Explore the vast range of titles available.

CD59 is an abundant immuno-regulatory receptor that protects human cells from damage during complement activation. Here we show how the receptor binds complement proteins C8 and C9 at the membrane to prevent insertion and polymerization of membrane attack complex (MAC) pores. We present cryo-electron microscopy structures of two inhibited MAC precursors known as C5b8 and C5b9. We discover that in both complexes, CD59 binds the pore-forming β-hairpins of C8 to form an intermolecular β-sheet that prevents membrane perforation. While bound to C8, CD59 deflects the cascading C9 β-hairpins, rerouting their trajectory into the membrane. Preventing insertion of C9 restricts structural transitions of subsequent monomers and indirectly halts MAC polymerization. We combine our structural data with cellular assays and molecular dynamics simulations to explain how the membrane environment impacts the dual roles of CD59 in controlling pore formation of MAC, and as a target of bacterial virulence factors which hijack CD59 to lyse human cells. CD59 protects human cells from damage by the MAC immune pore. The authors show how CD59 inhibits MAC, by deflecting pore-forming β-hairpins of complement proteins. As well as how the membrane environment influences the role of CD59 in complement regulation and in host-pathogen interactions.

The abnormal formation of the membrane attack complex (MAC) is intrinsically linked to a range of acute and chronic immune diseases. The insertion of complement C9 into the membrane is the final step and kinetic bottleneck of MAC formation. However, research on blocking the MAC formation of C9 is currently limited. Given its broad, flat, and polar functional interface, complement C9 is a challenging target for rational design. Here, we utilize deep learning-based methods for protein scaffold generation, sequence design, and complex structure prediction to de novo design mini-protein inhibitors that specifically block the membrane insertion of soluble complement C9. The binding affinity of the mini-protein inhibitor is further optimized to 700 pM via partial diffusion. Design accuracy and binding specificity are verified through X-ray crystallography and biochemical studies. An in vivo acute hemolysis inhibition assay reveals that the C9 mini-protein inhibitors remain effective against hemolysis even 8 minutes after complement activation, outperforming the complement C5 inhibitor eculizumab. The de novo designed C9 mini-protein inhibitors can offer an optional therapeutic approach for the prevention and treatment of acute or chronic immune diseases associated with abnormal complement activation. In this work the authors employed deep learning-based methods to design mini-protein that block membrane insertion of complement C9. These well performed inhibitors provide an alternative approach for preventing diseases associated with abnormal complement activation.

The Gram-negative bacterium Klebsiella pneumoniae is an important human pathogen. Its treatment has been complicated by the emergence of multi-drug resistant strains. The human complement system is an important part of our innate immune response that can directly kill Gram-negative bacteria by assembling membrane attack complex (MAC) pores into the bacterial outer membrane. To resist this attack, Gram-negative bacteria can modify their lipopolysaccharide (LPS). Especially the decoration of the LPS outer core with the O-antigen polysaccharide has been linked to increased bacterial survival in serum, but not studied in detail. In this study, we characterized various clinical Klebsiella pneumoniae isolates and show that expression of the LPS O1-antigen correlates with resistance to complement-mediated killing. Mechanistic data reveal that the O1-antigen does not inhibit C3b deposition and C5 conversion. In contrast, we see more efficient formation of C5a, and deposition of C6 and C9 when an O-antigen is present. Further downstream analyses revealed that the O1-antigen prevents correct insertion and polymerization of the final MAC component C9 into the bacterial membrane. Altogether, we show that the LPS O1-antigen is a key determining factor for complement resistance by K. pneumoniae and provide insights into the molecular basis of O1-mediated MAC evasion.

Complement component 9 (C9) functions as the pore-forming component of the Membrane Attack Complex (MAC). During MAC assembly, multiple copies of C9 are sequentially recruited to membrane associated C5b8 to form a pore. Here we determined the 2.2 Å crystal structure of monomeric murine C9 and the 3.9 Å resolution cryo EM structure of C9 in a polymeric assembly. Comparison with other MAC proteins reveals that the first transmembrane region (TMH1) in monomeric C9 is uniquely positioned and functions to inhibit its self-assembly in the absence of C5b8. We further show that following C9 recruitment to C5b8, a conformational change in TMH1 permits unidirectional and sequential binding of additional C9 monomers to the growing MAC. This mechanism of pore formation contrasts with related proteins, such as perforin and the cholesterol dependent cytolysins, where it is believed that pre-pore assembly occurs prior to the simultaneous release of the transmembrane regions. The Complement component 9 (C9) is the pore-forming component of the Membrane Attack Complex which targets pathogens. Here authors use structural biology to compare monomeric C9 to C9 within the polymeric assembly and identify the element which inhibits C9 self-assembly in the absence of the target membrane.

