Search

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 Dnase1L3in 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.Secretion of the endonuclease Dnase1L3 by macrophages limits lupus because its loss from macrophages induces autoantibodies, including anti-double-stranded DNA antibodies. Myeloid-derived DnaselL3 limits serum lupus phenotypes.

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 vivogene 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 vivoAAV8-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.

Dysferlinopathy is an autosomal recessive muscular dystrophy characterized by the progressive loss of motility that is caused by mutations throughout the DYSF gene. There are currently no approved therapies that ameliorate or reverse dysferlinopathy. Gene delivery using adeno-associated vectors (AAVs) is a leading therapeutic strategy for genetic diseases; however, the large size of dysferlin cDNA (6.2 kB) precludes packaging into a single AAV capsid. Therefore, using 3D structural modeling and hypothesizing dysferlin C2 domain redundancy, a 30% smaller, dysferlin-like molecule amenable to single AAV vector packaging was engineered (termed Nano-Dysferlin). The intracellular distribution of Nano-Dysferlin was similar to wild-type dysferlin and neither demonstrated toxicity when overexpressed in dysferlin-deficient patient myoblasts. Intramuscular injection of AAV-Nano-Dysferlin in young dysferlin-deficient mice significantly improved muscle integrity and decreased muscle turnover 3 weeks after treatment, as determined by Evans blue dye uptake and central nucleated fibers, respectively. Systemically administered AAV-Nano-Dysferlin to young adult dysferlin-deficient mice restored motor function and improved muscle integrity nearly 8 months after a single injection. These preclinical data are the first report of a smaller dysferlin variant tailored for AAV single particle delivery that restores motor function and, therefore, represents an attractive candidate for the treatment of dysferlinopathy.

Synaptotagmin (syt)-1 is a Ca2+sensor that triggers rapid synaptic vesicle exocytosis. Mutations that disrupt physical interactions between the tandem Ca2+-sensing modules (C2 domains) of syt-1 disrupt regulated membrane fusion in reconstituted fusion reactions and in neurons. Hence contacts between these domains are important for function.

The C2A domain is one of two calcium ion (Ca2+)- and membrane-binding domains within synaptotagmin I (Syt I), the identified Ca2+sensor for regulated exocytosis of neurotransmitter. We propose that the mechanistic basis for C2A's response to Ca2+and cellular function stems from marginal stability and ligand-induced redistributions of protein conformers. To test this hypothesis, we used a combination of calorimetric and fluorescence techniques. We measured free energies of stability by globally fitting differential scanning calorimetry and fluorescence lifetime spectroscopy denaturation data, and found that C2A is weakly stable. Additionally, using partition functions in a fluorescence resonance energy transfer approach, we found that the Ca2+- and membrane-binding sites of C2A exhibit weak cooperative linkage. Lastly, a dye-release assay revealed that the Ca2+- and membrane-bound conformer subset of C2A promote membrane disruption. We discuss how these phenomena may lead to both cooperative and functional responses of Syt I.

Synaptotagmin 1 (Syt1) is the Ca+2 receptor for fast, synchronous vesicle fusion in neurons. Because membrane fusion is an inherently mechanical, force-driven event, Syt1 must be able to adapt to the energetics of the fusion apparatus. Syt1 contains two C2 domains (C2A and C2B) that are homologous in sequence and three-dimensional in structure; yet, a number of observations have suggested that they have distinct biochemical and biological properties. In this study, we analyzed the mechanical stability of the C2A and C2B domains of human Syt1 using single-molecule atomic force microscopy. We found that stretching the C2AB domains of Syt1 resulted in two distinct unfolding force peaks. The larger force peak of ∼100 pN was identified as C2B and the second peak of ∼50 pN as C2A. Furthermore, a significant fraction of C2A domains unfolded through a low force intermediate that was not observed in C2B. We conclude that these domains have different mechanical properties. We hypothesize that a relatively small stretching force may be sufficient to deform the effector-binding regions of the C2A domain and modulate the affinity for soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptors (SNAREs), phospholipids, and Ca+2.

Myofibril assembly and disassembly are complex processes that regulate overall muscle mass. Titin kinase has been implicated as an initiating catalyst in signaling pathways that ultimately result in myofibril growth. In titin, the kinase domain is in an ideal position to sense mechanical strain that occurs during muscle activity. The enzyme is negatively regulated by intramolecular interactions occurring between the kinase catalytic core and autoinhibitory/regulatory region. Molecular dynamics simulations suggest that human titin kinase acts as a force sensor. However, the precise mechanism(s) resulting in the conformational changes that relieve the kinase of this autoinhibition are unknown. Here we measured the mechanical properties of the kinase domain and flanking Ig/Fn domains of the Caenorhabditis elegans titin-like proteins twitchin and TTN-1 using single-molecule atomic force microscopy. Our results show that these kinase domains have significant mechanical resistance, unfolding at forces similar to those for Ig/Fn β-sandwich domains (30–150pN). Further, our atomic force microscopy data is consistent with molecular dynamic simulations, which show that these kinases unfold in a stepwise fashion, first an unwinding of the autoinhibitory region, followed by a two-step unfolding of the catalytic core. These data support the hypothesis that titin kinase may function as an effective force sensor.