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The membrane attack complex (MAC) is one of the immune system’s first responders. Complement proteins assemble on target membranes to form pores that lyse pathogens and impact tissue homeostasis of self-cells. How MAC disrupts the membrane barrier remains unclear. Here we use electron cryo-microscopy and flicker spectroscopy to show that MAC interacts with lipid bilayers in two distinct ways. Whereas C6 and C7 associate with the outer leaflet and reduce the energy for membrane bending, C8 and C9 traverse the bilayer increasing membrane rigidity. CryoEM reconstructions reveal plasticity of the MAC pore and demonstrate how C5b6 acts as a platform, directing assembly of a giant β-barrel whose structure is supported by a glycan scaffold. Our work provides a structural basis for understanding how β-pore forming proteins breach the membrane and reveals a mechanism for how MAC kills pathogens and regulates cell functions. The complement membrane attack complex (MAC) is a lytic immune pore that kills pathogens. Here the authors use cryoEM to provide a structural and biophysical mechanism for how β-pore forming proteins breach the lipid bilayer, providing pathways to explore pore-formation in molecular detail.

The complement system is a major component of the innate immune system that works through the cytolytic effect of the membrane attack complex (MAC). Complement component 7 (C7) is essential for MAC assembly and its precisely regulated expression level is crucial for the cytolytic activity of MAC. We show that C7 is specifically expressed by the stromal cells in both mouse and human prostates. The expression level of C7 inversely correlates with clinical outcomes in prostate cancer. C7 is positively regulated by androgen signaling in the mouse prostate stromal cells. The androgen receptor directly transcriptionally regulates the mouse and human C7 . Increasing C7 expression in the C57Bl/6 syngeneic RM-1 and Pten-Kras allografts suppresses tumor growth in vivo. Conversely, C7 haploinsufficiency promotes tumor growth in the transgenic adenocarcinoma of the mouse prostate (TRAMP) model. Interestingly, replenishing C7 in androgen-sensitive Pten-Kras tumors during androgen depletion only slightly enhances cellular apoptosis, highlighting the diverse mechanisms employed by tumors to counteract complement activity. Collectively, our research indicates that augmenting complement activity could be a promising therapeutic approach to impede the development of castration resistance in prostate cancer.

HIV-1 genetic differences influence viral replication and progression to AIDS. HIV-1 circulating recombinant form (CRF)02_AG is the predominant viral subtype infecting humans in West and Central Africa, but its effects on HIV neuropathogenesis are not known. In the present study, we investigated the effects of Tat proteins from HIV-1 subtype B (Tat.B) and HIV-1 CRF02_AG (Tat.AG) on primary human brain microvascular endothelial cells (HBMEC), the major component of the blood–brain barrier (BBB). Using Affymetrix GeneChip Human Gene 1.0.ST arrays, we showed that Tat.AG had minimal effects while Tat.B induced transcriptional upregulation of 90 genes in HBMEC, including proinflammatory chemokines, complement components C3, C7, and complement factor B, matrix metalloproteinases (MMP)-3, MMP-10, and MMP-12. These results were confirmed by real-time PCR. Compared with Tat.AG, Tat.B significantly increased MMP-3, MMP-10, and MMP-12 activities in HBMEC, and the MMPs tissue inhibitor of metalloproteinase-2 blocked Tat-induced increase in MMPs activity. Western blot analyses also showed that Tat increased the expression of C3 and its cleaved fragment C3b in HBMEC. These data suggest that genetic differences between HIV-1 subtypes B and CRF02_AG influence the effects of Tat proteins from these two clades on HBMEC, including molecular and cellular functions, and canonical pathways, which would affect BBB dysfunction and viral neuropathogenesis.

