Semicarbazide-sensitive amine oxidase (SSAO) belongs to a ubiquitous family of copper-containing amine oxidases (CuAOs). SSAO is also known as vascular adhesion protein-1 (VAP-1) and has been identified as one of the adhesion molecules involved in the leukocyte-extravasation process. The structure of a truncated soluble form of human SSAO has been solved and refined to 2.5 A. As expected, SSAO is a homodimer with a fold typical of the CuAO family. The topaquinone (TPQ) cofactor and a copper ion characteristic of CuAOs are present in the active site, with the TPQ in the active ;off-copper' conformation. The structure reveals that a leucine residue (Leu469) located adjacent to the active site could function as a gate controlling its accessibility. An RGD motif is displayed on the surface, where it could be involved in integrin binding and possibly play a role in the shedding of SSAO from the membrane. Carbohydrate moieties are observed at five of six potential N-glycosylation sites. Carbohydrates attached to Asn232 flank the active-site entrance and might influence substrate specificity. The structure of an adduct of SSAO and the irreversible inhibitor 2-hydrazinopyridine has been solved and refined to 2.9 A resolution. Together, these structures will aid efforts to identify natural substrates, provide valuable information for the design of specific inhibitors and direct further studies.

Although mesenchymal stromal cells (MSCs) possess the capacity to modulate immune responses, little is known about the mechanisms that underpin these processes. In this study, we show that immunosupression is mediated by activation of nuclear factor kappa B (NF-κB) in human MSCs. This pathway is activated by TNF-α that is generated following TCR stimulation of T cells. Inhibition of NF-κB through silencing of IκB kinase β or the TNF-α receptor abolishes the immunosuppressive capacity of MSCs. Our data also indicate that MSC-associated NF-κB activation primarily leads to inhibition of T-cell proliferation with little effect on expression of the activation markers CD69 and CD25. Thus, our data support the hypothesis that the TNF-α/NF-κB signalling pathway is required for the initial priming of immunosuppressive function in human MSCs. Interestingly, drugs that interfere with NF-κB activation significantly antagonise the immunoregulatory effect of MSCs, which could have important implications for immunosuppression regimens in the clinic. Keywords:Immunoregulation r MSC r NF-κB r TNF-α See accompanying Commentary by Pistoia and RaffaghelloAdditional supporting information may be found in the online version of this article at the publisher's web-site IntroductionMesenchymal stromal cells (MSCs) are multipotent progenitor cells that have the capacity to differentiate into multiple lineages. These cells are found in a variety of tissues during development, of which BM represents the most common source for research purposes. From a clinical perspective, MSCs are considered to Correspondence: Dr. César Trigueros e-mail: ctrigueros@inbiomed.org have a potential use in tissue repair for bone, cartilage and tendon. However, due to their immunomodulatory properties and their inclusion as a stromal component of the marrow microenvironment, MSCs are currently utilised in other therapeutic scenarios, such as those encountered in hematopoietic stem cell transplantation, GVH disease or chronic inflammatory diseases [1,2]. These characteristics, together with their low immunogenicity [1,2], have opened up promising new avenues of research for the use of MSCs not only in autologous but also in allogeneic settings.C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu Eur. J. Immunol. 2014. 44: 480-488 Immunomodulation 481 The immunomodulatory activity of MSCs, directed against a wide range of effector cells of both the innate and adaptive immune system, has been described. Communication between MSCs and immune cells, through cell-to-cell contact-dependent and/or contact-independent mechanisms, has been shown to lead to increased production of soluble immunomodulatory factors such as indoleamine 2,3-deoxigenase [3,4], prostaglandin E2 [5][6][7], iNOS [8], transforming growth factor β (TGF-β), hepatocyte growth factor [9], human lymphocytes Ag molecule 5 and IL-10 [10]. Thus, the picture is complex, as it is likely that multiple regulatory mechanisms exist without an obvious hierarchy of importance.The inflammatory e...

