The complement system is a critical component of innate immunity comprised of serum proteins, which can be activated by three different pathways (classical, lectin and alternative) causing a progressively amplifying inflammatory cascade. Activation of the complement system is generally tightly controlled by an array of down- regulators to minimize host-tissue damage. However, when unregulated complement activation occurs, it contributes to tissue damage in a wide range of inflammatory disease processes including myocardial infarction, rheumatoid arthritis and transplant rejection. The long-range goal of this research program is to elucidate the molecular basis of complement activation upon initial recognition of ligand by the C1q (classical pathway) and MBL (lectin pathway) complex as a prerequisite to the development of therapeutic methods to attenuate or prevent host tissue destruction by unregulated complement activation. We previously discovered that the coat protein (CP) of human astrovirus potently suppresses both the classical and lectin pathways of complement at the level of C1q and MBL, respectively. Recently, a 30 amino acid peptide derived from the wild-type CP mediating this activity has been identified. In addition, preliminary data demonstrates that this coat protein peptide (CPP) derivative binds C1q to inhibit interaction with the C1q/MBL receptor, calreticulin (CRT/cC1qR). The discovery of a small molecule inhibitor of C1q and MBL activation provides a novel reagent to decipher how these complexes function. Our specific hypothesis is that CPP directly binds to the collagen-like region of C1q and MBL that is critical for interaction between the associated serine proteases required for initiation of classical and lectin pathway activity as well as CRT/cC1qR interaction. The experimental focus of this application is to define the precise interactions between CPP and C1q/MBL required to inhibit complement activation as well as the functional consequence of CPP inhibition of the C1q/MBL-CRT/cC1qR interaction on cellular function.
Specific Aim 1 will characterize the interactions between the CPP and C1q/MBL and measure functional effects on cognate serine protease activation by: (a) defining the interaction domains of CPP/C1q and CPP/MBL via binding (ELISA) assays, functional assays and mass spectrometric protein footprinting utilizing synthetic derivatives of CPP, (b) characterizing the kinetics of CPP binding C1q/MBL by surface plasmon resonance and isothermal titration calorimetry and (c) assaying the extent to which CPP interaction with C1q alters interaction with its cognate serine protease complex by utilizing a competitive binding assay developed in our laboratory (ELISA).
Specific Aim 2 will evaluate the extent to which CPP can alter the interaction with C1q-CRT/cC1qR on human neutrophils and its effect on cellular function by: (a) determining the ability of CPP to inhibit C1q binding to CRT/cC1qR on neutrophils utilizing flow cytometry and (b) characterizing the biological consequence of CPP disruption of the C1q-CRT/cC1qR interaction on respiratory burst activity in neutrophils using a SOD-inhibitable ferricytochrome C reduction assay.
Successful execution of this research project will define both the mechanism of CPP inhibition of C1 and MBL activation of the complement cascade and the consequence of impeding C1q-CRT/cC1qR interaction on neutrophil function. Dysregulated complement activation contributes to inflammatory diseases in humans such as rheumatoid arthritis, myocardial infarction and transplant rejection, however very few complement inhibitors are currently available for therapeutic use. Understanding the mechanism of action of this novel peptidic complement inhibitor will pave the way for its development as a therapeutic compound to prevent complement- mediated immunopathology.
|Shah, Tushar A; Mauriello, Clifford T; Hair, Pamela S et al. (2014) Clinical hypothermia temperatures increase complement activation and cell destruction via the classical pathway. J Transl Med 12:181|
|Douradinha, Bruno; McBurney, Sean P; Soares de Melo, Klecia M et al. (2014) C1q binding to dengue virus decreases levels of infection and inflammatory molecules transcription in THP-1 cells. Virus Res 179:231-4|