Summary: Inflammatory bowel diseases are characterized by a deregulated immune response targeting the gut bacterial flora. Mucosal-associated invariant T (MAIT) cells are major histocompatibility complex (MHC) class Ib-restricted innate-like lymphocytes with anti-bacterial functions. They display an effector/memory phenotype and are found in large numbers in the blood, mucosae and liver. They have also been implicated in inflammatory diseases such as multiple sclerosis. Therefore, we aimed to analyse the possible involvement of MAIT cells in Crohn's disease (CD) and ulcerative colitis (UC). To this end, a phenotypical and functional analysis of MAIT cells isolated from the blood of healthy subjects, CD and UC patients was undertaken. MAIT cells were also quantified in ileal biopsies of CD patients. The frequency of blood MAIT cells was specifically reduced in IBD patients compared with healthy donors, whereas it was dramatically greater in the inflamed versus healthy tissue. MAIT cells were activated as they expressed significantly more the Ki67 antigen, and this was accompanied by phenotypical changes such as increased expression of natural killer (NK)G2D and B and T lymphocyte attenuator (BTLA). Finally, in-vitro-activated MAIT cells from CD and UC patients secreted significantly more interleukin (IL)-17, together with a decreased interferon (IFN)-γ in CD but an increased IL-22 in UC. These data show that MAIT cells are activated in IBD, which results in an increased recruitment towards the inflamed tissues, an altered phenotype and a switch in the pattern of cytokine secretion. This is the first demonstration that MAIT cells are immune players in IBD, whose precise functions in this context need to be addressed.

In Arabidopsis thaliana, the degree of methylesterification (DM) of homogalacturonans (HGs), the main pectic constituent of the cell wall, can be modified by pectin methylesterases (PMEs). In all organisms, two types of protein structure have been reported for PMEs: group 1 and group 2. In group 2 PMEs, the active part (PME domain, Pfam01095) is preceded by an N-terminal extension (PRO part), which shows similarities to PME inhibitors (PMEI domain, Pfam04043). This PRO part mediates retention of unprocessed group 2 PMEs in the Golgi apparatus, thus regulating PME activity through a post-translational mechanism. This study investigated the roles of a subtilisin-type serine protease (SBT) in the processing of a PME isoform. Using a combination of functional genomics, biochemistry and proteomic approaches, the role of a specific SBT in the processing of a group 2 PME was assessed together with its consequences for plant development. A group 2 PME, AtPME17 (At2g45220), was identified, which was highly co-expressed, both spatially and temporally, with AtSBT3.5 (At1g32940), a subtilisin-type serine protease (subtilase, SBT), during root development. PME activity was modified in roots of knockout mutants for both proteins with consequent effects on root growth. This suggested a role for SBT3.5 in the processing of PME17 in planta. Using transient expression in Nicotiana benthamiana, it was indeed shown that SBT3.5 can process PME17 at a specific single processing motif, releasing a mature isoform in the apoplasm. By revealing the potential role of SBT3.5 in the processing of PME17, this study brings new evidence of the complexity of the regulation of PMEs in plants, and highlights the need for identifying specific PME-SBT pairs.

Pectin methylesterases (PMEs) catalyze the demethylesterification of homogalacturonan domains of pectin in plant cell walls and are regulated by endogenous pectin methylesterase inhibitors (PMEIs). In Arabidopsis dark-grown hypocotyls, one PME (AtPME3) and one PMEI (AtPMEI7) were identified as potential interacting proteins. Using RT-quantitative PCR analysis and gene promoter::GUS fusions, we first showed that AtPME3 and AtPMEI7 genes had overlapping patterns of expression in etiolated hypocotyls. The two proteins were identified in hypocotyl cell wall extracts by proteomics. To investigate the potential interaction between AtPME3 and AtPMEI7, both proteins were expressed in a heterologous system and purified by affinity chromatography. The activity of recombinant AtPME3 was characterized on homogalacturonans (HGs) with distinct degrees/patterns of methylesterification. AtPME3 showed the highest activity at pH 7.5 on HG substrates with a degree of methylesterification between 60 and 80% and a random distribution of methyl esters. On the best HG substrate, AtPME3 generates long non-methylesterified stretches and leaves short highly methylesterified zones, indicating that it acts as a processive enzyme. The recombinant AtPMEI7 and AtPME3 interaction reduces the level of demethylesterification of the HG substrate but does not inhibit the processivity of the enzyme. These data suggest that the AtPME3·AtPMEI7 complex is not covalently linked and could, depending on the pH, be alternately formed and dissociated. Docking analysis indicated that the inhibition of AtPME3 could occur via the interaction of AtPMEI7 with a PME ligand-binding cleft structure. All of these data indicate that AtPME3 and AtPMEI7 could be partners involved in the fine tuning of HG methylesterification during plant development.

The fine-tuning of the degree of methylesterification of cell wall pectin is a key to regulate cell elongation and ultimately the shape of plant body. Pectin methylesterification is spatio-temporally controlled by pectin methylesterases (PMEs, 66 members in Arabidopsis). The comparably large number of proteinaceous pectin methylesterase inhibitors (PMEIs, 76 members in Arabidopsis) questions the specificity of the PME-PMEI interaction and the functional role of such abundance. To understand the difference, or redundancy, between PMEIs, we used Molecular Dynamics (MD) to predict the behaviour of two PMEIs that are co-expressed and have distinct effects on plant development: AtPMEI4 and AtPMEI9. Simulations revealed the structural determinants of the pH-dependence for the interaction of these inhibitors with AtPME3, a major PME expressed in roots. Key residues that are likely to play a role in the pH-dependence were identified. The predictions obtained from MD simulations were confirmed in vitro, showing that AtPMEI9 is a stronger, less pH-independent, inhibitor as compared to AtPMEI4. Using pollen tubes as a developmental model, we showed that these biochemical differences have a biological significance. Application of purified proteins at pH ranges in which PMEI inhibition differed between AtPMEI4 and AtPMEI9 had distinct consequences on pollen tube elongation. MD simulations have therefore proven to be powerful tool to predict functional diversity between PMEIs, allowing the discovery of a strategy that may be used by PMEIs to inhibit PMEs in different micro-environmental conditions and paving the way to identify the specific role of PMEIs' diversity in muro.