Human glutathione transferase A1-1 is a well studied enzyme, but despite a wealth of structural and biochemical data a number of aspects of its catalytic function are still poorly understood. Here, five new crystal structures of this enzyme are described that provide several insights. Firstly, the structure of a complex of the wild-type human enzyme with glutathione was determined for the first time at 2.0 angstroms resolution. This reveals that glutathione binds in the G site in a very similar fashion as the glutathione portion of substrate analogues in other structures and also that glutathione binding alone is sufficient to stabilize the C-terminal helix of the protein. Secondly, we have studied the complex with a decarboxylated glutathione conjugate that is known to dramatically decrease the activity of the enzyme. The T68E mutant of human glutathione transferase A1-1 recovers some of the activity that is lost with the decarboxylated glutathione, but our structures of this mutant show that none of the earlier explanations of this phenomenon are likely to be correct. Thirdly, and serendipitously, the apo structures also reveal the conformation of the crucial C-terminal region that is disordered in all previous apo structures. The C-terminal region can adopt an ordered helix-like structure even in the apo state, but shows a strong tendency to unwind. Different conformations of the C-terminal regions were observed in the apo states of the two monomers, which suggests that cooperativity could play a role in the activity of the enzyme.

Mesenchymal stromal cells (MSCs) are multipotent cells found in connective tissues that can differentiate into bone, cartilage, and adipose tissue. Interestingly, they can regulate immune responses in a paracrine way and allogeneic MSCs do not elicit immune response. These properties have encouraged a number of clinical trials in a broad range of regenerative therapies. Although these trials were first focused on their differentiation properties, in the last years, the immunosuppressive features have gained most of the attention. In this review, we will summarize the up-to-date knowledge about the immunosuppressive mechanisms of MSCs in vivo and in vitro and the most promising approaches in clinical investigation.

A strategy for rational enzyme design is reported and illustrated by the engineering of a protein catalyst for thiol-ester hydrolysis. Five mutants of human glutathione (GSH; ␥-Glu-Cys-Gly) transferase A1-1 were designed in the search for a catalyst and to provide a set of proteins from which the reaction mechanism could be elucidated. The single mutant A216H catalyzed the hydrolysis of the S-benzoyl ester of GSH under turnover conditions with a kcat͞KM of 156 M ؊1 ⅐min ؊1 , and a catalytic proficiency of >10 7 M ؊1 when compared with the first-order rate constant of the uncatalyzed reaction. The wild-type enzyme did not hydrolyze the substrate, and thus, the introduction of a single histidine residue transformed the wild-type enzyme into a turnover system for thiol-ester hydrolysis. By kinetic analysis of single, double, and triple mutants, as well as from studies of reaction products, it was established that the enzyme A216H catalyzes the hydrolysis of the thiol-ester substrate by a mechanism that includes an acyl intermediate at the side chain of Y9. Kinetic measurements and the crystal structure of the A216H GSH complex provided compelling evidence that H216 acts as a general-base catalyst. The introduction of a single His residue into human GSH transferase A1-1 created an unprecedented enzymatic function, suggesting a strategy that may be of broad applicability in the design of new enzymes. The protein catalyst has the hallmarks of a native enzyme and is expected to catalyze various hydrolytic, as well as transesterification, reactions.T he quest for new enzymes extends the boundaries of our understanding of catalysis and protein structure and is expected, when successful, to generate new biocatalysts for reactions that are not catalyzed by nature (1-3). It allows for unprecedented opportunities in chemistry, but the implementation of nonnative catalytic functions in protein scaffolds remains a challenge. The redesign of protein macromolecules provides a powerful strategy for engineering nonnatural reactive sites and determining structure-function relationships. For example, the functional swapping between native enzymes has been instructive (4-8), and new catalytic activities have been introduced by chemical modification of amino acid side chains (9). In addition, the incorporation of amino acid residues in the active sites of native enzymes has been used to alter the fate of the natural substrates or inhibitors (10-14). However, although it is possible in principle to modify the active site of any native enzyme to generate nonnatural activity, this approach has met with only limited success because of the difficulties involved in predicting the effect of sequence modifications on structure and function. Catalytic antibodies based on the properties of the immune system in generating binding sites that are complementary to transition-state analogues have been shown to be efficient catalysts for numerous chemical reactions (15)(16). Their catalytic efficiencies, based mainly on transition-state stabilization...