Biology subjects

Keyel, P. A.

Publications and source records attributed to Keyel, P. A..

5 recordsLinked to original sources

bioRxiv2023

Pathogenic Mutations in the C2A Domain of Dysferlin form Amyloid that Activates the Inflammasome.

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 dysferlins C2A domain, namely C2AW52R and C2AV67D. 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 dysferlins ability to repair cells. The failure of C2AW52R and C2AV67D to initiate membrane repair arises from their propensity to form stable amyloid. The misfolding of the C2A domain caused by either mutation exposes {beta}-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-1{beta}. The present study suggests that the muscle dysfunction and inflammation evident in Limb-Girdle Muscular Dystrophy types-2B/2R, specifically in cases involving C2AW52R and C2AV67D, as well as other C2 domain mutations with considerable hydrophobic core involvement, may be attributed to this mechanism.

biophysics↗

bioRxiv2023

Deficiency of macrophage-derived Dnase1L3 causes lupus-like phenotypes in mice

Systemic Lupus Erythematosus (SLE) is a chronic autoimmune disease caused by environmental factors and loss of key proteins. One such protein is a serum endonuclease secreted by macrophages and dendritic cells, Dnase1L3. Loss of Dnase1L3 causes pediatric-onset lupus in humans is Dnase1L3. Reduction in Dnase1L3 activity occurs in adult-onset human SLE. However, the amount of Dnase1L3 necessary to prevent lupus onset, if the impact is continuous or requires a threshold, and which phenotypes are most impacted by Dnase1L3 remain unknown. To reduce Dnase1L3 protein levels, we developed a genetic mouse model with reduced Dnase1L3 activity by deleting Dnase1L3 from macrophages (cKO). Serum Dnase1L3 levels were reduced 67%, though Dnase1 activity remained constant. Sera were collected weekly from cKO and littermate controls until 50 weeks of age. Homogeneous and peripheral anti-nuclear antibodies were detected by immunofluorescence, consistent with anti-dsDNA antibodies. Total IgM, total IgG, and anti-dsDNA antibody levels increased in cKO mice with increasing age. In contrast to global Dnase1L3-/- mice, anti-dsDNA antibodies were not elevated until 30 weeks of age. The cKO mice had minimal kidney pathology, except for deposition of immune complexes and C3. Based on these findings, we conclude that an intermediate reduction in serum Dnase1L3 causes mild lupus phenotypes. This suggest that macrophage-derived DnaselL3 is critical to limiting lupus.

immunology↗

bioRxiv2022

Patch repair protects cells from the small pore-forming toxin aerolysin

Small pore-forming toxins in the aerolysin family lyse cells by damaging the membrane, but membrane repair responses used to resist them, if any, remain controversial. Four membrane repair mechanisms have been proposed: toxin removal by caveolar endocytosis, clogging by annexins, microvesicle shedding catalyzed by MEK, and patch repair. Which of these repair mechanisms aerolysin triggers is unknown. Furthermore, Ca2+ flux triggered by aerolysin is controversial, yet membrane repair responses require Ca2+. Here, we determined Ca2+ influx and repair mechanisms activated by aerolysin. In contrast to cholesterol-dependent cytolysins (CDCs), removal of extracellular Ca2+ protected cells from aerolysin. Aerolysin triggered sustained Ca2+ influx. Since aerolysin triggered Ca2+ flux, we investigated Ca2+-dependent repair pathways. Caveolar endocytosis failed to protect cells from aerolysin or CDCs. MEK-dependent repair did not protect against aerolysin. Aerolysin triggered slower annexin A6 membrane recruitment compared to CDCs. In contrast to CDCs, expression of the patch repair protein dysferlin potently protected cells from aerolysin. We propose that aerolysin triggers a Ca2+-dependent death mechanism that obscures repair responses, and the primary repair mechanism used to resist aerolysin is patch repair. We conclude that different classes of bacterial toxins trigger distinct repair mechanisms.

cell biology↗

bioRxiv2022

Sphingolipids protect ergosterol in the Leishmania major membrane from sterol-binding toxins

Susceptibility of Leishmania to the first line treatment amphotericin B remains poorly understood. Amphotericin B targets ergosterol, so one approach to improving drug efficacy and reducing side effects could be improving access to ergosterol. While the surface exposure of ergosterol in Leishmania is unknown, sterols in mammalian cells can be sheltered from sterol-binding agents by membrane components, including sphingolipids. Here, we tested the ability of the Leishmania major sphingolipids inositol phosphorylceramide (IPC), and ceramide to shelter ergosterol by preventing binding and cytotoxicity of the sterol-specific toxins streptolysin O and perfringolysin O using flow cytometry. In contrast to mammalian systems, Leishmania sphingolipids did not preclude toxin binding to sterols in the membrane. However, IPC interfered with cytotoxicity. Ceramide reduced perfringolysin O, but not streptolysin O, cytotoxicity in cells. Ceramide sensing was controlled by the toxin L3 loop. Ceramide was sufficient to protect L. major promastigotes from amphotericin B. We propose a mechanism whereby pore-forming toxins engage additional lipids like ceramide to determine the optimal environment to sustain pore formation. Thus, L. major offers a genetically tractable model organism for understanding toxin-membrane interactions. Furthermore, our findings suggest targeting ceramide may enhance the efficacy of ergosterol-targeting anti-leishmanial drugs. Abstract ImportanceLeishmaniasis is a neglected tropical disease with [~]1.5-2 million new cases and [~]70,000 deaths annually. One first-line treatment for leishmaniasis is liposomal amphotericin B, which is expensive and damages the kidneys. Cost and side effects can be minimized by improving efficacy. To improve efficacy, we must learn how amphotericins target--ergosterol--is protected by other components of Leishmania. The human ergosterol equivalent is protected by components called sphingolipids. We tested the ability of sphingolipids to protect ergosterol using pore-forming toxins. Pore-forming toxins use ergosterol to bind and kill Leishmania. Unlike human cells, toxins bound to ergosterol--indicating that they had access--when sphingolipids were present. However, sphingolipids protected Leishmania from toxins and amphotericin. Thus, Leishmania organizes sterol-protective components differently from humans. Further, toxins and Leishmania serve as a system to understand fundamental rules governing sterol-protecting component membrane organization. We can use this information to help improve drugs targeting sterols.

microbiology↗

bioRxiv2022

Structural features of Dnase1L3 responsible for serum antigen clearance

Autoimmunity develops when extracellular DNA released from dying cells is not cleared from serum. While serum DNA is primarily digested by Dnase1 and Dnase1L3, Dnase1 does not rescue autoimmunity arising from Dnase1L3 deficiencies. Dnase1L3 uniquely degrades antigenic forms of cell-free DNA, including DNA complexed with lipids and proteins. The distinct activity of Dnase1L3 relies on its unique C-terminal Domain (CTD), but the mechanism is unknown. We used multiple biophysical techniques and functional assays to study the interplay between the core catalytic domain and the CTD. While the core domain resembles Dnase1, there are several key differences between the two enzymes. Dnase1L3 is not inhibited by actin due to multiple differences in the actin recognition site. The CTD augments the ability of the core to bind DNA, thereby facilitating the degradation of complexed DNA to prevent autoimmune pathology. Together, these structural insights will inform the development of Dnase1L3-based therapies for autoimmunity.

biochemistry↗