Structure of homo- and hetero-oligomeric meprin metalloproteases. Dimers,tetramers, and high molecular mass multimers

Meprin A and B, metalloproteases consisting of evolutionarily related alpha and/or beta subunits, are membrane-bound and secreted enzymes expressed by kidney and intestinal epithelial cells, leukocytes, and cancer cells. Previous work established that the multidomain meprin subunits (each approximately 80 kDa) form disulfide-bridged homo- and heterodimers, and differ in substrate and peptide bond specificities. The work herein clearly demonstrates that meprin dimers differ markedly in their ability to oligomerize. Electrophoresis, light scattering, size exclusion chromatography, and electron microscopy were used to characterize quaternary structures of recombinant rat meprins. Meprin B, consisting of meprin beta subunits only, was dimeric under a wide range of conditions. By contrast, meprin alpha homodimers formed heterogeneous multimers (ring-, circle-, spiral-, and tube-like structures) containing up to 100 subunits, with molecular masses at protein peaks ranging from approximately 1.0 to 6.0 MDa. The size of the meprin alpha homo-oligomers was dependent on protein concentration, ionic strength, and activation state. Meprin alphabeta heterodimers tended to form tetramers but not higher oligomers. Thus, the presence of meprin beta, which has a transmembrane domain in vivo, restricts the oligomerization potential of meprin molecules and localizes meprins to the plasma membrane. By contrast, the propensity of secreted meprin alpha homodimers to self-associate concentrates proteolytic potential into high molecular mass multimers and thus allows for autocompartmentalization. The work indicates that different mechanisms exist to localize and concentrate the proteolytic activity of membrane-bound and secreted meprin metalloproteinases.     

Alzheimer's disease beta-amyloid peptide is increased in mice deficient in endothelin-converting enzyme

The abnormal accumulation of beta-amyloid (Abeta) in the brain is an early and invariant feature in Alzheimer's disease (AD) and is believed to play a pivotal role in the etiology and pathogenesis of the disease. As such, a major focus of AD research has been the elucidation of the mechanisms responsible for the generation of Abeta. As with any peptide, however, the degree of Abeta accumulation is dependent not only on its production but also on its removal. In cell-based and in vitro models we have previously characterized endothelin-converting enzyme-1 (ECE-1) as an Abeta-degrading enzyme that appears to act intracellularly, thus limiting the amount of Abeta available for secretion. To determine the physiological significance of this activity, we analyzed Abeta levels in the brains of mice deficient for ECE-1 and a closely related enzyme, ECE-2. Significant increases in the levels of both Abeta40 and Abeta42 were found in the brains of these animals when compared with age-matched littermate controls. The increase in Abeta levels in the ECE-deficient mice provides the first direct evidence for a physiological role for both ECE-1 and ECE-2 in limiting Abeta accumulation in the brain and also provides further insight into the factors involved in Abeta clearance in vivo.     

Evolution of amino acid frequencies in proteins over deep time: inferred order of introduction of amino acids into the genetic code

To understand more fully how amino acid composition of proteins has changed over the course of evolution, a method has been developed for estimating the composition of proteins in an ancestral genome. Estimates are based upon the composition of conserved residues in descendant sequences and empirical knowledge of the relative probability of conservation of various amino acids. Simulations are used to model and correct for errors in the estimates. The method was used to infer the amino acid composition of a large protein set in the Last Universal Ancestor (LUA) of all extant species. Relative to the modern protein set, LUA proteins were found to be generally richer in those amino acids that are believed to have been most abundant in the prebiotic environment and poorer in those amino acids that are believed to have been unavailable or scarce. It is proposed that the inferred amino acid composition of proteins in the LUA probably reflects historical events in the establishment of the genetic code.     

Multiple HLA class II-restricted melanocyte differentiation antigens are recognized by tumor-infiltrating lymphocytes from a patient with melanoma

Dramatic clinical responses were observed in patient 888 following the adoptive transfer of autologous tumor-infiltrating lymphocytes (TIL). Previously, extensive analysis of the specificity of class I-restricted T cells from patient 888 TIL has revealed that these T cells recognize a mutated, as well as several nonmutated tumor Ags. Additional studies that were conducted on TIL from patient 888 indicated that they contained CD4-positive T cells that recognized the autologous tumor that had been induced to express HLA class II molecules. Tumor-reactive CD4-positive T cell clones were isolated from TIL and tested for their ability to react with Ags that are recognized by HLA class I-restricted, melanoma-reactive T cells. Using this approach, T cell clones were identified that recognized an epitope expressed in both the tyrosinase-related protein 1 and tyrosinase-related protein 2 Ags in the context of the HLA-DRbeta1*1502 class II gene product. Additional clones were found to recognize an epitope of gp100 in the context of the same HLA-DR restriction element. These observations provide an impetus to develop strategies directed toward generating HLA class II-restricted tumor-reactive T cells.     

Cyclosporine A enhances leukocyte binding by human intestinal microvascular endothelial cells through inhibition of p38 MAPK and iNOS. Paradoxical proinflammatory effect on the microvascular endothelium

The calcineurin inhibitor cyclosporine A (CsA) modulates leukocyte cytokine production but may also effect nonimmune cells, including microvascular endothelial cells, which regulate the inflammatory process through leukocyte recruitment. We hypothesized that CsA would promote a proinflammatory phenotype in human intestinal microvascular endothelial cells (HIMEC), by inhibiting inducible nitric-oxide synthase (iNOS, NOS2)-derived NO, normally an important mechanism in limiting endothelial activation and leukocyte adhesion. Primary cultures of HIMEC were used to assess CsA effects on endothelial activation, leukocyte interaction, and the expression of iNOS as well as cell adhesion molecules. CsA significantly increased leukocyte binding to activated HIMEC, but paradoxically decreased endothelial expression of cell adhesion molecules (E-selectin, intercellular adhesion molecule 1, and vascular cell adhesion molecule-1). In contrast, CsA completely inhibited the expression of iNOS in tumor necrosis factor-alpha/lipopolysaccharide-activated HIMEC. CsA blocked p38 MAPK phosphorylation in activated HIMEC, a key pathway in iNOS expression, but failed to inhibit NFkappaB activation. These studies demonstrate that CsA exerts a proinflammatory effect on HIMEC by blocking iNOS expression. CsA exerts a proinflammatory effect on the microvascular endothelium, and this drug-induced endothelial dysfunction may help explain its lack of efficacy in the long-term treatment of chronically active inflammatory bowel disease.