Multiple functions of CARD14 in keratinocyte differentiation, response to TLR-activation, ferroptosis and psoriasis pathogenesis

Description

In skin homeostasis dynamic interactions occur between the epidermis, microbiome and immune cells. The disruption of such a balanced interplay is the basis of many skin disorders (atopic dermatitis; psoriasis; tumors). These pathological events can be caused by alteration of the composition of microbial communities, physical damage to the epidermal barrier, abnormal keratinocyte differentiation process, aberrant self-produced and self-sustaining immune response. A crucial role in epidermal homeostasis is played by CARD14, a multi-domain adapter protein, which confers susceptibility to psoriasis. CARD14 is specifically expressed in skin and forms a signalling complex with BCL10 and MALT1, (CBM complex), which activates NF-κB and regulates the balance between inflammation and innate immune response to Pathogen Associated Molecular Patterns (PAMPs). However, the molecular mechanisms underlying CARD14 regulation of skin-specific signalling pathways are still unclear. Therefore, the project proposal is aimed to investigate the involvement of CARD14 and the CBM complex in: keratinocyte differentiation (I), TLR-Induced innate response (II) and the regulation of keratinocyte-specific redox status and lipid peroxidation (III), with specific attention towards perturbing events that cause psoriasis. To clarify the epidermis-specific functions of CARD14, our secondary aims will be:

  • the structural analysis of the interactions that CARD14 and its variants, both natural and pathogenic, tightens with antiviral signal transducers recently identified as new molecular partners, TANK and MAVS;
  • the functional study of the role of the ferroptosis, a new form of regulated iron-dependent cell death caused by a lethal level of intracellular ROS, in the pathogenesis of psoriasis. These goals will be achieved through a multidisciplinary approach, which will integrate the expertise of research units in cellular and molecular biology, cell metabolism, dermatology, mucosal immunity, and biochemistry. The proposed activities are organized in work packages that each research unit will carry out autonomously, according to specific skills. The project will contribute in the definition of new therapeutic approaches, through the identification of novel targets and reliable biomarkers in the treatment of inflammatory skin disorders.

Objectives and expected results

Preliminary data produced by the BN-RU suggest that CARD14sh, might interact and affect the aggregation of the CARD-domain containing protein MAVS. Co-IP experiments and two-hybrid assay in yeast have also demonstrated that CARD14sh associates to the immunomodulatory adapter protein TANK. Notably, psoriasis mutants CARD14shE138A and E142G showed less efficacy in binding TANK when compared to CARD14shWT. Therefore, in vitro analysis of CARD14sh/MAVS and CARD14sh/TANK putative direct interactions will provide important information regarding CARD14sh functions. Furthermore, the effects of psoriasis-associated mutations, both activating (E138A and E142G) and not-activating (R38C) NF-κB, on these bindings might serve to clarify the molecular mechanisms at the basis of the pathological processes in which CARD14 is implicated. IBB unit will express CARD14sh and its single-point mutated isoforms (E138A, E142G and R38C), MAVS and TANK proteins in E.coli, purify and biochemically characterize these proteins and investigate the capability of CARD14sh and its mutants to bind MAVS and TANK proteins. In addition, crystallographic studies on the putative obtained protein-protein complexes will be performed. In particular, the proteins object of the study will be produced in E. coli screening different expression conditions. Proteins will be purified by means of chromatographic techniques. Molecular weight, oligomeric state and secondary structure will be evaluated by means of Mass Spectrometry (MS), Light Scattering (LS) and Circular Dichroism (CD). For the binding assays, Enzyme Linked Immunosorbent Assay (ELISA), Isothermal Titration Calorimetry (ITC), Micro Scale Thermophoresis (MST) and Bio-Layer Interferometry (BLI) will be employed. ELISA and BLI measurements will allow the investigation of protein-protein binding by immobilizing one of the two binding partners on a plate or a biosensor, respectively. By MST, temperature-induced changes in motion of fluorescent molecules along a microscopic temperature gradient as a function of the concentration of a label-free ligand will be detected. Finally, ITC a label-free technique will provide information regarding the thermodynamic parameters of the protein-protein binding in solution. All the described methods will allow the estimation of the binding affinity constants, thus obtaining quantitative analysis. If a direct binding between the proteins object of this study will be observed, a structural characterization of the complexes by X-ray crystallography will be performed. Crystallization trials on CARD14sh/protein adducts will be carried out by using an automatic crystallization system. Data collection will be carried out either by means of the rotate anode source available at the IBB, or by means of the synchrotron. Finally, the complex structure will be resolved by molecular replacement method.

Project proponents and involved entities

  • Università degli Studi del Sannio di Benevento
  • Università degli Studi “Magna Graecia” di Catanzaro

Project leader

Dr. Martina Buonanno (IBB Unit leader)

Project code

P2022RBPHH_LS1_PRIN2022PNRR

Received funds

€ 65.559,00

Time frame

30/11/2023 - 29/11/2025

Progress status

70%
  • Post category:PRIN / Projects