Periodic Reporting for period 2 - Controlling MAC (Structural basis of controlling the membrane attack complex)
Periodo di rendicontazione: 2022-01-01 al 2023-06-30
Complement is a blood based immune network that plays an important role in innate and adaptive immune responses. Activation of complement culminates in the formation of a large lytic pore called the Membrane Attack Complex (MAC), which assembles on lipid bilayers and punches holes in target cells. MAC is a versatile and highly effective immune effector as there is no specific receptor directing MAC to pathogens. However, human cells are susceptible to damage by MAC if not properly controlled. Dysregulation of MAC on human cells can have devastating impact for disease pathologies including haemolytic anaemia and immune evasion strategies by cancer cells. Understanding how MAC is controlled during an immune response will enable the design of therapeutics that have the potential to precisely regulate complement activity and improve human health.
The objective of CONTROLLING MAC is to uncover the fundamental molecular principles underpinning MAC regulation on human cells. To achieve this ambitious goal, Controlling MAC uses a range of structural biology and biophysical tools to understand how complement activation impacts cellular pathways that modulate inflammation. The project develops an innovative approach to exploring the interactions between complement proteins and their local lipid environment. The novel insights from these experiments will explain how conformational changes in protein structure are linked with changes in physical properties of cellular membranes. Together, these data will explain how MAC is regulated and define a new role for lipids in immune homeostasis.
The objective of CONTROLLING MAC is to uncover the fundamental molecular principles underpinning MAC regulation on human cells. To achieve this ambitious goal, Controlling MAC uses a range of structural biology and biophysical tools to understand how complement activation impacts cellular pathways that modulate inflammation. The project develops an innovative approach to exploring the interactions between complement proteins and their local lipid environment. The novel insights from these experiments will explain how conformational changes in protein structure are linked with changes in physical properties of cellular membranes. Together, these data will explain how MAC is regulated and define a new role for lipids in immune homeostasis.
In the first half of the project, CONTROLLING MAC has delivered on three main major research initiatives. The first focuses on the role of a cell surface receptor, CD59, in stopping MAC assembly on human cells. The second investigates removal of MAC from the plasma membrane. Finally, the third research stream uncovers the role of blood-based chaperones in capturing and clearing potentially harmful MAC activation by-products. The major achievement of CONTROLLING MAC thus far has been in the delivery of the third research stream. Using a structural biology technique called cryo electron microscopy, the PI and team solved the structure of an innate-immune activation complex, which is regulated by blood-based chaperones to prevent bystander damage. sMAC (also called sC5b9), is a soluble form of the MAC regulated by clusterin and vitronectin in blood plasma. Our structural data explain how clusterin blocks polymerizing cargo through electrostatic interactions and provide evidence into how cell clearance pathways mediate immune homeostasis.
The results achieved by the third research stream have provided novel insight into complement regulation and the role of blood-based chaperones in modulating inflammation during an immune response. By developing creative strategies in image processing, we were able to solve multiple structures of what was previously thought to be a single immune activation complex. We showed that sMAC is a heterogeneous assembly in which the main pore forming protein, C9, is caught in an intermediate state. By combining information from crosslinking mass spectrometry, we were also able to identify the location of clusterin within our maps and show how clusterin binds a negatively charged surface on C9 to block polymerization of the pore. These data provide a structural framework for understanding pore formation and the molecular details for a complement control mechanism.