Molecular Basis for the Recognition and Cleavages of IGF-II, TGF-α, and Amylin by Human Insulin-Degrading Enzyme

Insulin-degrading enzyme (IDE) is involved in the clearance of many bioactive peptide substrates, including insulin and amyloid-β, peptides vital to the development of diabetes and Alzheimer's disease, respectively. IDE can also rapidly degrade hormones that are held together by intramolecular disulfide bond(s) without their reduction. Furthermore, IDE exhibits a remarkable ability to preferentially degrade structurally similar peptides such as the selective degradation of insulin-like growth factor (IGF)-II and transforming growth factor-α (TGF-α) over IGF-I and epidermal growth factor, respectively. Here, we used high-accuracy mass spectrometry to identify the cleavage sites of human IGF-II, TGF-α, amylin, reduced amylin, and amyloid-β by human IDE. We also determined the structures of human IDE-IGF-II and IDE-TGF-α at 2.3 Å and IDE-amylin at 2.9 Å. We found that IDE cleaves its substrates at multiple sites in a biased stochastic manner. Furthermore, the presence of a disulfide bond in amylin allows IDE to cut at an additional site in the middle of the peptide (amino acids 18-19). Our amylin-bound IDE structure offers insight into how the structural constraint from a disulfide bond in amylin can alter IDE cleavage sites. Together with NMR structures of amylin and the IGF and epidermal growth factor families, our work also reveals the structural basis of how the high dipole moment of substrates complements the charge distribution of the IDE catalytic chamber for the substrate selectivity. In addition, we show how the ability of substrates to properly anchor their N-terminus to the exosite of IDE and undergo a conformational switch upon binding to the catalytic chamber of IDE can also contribute to the selective degradation of structurally related growth factors.     

Structural and Biochemical Characterization of Yeast Monothiol Glutaredoxin Grx6

Glutaredoxins (Grxs) are a ubiquitous family of proteins that reduce disulfide bonds in substrate proteins using electrons from reduced glutathione (GSH). The yeast Saccharomyces cerevisiae Grx6 is a monothiol Grx that is localized in the endoplasmic reticulum and Golgi compartments. Grx6 consists of three segments, a putative signal peptide (M1-I36), an N-terminal domain (K37-T110), and a C-terminal Grx domain (K111-N231, designated Grx6C). Compared to the classic dithiol glutaredoxin Grx1, Grx6 has a lower glutathione disulfide reductase activity but a higher glutathione S-transferase activity. In addition, similar to human Grx2, Grx6 binds GSH via an iron-sulfur cluster in vitro. The N-terminal domain is essential for noncovalent dimerization, but not required for either of the above activities. The crystal structure of Grx6C at 1.5 Å resolution revealed a novel two-strand antiparallel β-sheet opposite the GSH binding groove. This extra β-sheet might also exist in yeast Grx7 and in a group of putative Grxs in lower organisms, suggesting that Grx6 might represent the first member of a novel Grx subfamily.     

The Recombinant Amyloid-β Peptide Aβ1-42 Aggregates Faster and Is More Neurotoxic than Synthetic Aβ1-42

Aggregation of the amyloid-β (Aβ) peptide is considered a central event in the pathogenesis of Alzheimer's disease (AD). In order to bypass methodological bias related to a variety of impurities commonly present in typical preparations of synthetic Aβ, we developed a simple, generally applicable method for recombinant production of human Aβ and Aβ variants in Escherichia coli that provides milligram quantities of Aβ in very high purity and yield. Amyloid fibril formation in vitro by human Aβ1-42, the key amyloidogenic Aβ species in AD, was completed threefold faster with recombinant Aβ1-42 compared to synthetic preparations. In addition, recombinant Aβ1-42 was significantly more toxic to cultured rat primary cortical neurons, and it was more toxic in vivo, as shown by strongly increased induction of abnormal phosphorylation of tau and tau aggregation into neurofibrillary tangles in brains of P301L tau transgenic mice. We conclude that even small amounts of impurities in synthetic Aβ—including a significant fraction of racemized peptides that cannot be avoided due to the technical limitations of peptide synthesis—prevent or slow Aβ incorporation into the regular quaternary structure of growing β-amyloid fibrils. The results validate the use of recombinant Aβ1-42 for both in vitro and in vivo studies addressing the mechanisms underlying Aβ aggregation and its related biological consequences for the pathophysiology, therapy, and prevention of AD.     

