β-glycoprotein I (βGPI) is an abundant multidomain plasma protein that plays various roles in the clotting and complement cascades. It is also the main target of antiphospholipid antibodies (aPL) in the acquired coagulopathy known as antiphospholipid syndrome (APS). Previous studies have shown that βGPI adopts two interconvertible biochemical conformations, oxidized and reduced, depending on the integrity of the disulfide bonds. However, the precise contribution of the disulfide bonds to βGPI structure and function is unknown. Here, we substituted cysteine residues with serine to investigate how the disulfide bonds C32-C60 in domain I (DI) and C288-C326 in domain V (DV) regulate βGPI’s structure and function. Results of our biophysical and biochemical studies support the hypothesis that the C32-C60 disulfide bond plays a structural role, whereas the disulfide bond C288-C326 is allosteric. We demonstrate that absence of the C288-C326 bond, unlike absence of the C32-C60 bond, diminishes membrane binding without affecting the thermodynamic stability and overall structure of the protein, which remains elongated in solution. We also document that, while absence of the C32-C60 bond directly impairs recognition of βGPI by pathogenic anti-DI antibodies, absence of the C288-C326 disulfide bond is sufficient to abolish complex formation in the presence of anionic phospholipids. We conclude that the disulfide bond C288-C326 operates as a molecular switch capable of regulating βGPI’s physiological functions in a redox-dependent manner. We propose that in APS patients with anti-DI antibodies, selective rupture of the C288-C326 disulfide bond may be a valid strategy to lower the pathogenic potential of aPL.

Increased levels of circulating complement activation products have been reported in COVID-19 patients, but only limited information is available on complement involvement at the tissue level. The mechanisms and pathways of local complement activation remain unclear. The aim of this study was to investigate the deposition of complement components in the lungs, kidneys, and liver in patients with COVID-19 patients and to determine the pathway/s of complement activation. We performed immunofluorescence analyses of autopsy specimens of lungs, kidney, and liver from 12 COVID-19 patients who died of acute respiratory failure. Snap-frozen samples embedded in OCT were stained with antibodies against complement components and activation products, IgG, and spike protein of SARS-CoV-2. Lung deposits of C1q, C4, C3, and C5b-9 were localized in the capillaries of the interalveolar septa and on alveolar cells. IgG displayed a similar even distribution, suggesting classical pathway activation. The spike protein is a potential target of IgG, but its uneven distribution suggests that other viral and tissue molecules may be targeted by IgG. FB deposits were also seen in COVID-19 lungs and are consistent with activation of the alternative pathway, whereas MBL and MASP-2 were hardly detectable. Analysis of kidney and liver specimens mirrored findings observed in the lung. Complement deposits were seen on tubules and vessels of the kidney with only mild C5b-9 staining in glomeruli, and on the hepatic artery and portal vein of the liver. Complement deposits in different organs of deceased COVID-19 patients caused by activation of the classical and alternative pathways support the multi-organ nature of the disease and the contribution of the complement system to inflammation and tissue damage.

Nanocomposite hydrogels containing two-dimensional nanosilicates (NS) have emerged as a new technology for the prolonged delivery of biopharmaceuticals. However, little is known about the physical-chemical properties governing the interaction between NS and proteins and the release profiles of NS-protein complexes in comparison to traditional poly(ethylene glycol) (PEG) hydrogel technologies. To fill this gap in knowledge, we fabricated a nanocomposite hydrogel composed of PEG and laponite and identified simple but effective experimental conditions to obtain sustained protein release, up to 23 times slower as compared to traditional PEG hydrogels, as determined by bulk release experiments and fluorescence correlation spectroscopy. Slowed protein release was attributed to the formation of NS-protein complexes, as NS-protein complex size was inversely correlated with protein diffusivity and release rates. While protein electrostatics, protein concentration, and incubation time were important variables to control protein-NS complex formation, we found that one of the most significant and less appreciated variable to obtain a sustained release of bioactive proteins was the buffer chosen for preparing the initial suspension of NS particles. The buffer was found to control the size of nanoparticles, the absorption potential, morphology, and stiffness of hydrogels. From these studies, we conclude that the PEG-laponite composite fabricated is a promising new platform for sustained delivery of positively charged protein therapeutics.

(Mtb), the pathological agent that causes tuberculosis (TB) is the number one infectious killer worldwide with one fourth of the world’s population currently infected. Data indicate that γδ T cells secrete Granzyme A (GzmA) in the extracellular space triggering the infected monocyte to inhibit growth of intracellular mycobacteria. Accordingly, deletion of from γδ T cells reverses their inhibitory capacity. Through mechanistic studies, GzmA’s action was investigated in monocytes from human PBMCs. The use of recombinant human GzmA expressed in a mammalian system induced inhibition of intracellular mycobacteria to the same degree as previous human native protein findings. Our data indicate that: 1) GzmA is internalized within mycobacteria-infected cells, suggesting that GzmA uptake could prevent infection and 2) that the active site is not required to inhibit intracellular replication. Global proteomic analysis demonstrated that the ER stress response and ATP producing proteins were upregulated after GzmA treatment, and these proteins abundancies were confirmed by examining their expression in an independent set of patient samples. Our data suggest that immunotherapeutic host interventions of these pathways may contribute to better control of the current TB epidemic.

Autoantibodies targeting prothrombin (aPT) can be found in antiphospholipid syndrome (APS) patients. However, their detection has proven difficult to standardize. Here, we developed a new ELISA assay to improve the identification of aPT and compared its performance with currently available anti-phosphatidylserine/prothrombin antibodies (aPS/PT) and autoantibodies targeting prothrombin bound to the plastic plate (aPT-A) assays using a cohort of 27 APS patients at high risk of thrombosis. We generated a novel prothrombin variant, ProTS525A-Biot, carrying an artificial tag at the C-terminus suitable for site-specific biotinylation and added the mutation S525A to improve stability. ProTS525A-Biot was immobilized to neutravidin-coated plates at the desired density and with a defined orientation, i.e., pointing the N-terminal fragment-1 toward the solvent. Antibodies against ProTS525A-Biot (aPT-Bio) were found in 24 out of 27 triple-positive APS patients (88%). When compared to aPS/PT and aPT-A, aPT-Bio showed an excellent linear correlation with aPS/PT (R 0.85) but not with aPT-A (R 0.40). Since aPS/PT but not aPT-A are an emerging biomarker of thrombosis in APS, this method may find utility for detecting pathogenic aPT in APS but also other prothrombotic conditions such as COVID-19.