The loss of photoreceptors is the defining characteristic of many retinal degenerative diseases, but the mechanisms that regulate photoreceptor cell death are not fully understood. Here we have used the 661W cone photoreceptor cell line to ask whether exposure to the terminal complement complex C5b-9 induces cell death and/or modulates the sensitivity of these cells to other cellular stressors. 661W cone photoreceptors were exposed to complete normal human serum following antibody blockade of CD59. Apoptosis induction was assessed morphologically, by flow cytometry, and on western blotting by probing for cleaved PARP and activated caspase-3. Necroptosis was assessed by flow cytometry and Sirtuin 2 inhibition using 2-cyano-3-[5-(2,5-dichlorophenyl)-2-furyl]- N -5-quinolinylacrylamide (AGK2). The sensitivity of 661W cells to ionomycin, staurosporine, peroxide and chelerythrine was also investigated, with or without prior formation of C5b-9. 661W cells underwent apoptotic cell death following exposure to C5b-9, as judged by poly(ADP-ribose) polymerase 1 cleavage and activation of caspase-3. We also observed apoptotic cell death in response to staurosporine, but 661W cells were resistant to both ionomycin and peroxide. Interestingly, C5b-9 significantly increased 661W sensitivity to staurosporine-induced apoptosis and necroptosis. These studies show that low levels of C5b-9 on 661W cells can induce apoptosis, and that C5b-9 specifically sensitizes 661W cells to certain apoptotic and necroptotic pathways. Our observations provide new insight into the potential role of the complement system in photoreceptor loss, with implications for the molecular aetiology of retinal disease.

Defense mechanisms in developing fish are poorly known but before maturation of lymphoid organs and immunocompetence, innate mechanisms are essential. The complement system represents a major part of innate immunity. Our main objective was to map the presence of complement components early in fish development. Rainbow trout eggs, embryos, and hatchlings were assayed for the onset and duration of C3-1, C3-3, C3-4, C4, C5, C7, factor B, and factor D transcription using real-time reverse transcription-polymerase chain reaction. In general, complement transcript levels increased steadily from day 28 postfertilization to hatch, followed by a decrease during yolk-sac resorption. All the complement proteins studied were found in unfertilized eggs. There was no correlation between the transcript and protein levels throughout the study period. Complement proteins appeared in the liver, kidney, and intestine between day 7 and 35 but not until day 77 in the heart. This study is the first to address the ontogeny of several complement components and represents the first evidence that maternal transfer of complement components, other than C3, occurs in teleost fish.

Earlier studies have shown that sequential treatment of resealed erythrocyte ghosts with C5b6, C7, C8, and C9 leads to insertion of hydrophobic peptides from these complement proteins into the membrane and assembly of transmembrane channels. The number of molecules of each of the proteins required for assembly of the membrane-associated channel structure was evaluated by measuring the quantitative relationship between the doses of the individual proteins and the release of two trapped markers, sucrose and inulin, from ghosts after channel formation. The incubation period was sufficient to attain equilibrium of marker distribution between the ghosts and the extracellular fluid. Two markers of different size (sucrose and inulin, 0.9 and 3 nm molecular diameter, respectively) were used in order to develop information on the molecular composition of small and large channels, respectively. We found that participation of C5b6, C7, and C8 in channel formation displayed one-hit characteristics, regardless of marker size. By contrast, the participation of C9 was one-hit with respect to the sucrose marker, whereas with respect to the inulin marker the C9 reaction was multi-hit. Our results are compatible with the view that these markers are released through a channel structure in the membrane that is a monomer of C5b-9 of the composition C5b61C71C81C9n, in which n = 1 for channels permitting passage of sucrose and n = 2 for channels allowing transit of inulin.

The molecular basis of the membranolytic activity of the membrane attack complex (MAC) of complement was investigated. By using density gradient equilibrium ultracentrifugation, the binding of egg yolk lecithin to the isolated MAC and to its intermediate complexes and precursor proteins was measured. No stable phospholipid-protein complexes were formed with the MAC precursor components C5b-6, C7, C8, and C9. Stable complexes of phospholipid and protein were formed by C5b-7, C5b-8, C5b-9, and the MAC (C5b-9 dimer) and they exhibited densities of 1.2164, 1.184, 1.2055, and 1.2275 g/ml, respectively. The molar phospholipid/protein ratios for the four complexes were determined to be: C5b-7, 399:1; C5b-8, 841:1; C5b-9, 918:1, and C5b-9 dimer, 1460:1. Electron microscopy of the isolated phospholipid-protein complexes revealed no lipid bilayer structures. The magnitude of the phospholipid binding capacity of the MAC is consistent with the interpretation that the MAC forms phospholipid-protein mixed in micelles in lipid bilayers and biological membranes and thus causes formation of hydrophilic lipid channels.

