Studying protein–protein affinity and immobilized ligand–protein affinity interactions using MS-based methods

This review discusses the most important current methods employing mass spectrometry (MS) analysis for the study of protein affinity interactions. The methods are discussed in depth with particular reference to MS-based approaches for analyzing protein–protein and protein–immobilized ligand interactions, analyzed either directly or indirectly. First, we introduce MS methods for the study of intact protein complexes in the gas phase. Next, pull-down methods for affinity-based analysis of protein–protein and protein–immobilized ligand interactions are discussed. Presently, this field of research is often called interactomics or interaction proteomics. A slightly different approach that will be discussed, chemical proteomics, allows one to analyze selectivity profiles of ligands for multiple drug targets and off-targets. Additionally, of particular interest is the use of surface plasmon resonance technologies coupled with MS for the study of protein interactions. The review addresses the principle of each of the methods with a focus on recent developments and the applicability to lead compound generation in drug discovery as well as the elucidation of protein interactions involved in cellular processes. The review focuses on the analysis of bioaffinity interactions of proteins with other proteins and with ligands, where the proteins are considered as the bioactives analyzed by MS

Inefficient differentiation response to cell cycle stress leads to genomic instability and malignant progression of squamous carcinoma cells

Squamous cell carcinoma (SCC) or epidermoid cancer is a frequent and aggressive malignancy. However in apparent paradox it retains the squamous differentiation phenotype except for very dysplastic lesions. We have shown that cell cycle stress in normal epidermal keratinocytes triggers a squamous differentiation response involving irreversible mitosis block and polyploidisation. Here we show that cutaneous SCC cells conserve a partial squamous DNA damage-induced differentiation response that allows them to overcome the cell division block. The capacity to divide in spite of drug-induced mitotic stress and DNA damage made well-differentiated SCC cells more genomically instable and more malignant in vivo. Consistently, in a series of human biopsies, non-metastatic SCCs displayed a higher degree of chromosomal alterations and higher expression of the S phase regulator Cyclin E and the DNA damage signal γH2AX than the less aggressive, non-squamous, basal cell carcinomas. However, metastatic SCCs lost the γH2AX signal and Cyclin E, or accumulated cytoplasmic Cyclin E. Conversely, inhibition of endogenous Cyclin E in well-differentiated SCC cells interfered with the squamous phenotype. The results suggest a dual role of cell cycle stress-induced differentiation in squamous cancer: the resulting mitotic blocks would impose, when irreversible, a proliferative barrier, when reversible, a source of genomic instability, thus contributing to malignancy.AG is grateful to Jean-Jeaques Guilhou, Jean-Claude Rossi and the INSERM for professional support and to Renata Polakowska for the generous gift of precious BCCP cell line. We thank Lucía Barbier, Tania Lobato, Evelyn Andrades, Alicia Noriega and María Aramburu for technical assistance and Natalia Sanz for critical reading of the MS. To AG: National grants from Instituto de Salud Carlos III, Fondo de Investigación Sanitaria (ISCIII-FIS/FEDER, Spain): PI08/0890, PI11/02070, PI14/00900; Ligue Nationale Contre la Cancer (La Ligue; France). To AT: ISCIII-FIS PI10/00785. To JP: MINECO grant SAF2015-66015-R; AES grant ISCIII-RETIC RD12/0036/0009. VC was funded by a fellowship from La Ligue (France), PA by IDIVAL (Spain), RM and IdP by AG lab and ISCIII-FIS-FEDER PI11/02070 (Spain)