disruption. The DYSF gene encodes 7-8 C2 domains, several Fer and Dysf domains, and a C-terminal transmembrane sequence. Because its coding sequence is too large to package in adeno-associated virus, the full-length sequence is not amenable to current gene delivery methods. Thus, we have examined smaller versions of dysferlin, termed "nanodysferlins," designed to eliminate several C2 domains, specifically C2 domains D, E, and F; B, D, and E; and B, D, E, and F. We also generated a variant by replacing eight amino acids in C2G in the nanodysferlin missing domains D through F. We electroporated dysferlin-null A/J mouse myofibers with Venus fusion constructs of these variants, or as untagged nanodysferlins together with GFP, to mark transfected fibers We found that, although these nanodysferlins failed to concentrate in transverse tubules, three of them supported membrane repair after laser wounding while all four bound the membrane repair protein, TRIM72/MG53, similar to WT dysferlin. By contrast, they failed to suppress Ca2+ waves after myofibers were injured by mild hypoosmotic shock. Our results suggest that the internal C2 domains of dysferlin are required for normal t-tubule localization and Ca2+ signaling and that membrane repair does not require these C2 domains.

The DNase1 family of enzymes are endonucleases that are important in the removal of dsDNA debris. Null functioning mutations in DNase1 and DNase1L3 result in the development of systemic lupus erythematosus and related autoimmune conditions. Interestingly, despite the ubiquity of DNase1 and related enzymes, the catalytic mechanism of this family of nucleases has not been fully explored. Using X-ray crystallography we have solved the structure of recombinant human DNase1L3. Careful analysis of our results revealed a second magnesium ion coordinated in the active site of DNase1L3.

According to the Lupus Foundation of America, systemic lupus erythematosus (SLE) affects more than 1.5 million Americans. Long-term use of standard SLE therapies, such as hydroxychloroquine, often result in serious side effects; therefore, new treatments are required. The production of autoantibodies against dsDNA defines the inflammatory response in SLE. Autoantibodies to nucleic acids can produce immunological complexes (ICs), which accumulate in the kidney and cause lupus nephritis. Dnase1L3 degrades antigenic DNA complexes and could be used to treat SLE.

Systemic lupus erythematosus (SLE) is an autoimmune disease caused by environmental factors and loss of key proteins, including the endonuclease Dnase1L3. Dnase1L3 absence causes pediatric-onset lupus in humans, while reduced activity occurs in adult-onset SLE. The amount of Dnase1L3 that prevents lupus remains unknown. To genetically reduce Dnase1L3 levels, we developed a mouse model lacking Dnase1L3 in macrophages (conditional knockout [cKO]). Serum Dnase1L3 levels were reduced 67%, though Dnase1 activity remained constant. Homogeneous and peripheral antinuclear antibodies were detected in the sera by immunofluorescence, consistent with anti-double-stranded DNA (anti-dsDNA) antibodies. Total immunoglobulin M, total immunoglobulin G, and anti-dsDNA antibody levels increased in cKO mice with age. The cKO mice developed anti-Dnase1L3 antibodies. In contrast to global Dnase1L3(-/-) mice, anti-dsDNA antibodies were not elevated early in life. The cKO mice had minimal kidney pathology. Therefore, we conclude that an intermediate reduction in serum Dnase1L3 causes mild lupus phenotypes, and macrophage-derived DnaselL3 helps limit lupus.

Limb-Girdle Muscular Dystrophy Type-2B/2R is caused by mutations in the dysferlin gene ( DYSF ). This disease has two known pathogenic missense mutations that occur within dysferlin’s C2A domain, namely C2A W52R and C2A V67D . Yet, the etiological rationale to explain the disease linkage for these two mutations is still unclear. In this study, we have presented evidence from biophysical, computational, and immunological experiments which suggest that these missense mutations interfere with dysferlin’s ability to repair cells. The failure of C2A W52R and C2A V67D to initiate membrane repair arises from their propensity to form stable amyloid. The misfolding of the C2A domain caused by either mutation exposes β-strands, which are predicted to nucleate classical amyloid structures. When dysferlin C2A amyloid is formed, it triggers the NLRP3 inflammasome, leading to the secretion of inflammatory cytokines, including IL-1β. The present study suggests that the muscle dysfunction and inflammation evident in Limb-Girdle Muscular Dystrophy types-2B/2R, specifically in cases involving C2A W52R and C2A V67D , as well as other C2 domain mutations with considerable hydrophobic core involvement, may be attributed to this mechanism.