To evaluate the effect of membrane lipid acylchain packing on the efficiency of cell lysis by complement, we have studied membrane modulation by 2-(2-methoxy)-ethoxyethyl-8-(cis-2-n-octylcyclopropyl)-octanoate (A2C) and by myristoleyl alcohol, the cis isomer of a C14:1 aliphatic alochol. These substances are known to increase the membrane lipid disorder by virtue of the bend in their acyl chains, which is believed to loosen the phospholipid acyl-chain packing. We have found that both of these compounds markedly enhance the lysis of erythrocytes by the terminal complement proteins C5b--9. The enhancing effect by A2C is operative in the formation of erythrocytes carrying complement components C5b, C6, and C7, as well as in the subsequent reactions with complement components C8 and C9. We have also found that A2C-treated erythrocytes bind C5b6 to a measurable extent, whereas untreated erythrocytes do not. We attribute this to a shift in the partition equilibrium of C5b6 toward membrane association, which would improve lytic efficiency. The increase of membrane lipid disorder by these agents would also be expected to increase insertion of hydrophobic peptides from C7, C8, and C9, with consequent gain in lytic efficiency. Treatment of erythrocytes with sublytic doses of NaDodSO4or Triton X-100 did not enhance lysis by C5b--9 appreciably, suggesting that enhancement of lysis by C5b--9 is not a general property of amphiphiles.

Lipid vesicles carrying the purified membrane C5b-9 complex [C5b-9(m)] of complement were analyzed immunochemically and in the electron microscope after treatment with a combination of trypsin and $\\alpha $-chymotrypsin. Under reducing conditions, the externally oriented annulus was removed. The remaining part of the C5b-9(m), representing approximately half of the total mass of the macromolecular complex, was visualized in the electron microscope as a hollow cylindrical structure with walls of 1-nm thickness. This structure remained tenaciously attached to the lipid bilayer, projecting 8-9 nm from the external membrane surface into the aqueous environment. Cleavage of C5b-9(m) by proteolysis and reduction resulted in a sharp reduction of its antigenic determinants. One hydrophilic protease-resistant C5 derivative was released from the membrane and recovered in the fluid phase. The membrane-bound residue almost totally lacked antigens precipitable with antisera to C5, C6, C9, and C5b-9(m).

Diabetic nephropathy (DN) is the leading cause of end-stage renal disease, but it remains relatively underdiagnosed. In this study, we aimed to explore the key regulatory pathways and potential biomarkers related to DN using integrated bioinformatics analysis and validation. First, the microarray data of the GSE30528 and GSE96804 datasets were downloaded from the Gene Expression Omnibus (GEO) database, and differentially expressed genes (DEGs) were screened. Then, weighted gene coexpression network analysis (WGCNA), gene ontology (GO) annotation, gene set enrichment analysis (GSEA) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed to identify key pathways and genes. qRT-PCR and receiver operating characteristic (ROC) curves were used to validate our results. Furthermore, single-cell RNA sequencing (scRNA-seq) data were reanalyzed to investigate the expression specificity of C7 in DN cells. An online database search and luciferase reporter assay identified the target relationship between miRNAs and C7. The \"complement and coagulation cascades\" were significantly enriched, and complement C3 and C7 were candidate markers. The receiver operating characteristic (ROC) curve revealed that C7 had significant diagnostic value (AUC=0.865) in DN. Through scRNA-seq reanalysis, we found that C7 was specifically elevated in mesangial (MES) cells of DN. Moreover, we found that the expression of C7 was regulated by miR-494-3p and miR-574-5p. This is the first study to reveal that C7 is specifically expressed in mesangial cells, is a potential diagnostic biomarker for diabetic nephropathy, and is regulated by miR-494-3p and miR-574-5p.