Prospective Validation of CD-62L (L-Selectin) as Marker of Durable Response to Infliximab Treatment in Patients With Inflammatory Bowel Disease: A 5-Year Clinical Follow-up.

INTRODUCTION: The development of biomarkers to guide management of anti-tumor necrosis factor (TNF) agents in patients with inflammatory bowel disease (IBD) is an unmet need. We developed an in vitro blood assay to predict patient long-term outcome with the anti-TNFα agent infliximab (IFX). METHODS: Patients with IBD were classified according to the shedding of an L-selectin (CD62L) from the surface of their granulocytes in whole blood. CD62L shedding was quantified by flow cytometry before and after drug administration. A clinical data collection from June 2012 to August 2017 with blinded IFX management was aimed at validating the long-term predictive value of this test. RESULTS: Among 33 patients with IBD (17 Crohn's disease and 5 ulcerative colitis), 22 were predicted functional responders (PFR) and 11 were predicted as nonresponders (NR) according to the in vitro test. Five years after study initiation, 72% of PFR were still treated with IFX (vs 27% in the NR group; P < 0.05), with a median time spent under IFX of 45 vs 12 months (P = 0.019), respectively. Thirty-five medicosurgical events occurred with a median time to first event of 3 vs 30 months (P = 0.023), respectively. Our assay was the best independent predictor of staying long term on IFX (P = 0.056). DISCUSSION: An assay-based in vitro test for functional blockade of TNFα (CD62L shedding) provides an excellent long-term (at 3-5 years) independent predictor of durable use of IFX in patients with IBD. Testing patients could personalize decision making to significantly reduce costs and risk of adverse events and complications.

Functional cross-talk between allosteric effects of activating and inhibiting ligands underlies PKM2 regulation.

Several enzymes can simultaneously interact with multiple intracellular metabolites, however, how the allosteric effects of distinct ligands are integrated to coordinately control enzymatic activity remains poorly understood. We addressed this question using, as a model system, the glycolytic enzyme pyruvate kinase M2 (PKM2). We show that the PKM2 activator fructose 1,6-bisphosphate (FBP) alone promotes tetramerisation and increases PKM2 activity, but addition of the inhibitor L-phenylalanine (Phe) prevents maximal activation of FBP-bound PKM2 tetramers. We developed a method, AlloHubMat, that uses eigenvalue decomposition of mutual information derived from molecular dynamics trajectories to identify residues that mediate FBP-induced allostery. Experimental mutagenesis of these residues identified PKM2 variants in which activation by FBP remains intact but cannot be attenuated by Phe. Our findings reveal residues involved in FBP-induced allostery that enable the integration of allosteric input from Phe and provide a paradigm for the coordinate regulation of enzymatic activity by simultaneous allosteric inputs.

An engineered photoswitchable mammalian pyruvate kinase.

Changes in allosteric regulation of glycolytic enzymes have been linked to metabolic reprogramming involved in cancer. Remarkably, allosteric mechanisms control enzyme function at significantly shorter time-scales compared to the long-term effects of metabolic reprogramming on cell proliferation. It remains unclear if and how the speed and reversibility afforded by rapid allosteric control of metabolic enzymes is important for cell proliferation. Tools that allow specific, dynamic modulation of enzymatic activities in mammalian cells would help address this question. Towards this goal, we have used molecular dynamics simulations to guide the design of mPKM2 internal light/oxygen/voltage-sensitive domain 2 (LOV2) fusion at position D24 (PiL[D24]), an engineered pyruvate kinase M2 (PKM2) variant that harbours an insertion of the light-sensing LOV2 domain from Avena Sativa within a region implicated in allosteric regulation by fructose 1,6-bisphosphate (FBP). The LOV2 photoreaction is preserved in the PiL[D24] chimera and causes secondary structure changes that are associated with a 30% decrease in the Km of the enzyme for phosphoenolpyruvate resulting in increased pyruvate kinase activity after light exposure. Importantly, this change in activity is reversible upon light withdrawal. Expression of PiL[D24] in cells leads to light-induced increase in labelling of pyruvate from glucose. PiL[D24] therefore could provide a means to modulate cellular glucose metabolism in a remote manner and paves the way for studying the importance of rapid allosteric phenomena in the regulation of metabolism and enzyme control.