Splashed white in horses is characterized by extensive white patterning on the legs, face and abdomen and may be accompanied by deafness. To date, seven variants in microphthalmia-associated transcription factor (MITF) and two variants in Paired Box 3 (PAX3) have been identified to explain this phenotype. A splashed white Thoroughbred stallion, whose sire and dam were not patterned, was hypothesized to have a de novo variant leading to his white coat pattern. A whole-genome sequencing candidate gene approach identified two single nucleotide variants (SNVs) in SOX10, four SNVs in MITF and a 2.3 kb deletion in MITF with the alternative allele present in this stallion but absent in the other 18 horses analyzed. All six SNVs were annotated as modifiers and were not further considered. The deletion in MITF (NC_009159.3:g.21555811_21558139delinsAAAT) encompasses exon 9 encoding a part of the helix-loop-helix domain required for DNA binding. Sanger sequencing and parentage testing confirmed that this deletion was a de novo mutation of maternal origin. Consistent with the published nomenclature, we denote this likely causal variant as SW8. Genotyping three of this stallion's offspring identified SW8 only in the nearly all-white foal that was confirmed deaf by brainstem auditory evoked response testing. This foal was also a compound heterozygote for dominant white variants (W20/W22), but to date, W variants alone have not been connected to deafness. SW8 marks the fourth de novo MITF variant in horses reported to cause white patterning. The link between deafness and all MITF variants with and without other variants impacting melanocyte development and function needs to be further explored.

In poikilotherms, temperature changes challenge the integration of physiological function. Within the complex nervous systems of the behaviorally sophisticated coleoid cephalopods, these problems are substantial. RNA editing by adenosine deamination is a well-positioned mechanism for environmental acclimation. We report that the neural proteome of Octopus bimaculoides undergoes massive reconfigurations via RNA editing following a temperature challenge. Over 13,000 codons are affected, and many alter proteins that are vital for neural processes. For two highly temperature-sensitive examples, recoding tunes protein function. For synaptotagmin, a key component of Ca2+-dependent neurotransmitter release, crystal structures and supporting experiments show that editing alters Ca2+ binding. For kinesin-1, a motor protein driving axonal transport, editing regulates transport velocity down microtubules. Seasonal sampling of wild-caught specimens indicates that temperature-dependent editing occurs in the field as well. These data show that A-to-I editing tunes neurophysiological function in response to temperature in octopus and most likely other coleoids.

Systemic lupus erythematosus (SLE) affects 1.5 million Americans. Lupus is a debilitating autoimmune disorder in which patients form antibodies that react to self-antigens. Anti-dsDNA antibodies bind to DNA, forming immune complexes (ICs) and deposit on organs. Over time accumulation of the ICs can lead to lupus nephritis, which will ultimately require a kidney transplant. Removing and breaking down the immune complexes from circulation and deposits would be beneficial in preventing further damage to organs. Some patients with SLE have a deficiency in Dnase1L3 (Dnase1-Like 3) enzyme caused by a pathogenic mutation in the Dnase1L3 gene. Dnase1L3 digests extracellular DNA in the peripheral circulation. In contrast to Dnase1, Dnase1L3 possesses the unique activity of digesting DNA complexed with other biological molecules in the form of microparticles and immune complexes; this activity is essential in preventing the creation of antibodies against self-antigens. Dnase1L3 degrades antigenic DNA complexes and is a potential therapeutic for SLE. Dnase1L3 has also been implicated in having a role in releasing inflammatory cytokines, but specific inhibitors block this action; it is still unclear what Dnase1L3 function is inhibited by these inhibitors. We have developed a unique bacterial expression system for Dnase1L3 and solved the X-ray crystal structure of the core enzyme without the C-terminal domain. I plan to determine the structure of the full-length enzyme bound to DNA to better understand the mechanism of action of Dnase1L3. To minimize any potential off-target side effects from a Dnase1L3 therapeutic, I am also investigating the inhibitors to elucidate the Dnase1L3 role that is blocked in the inflammatory pathway. The long-term goal of this project is to produce the most effective therapeutic for lupus nephritis.