Increasing evidence points toward an essential role for complement activation in the pathogenesis of diabetic kidney disease (DKD). However, the precise molecular mechanisms remain unclear, and the pathway predominantly contributing to complement activation in DKD is of particular interest. In this study, the glomerular proteome, especially the profiles of the complement proteins, was analyzed in kidney biopsies from 40 DKD patients and 10 normal controls using laser microdissection-assisted liquid chromatography-tandem mass spectrometry (LMD-LC-MS/MS). The glomerular abundances of three proteins related to classical pathway (CP) (C1q, C1r, C1s), five proteins related to alternative pathway (AP) (CFB, CFH, CFHR1, CFHR3, CFHR5), one common protein related to CP and lectin pathway (LP) (C4), and six proteins related to terminal complement pathway (C3, C5, C6, C7, C8, C9) were significantly increased in DKD. Notably, none of the proteins unique to the lectin complement pathway, including mannose-binding lectin (MBL) and its associated proteins, were detected in DKD glomeruli. Furthermore, the glomerular complement proteins of CP and AP were positively correlated with glomerular pathological grades and proteinuria, and negatively correlated with eGFR in DKD patients. Our results highlight a critical role for complement activation of the CP and AP, rather than the LP, in DKD progression.

Gastrointestinal (GI) cancer patients undergoing surgery often face immunosuppression, increasing postoperative risk. Immunomodulatory enteral nutrition (IEN) may enhance immune function and recovery, but mechanisms remain unclear. This study compared plasma proteomic profiles of patients receiving IEN versus standard enteral nutrition (SEN) to explore pathways linked to outcomes. This analysis extended a previously published randomized clinical trial in GI cancer patients who received SEN or IEN postoperatively, with 50 patients in each group. The IEN was rich in arginine, nucleotides, vitamin B12, chloride, vitamin C, selenium, chromium, and molybdenum. Plasma samples were analyzed using mass spectrometry–based proteomics (MassLynx v4.1 and Progenesis v4.1). Proteins consistently detected across replicates (database search P < 0.05) were identified. Clinical outcomes, including complications and biochemical markers, were integrated with proteomic findings to interpret biological mechanisms. Distinct proteomic profiles were observed. The IEN group showed higher levels of complement proteins (C3, C5, C9), inter-alpha-trypsin inhibitor heavy chain H2, pregnancy zone protein, immunoglobulins, and apolipoproteins—proteins linked to immune modulation, tissue repair, and inflammation control. The SEN group displayed elevated acute-phase proteins and coagulation factors, including fibrinogen and serum amyloid A-4, consistent with a pro-inflammatory, hypercoagulable state. These differences paralleled clinical results, with the IEN group experiencing fewer complications and improved albumin/globulin ratios. This exploratory study suggests that immunonutrition may modulate complement activation and immune pathways, supporting better postoperative outcomes in GI cancer patients. The proteomic profile provides evidence that supports a mechanistic hypothesis underlying the observed clinical benefits. Future quantitative proteomics with larger cohorts is warranted to validate these findings and optimize perioperative nutrition strategies. To summarize our findings, Figure 1 illustrates the key proteomic differences between the standard and immunomodulatory enteral nutrition groups, highlighting the pathways potentially underlying the improved clinical outcomes observed. [Display omitted] •Clinical-proteomic integration: IEN links outcomes to unique signatures.•IEN raises complement, immunoglobulins, and matrix for defense and repair.•IEN nutrients modulate immunity, oxidative stress, and metabolic pathways.•Standard nutrition increases acute-phase proteins and pro-thrombotic markers.•Findings support immunonutrition to enhance recovery in GI cancer surgery.

Background Emerging data suggest a complex pathophysiology of chronic subdural hematoma (CSDH) to which an inflammatory response might contribute. The complement system is activated in acute traumatic setting, although its role in CSDH is unknown. To investigate the complement system in CSDH pathophysiology, we analyzed blood and hematoma fluid biomarkers, as well as immunohistochemistry of the CSDH membrane and dura. Materials and Methods We simultaneously collected CSDH fluid and peripheral blood from 20 CSDH patients at the time of surgery. Biopsies of the dura mater and the CSDH capsule were obtained and analyzed by immunohistochemistry for C5b-C9 or C5a deposition. Biomarkers of inflammation and complement activation were analyzed by a 21-multiplex assay, including Adiponectin, Clusterin, Complement factor C9 and CRP. Complement factor C5a was analyzed separately by a commercial R-plex electrochemiluminescence assay. Results Ten biomarkers differed significantly between peripheral blood and paired CSDH of which two were significantly increased in CSDH fluid (Clusterin and Cystatin C). Eight of the significantly altered biomarkers were significantly decreased in CSDH fluid, including C5a, Complement 9 and Adiponectin. There was no immunoreactivity for C5a or the C5b-C9 membrane attack complex in the dura or CSDH membrane. Conclusions In CSDH levels of the complement inhibitor Clusterin were increased, whereas levels of C5a and C9 were decreased. Membrane attack complex C5b-C9 was not detected in the membrane or dura surrounding the CSDH. Inhibition of complement could lead to reduced clearance of debris in the CSDH as well as secondary inflammatory reactions.