Allosteric regulation of metabolism in cancer: endogenous mechanisms and considerations for drug design.

Alterations in metabolic processes have been linked to various diseases, including cancer. Although gene expression can dictate long-term metabolic adaptation, many metabolic changes found in cancer are associated with altered allosteric properties of the underlying enzymes. Small molecule-protein interactions and intracellular signalling converge to orchestrate these allosteric mechanisms, which, emerging evidence suggests, constitute a promising therapeutic avenue. In this review we focus on glucose and energy metabolism to illustrate the role of allostery in cancer physiology and we discuss approaches to streamline the process of targeting aberrant allosteric pathways with small molecules.

Surface Accessibility and Dynamics of Macromolecular Assemblies Probed by Covalent Labeling Mass Spectrometry and Integrative Modeling.

Mass spectrometry (MS) has become an indispensable tool for investigating the architectures and dynamics of macromolecular assemblies. Here we show that covalent labeling of solvent accessible residues followed by their MS-based identification yields modeling restraints that allow mapping the location and orientation of subunits within protein assemblies. Together with complementary restraints derived from cross-linking and native MS, we built native-like models of four heterocomplexes with known subunit structures and compared them with available X-ray crystal structures. The results demonstrated that covalent labeling followed by MS markedly increased the predictive power of the integrative modeling strategy enabling more accurate protein assembly models. We applied this strategy to the F-type ATP synthase from spinach chloroplasts (cATPase) providing a structural basis for its function as a nanomotor. By subjecting the models generated by our restraint-based strategy to molecular dynamics (MD) simulations, we revealed the conformational states of the peripheral stalk and assigned flexible regions in the enzyme. Our strategy can readily incorporate complementary chemical labeling strategies and we anticipate that it will be applicable to many other systems providing new insights into the structure and function of protein complexes.

Promiscuous antibodies characterised by their physico-chemical properties: From sequence to structure and back.

Human B cells produce antibodies, which bind to their cognate antigen based on distinct molecular properties of the antibody CDR loop. We have analysed a set of 10 antibodies showing a clear difference in their binding properties to a panel of antigens, resulting in two subsets of antibodies with a distinct binding phenotype. We call the observed binding multiplicity 'promiscuous' and selected physico-chemical CDRH3 characteristics and conformational preferences may characterise these promiscuous antibodies. To classify CDRH3 physico-chemical properties playing a role in their binding properties, we used statistical analyses of the sequences annotated by Kidera factors. To characterise structure-function requirements for antigen binding multiplicity we employed Molecular Modelling and Monte Carlo based coarse-grained simulations. The ability to predict the molecular causes of promiscuous, multi-binding behaviour would greatly improve the efficiency of the therapeutic antibody discovery process.

The outer mucus layer hosts a distinct intestinal microbial niche.

The overall composition of the mammalian intestinal microbiota varies between individuals: within each individual there are differences along the length of the intestinal tract related to host nutrition, intestinal motility and secretions. Mucus is a highly regenerative protective lubricant glycoprotein sheet secreted by host intestinal goblet cells; the inner mucus layer is nearly sterile. Here we show that the outer mucus of the large intestine forms a unique microbial niche with distinct communities, including bacteria without specialized mucolytic capability. Bacterial species present in the mucus show differential proliferation and resource utilization compared with the same species in the intestinal lumen, with high recovery of bioavailable iron and consumption of epithelial-derived carbon sources according to their genome-encoded metabolic repertoire. Functional competition for existence in this intimate layer is likely to be a major determinant of microbiota composition and microbial molecular exchange with the host.