The complement system comprises the frontline of the innate immune system. Triggered by pathogenic surface patterns in different pathways, the cascade concludes with the formation of a membrane attack complex (MAC; complement components C5b to C9) and C5a, a potent anaphylatoxin that elicits various inflammatory signals through binding to C5a receptor 1 (C5aR1). Despite its important role in pathogen elimination, priming and recruitment of myeloid cells from the immune system, as well as crosstalk with other physiological systems, inadvertent activation of the complement system can result in self-attack and overreaction in autoinflammatory diseases. Consequently, it constitutes an interesting target for specialized therapies. The paradigm of safe and efficacious terminal complement pathway inhibition has been demonstrated by the approval of eculizumab in paroxysmal nocturnal hematuria. In addition, complement contribution in rare kidney diseases, such as lupus nephritis, IgA nephropathy, atypical hemolytic uremic syndrome, C3 glomerulopathy, or antineutrophil cytoplasmic antibody-associated vasculitis has been demonstrated. This review summarizes the involvement of the terminal effector agents of the complement system in these diseases and provides an overview of inhibitors for complement components C5, C5a, C5aR1, and MAC that are currently in clinical development. Furthermore, a link between increased complement activity and lung damage in severe COVID-19 patients is discussed and the potential for use of complement inhibitors in COVID-19 is presented.

Complement proteins can form membrane attack complex (MAC) pores that directly kill Gram-negative bacteria. MAC pores assemble by stepwise binding of C5b, C6, C7, C8 and finally C9, which can polymerize into a transmembrane ring of up to 18 C9 monomers. It is still unclear if the assembly of a polymeric-C9 ring is necessary to sufficiently damage the bacterial cell envelope to kill bacteria. In this paper, polymerization of C9 was prevented without affecting binding of C9 to C5b-8, by locking the first transmembrane helix domain of C9. Using this system, we show that polymerization of C9 strongly enhanced damage to both the bacterial outer and inner membrane, resulting in more rapid killing of several Escherichia coli and Klebsiella strains in serum. By comparing binding of wildtype and ‘locked’ C9 by flow cytometry, we also show that polymerization of C9 is impaired when the amount of available C9 per C5b-8 is limited. This suggests that an excess of C9 is required to efficiently form polymeric-C9. Finally, we show that polymerization of C9 was impaired on complement-resistant E . coli strains that survive killing by MAC pores. This suggests that these bacteria can specifically block polymerization of C9. All tested complement-resistant E . coli expressed LPS O-antigen (O-Ag), compared to only one out of four complement-sensitive E . coli . By restoring O-Ag expression in an O-Ag negative strain, we show that the O-Ag impairs polymerization of C9 and results in complement-resistance. Altogether, these insights are important to understand how MAC pores kill bacteria and how bacterial pathogens can resist MAC-dependent killing.

Unregulated complement activation causes inflammatory and immunological pathologies with consequences for human disease. To prevent bystander damage during an immune response, extracellular chaperones (clusterin and vitronectin) capture and clear soluble precursors to the membrane attack complex (sMAC). However, how these chaperones block further polymerization of MAC and prevent the complex from binding target membranes remains unclear. Here, we address that question by combining cryo electron microscopy (cryoEM) and cross-linking mass spectrometry (XL-MS) to solve the structure of sMAC. Together our data reveal how clusterin recognizes and inhibits polymerizing complement proteins by binding a negatively charged surface of sMAC. Furthermore, we show that the pore-forming C9 protein is trapped in an intermediate conformation whereby only one of its two transmembrane β-hairpins has unfurled. This structure provides molecular details for immune pore formation and helps explain a complement control mechanism that has potential implications for how cell clearance pathways mediate immune homeostasis. To prevent unregulated complement activation, extracellular chaperones capture soluble precursors to the membrane attack complex (sMAC). Here, structural analysis of sMAC reveals how clusterin recognizes heterogeneous sMAC complexes and inhibits polymerization of complement protein C9.