Site-Directed Mutagenesis on Human Cystathionine-γ-Lyase Reveals Insights into the Modulation of H2S Production

In recent years, increased interest has been directed towards hydrogen sulfide (H₂S) as the third gasotransmitter and its role in various diseases. Cystathionine-γ-lyase (CSE) is one of the enzymes responsible for the endogenous production of H₂S in mammals. With the aid of the crystal structures of human CSE and site-directed mutagenesis studies, we have identified several amino acid residues in CSE that are actively involved in the catalysis of H₂S production. Contrary to reports suggesting that Tyr114 is required for substrate binding, our results reveal a significant increase in the production of H₂S upon mutation of Tyr114 to phenylalanine. This is attributed to an increased rate of pyridoxal 5′-phosphate (PLP) regeneration due to weakened π-stacking interactions between Phe114 and PLP. Thr189 is also identified as a crucial residue where hydrogen bonding to Asp187 keeps the latter in an optimal position for hydrogen bonding to the pyridoxal nitrogen of PLP. Furthermore, mutation of Glu339 to lysine, alanine or tyrosine reveals the importance of the hydrophobicity of the 339th amino acid in determining the specificity of the enzyme for the catalysis of α,γ-elimination or α,β-elimination reaction. Our study also shows that the rate of H₂S production is increased with increasing exogenous PLP concentration, hence supporting our hypothesis that apo-CSE is formed during the catalysis of H₂S production. Taken together, these findings suggest novel routes towards the design of activators or inhibitors that modulate the production of H₂S; these modulators may also serve as lead compounds in the development of drugs or mechanistic probes in the study of various H₂S-related diseases.     

Degradation of carbohydrate moieties of arabinogalactan-proteins by glycoside hydrolases from Neurospora crassa

Arabinogalactan-proteins (AGPs) are a family of plant proteoglycans having large carbohydrate moieties attached to core-proteins. The carbohydrate moieties of AGPs commonly have β-(1→3)(1→6)-galactan as the backbone, to which other auxiliary sugars such as l-Ara and GlcA are attached. For the present study, an α-l-arabinofuranosidase belonging to glycoside hydrolase family (GHF) 54, NcAraf1, and an endo-β-(1→6)-galactanase of GHF 5, Nc6GAL, were identified in Neurospora crassa. Recombinant NcAraf1 (rNcAraf1) expressed in Pichia pastoris hydrolyzed radish AGPs as well as arabinan and arabinoxylan, showing relatively broad substrate specificity toward polysaccharides containing α-l-arabinofuranosyl residues. Recombinant Nc6GAL (rNc6GAL) expressed in P. pastoris specifically acted on β-(1→6)-galactosyl residues. Whereas AGP from radish roots was hardly hydrolyzed by rNc6GAL alone, β-(1→6)-galactan side chains were reduced to one or two galactan residues by a combination of rNcAraf1 and rNc6GAL. These results suggest that the carbohydrate moieties of AGPs are degraded by the concerted action of NcAraf1 and Nc6GAL secreted from N. crassa.     

Expression of β-1,4-galactosyltransferase-I affects cellular adhesion in human peripheral blood CD4 T cells

β-1,4-galactosyltransferase-I (β-1,4-GalT-I) has two isoforms that differ only in the length of their cytoplasmic domains. In this study, we found that both the long and short isoforms of β-1,4-GalT-I were expressed in human CD4⁺ T lymphocytes, and localized in the cytoplasm and on the plasma membrane. The expression level of β-1,4-GalT-I was increased in CD4⁺ T cells after stimulation with interleukin (IL)-2, and was further increased after stimulation with IL-2 + IL-12, but decreased after stimulation with IL-2 + IL-4 when compared to stimulation with IL-2 alone. We also demonstrated that the cellular adhesion of CD4⁺ T cells was significantly increased upon cytokine stimulation, and was inhibited by α-lactalbumin, indicating that the increase in adhesion was positively correlated with the expression and activity of long β-1,4-GalT-I. Collectively, the data suggest that β-1,4-GalT-I plays a role in the cellular adhesion of CD4⁺ T cells.     

Synthesis and characterization of quaternized β-chitin

Water-soluble 2′-O-hydroxypropyltrimethylammoniumchitin chloride (2′-O-HTACCt) was prepared directly from β-chitin and 3-chloro-2-hydroxypropyltrimethylammonium chloride (CTA) in basic medium. The effect of alkali concentration, reaction temperature, reaction time, and dosage of CTA on yield and degree of substitution (DS) of 2′-O-HTACCt were studied. These quaternized chitin derivatives were characterized by FTIR and ¹H NMR spectroscopy, conductometric titration, and elemental analysis methods. Research results indicate that β-chitin can react directly with CTA to produce a water-soluble 2′-O-HTACCt derivative with a high DS. The optimal preparation conditions were determined to be 35-40 wt % (aq NaOH), 40 °C (reaction temperature), 6 h (reaction time), and 4 (molar ratio of CTA to β-chitin unit).     

Differential requirement of lipid rafts for FcγRIIA mediated effector activities

Immunoglobulin G (IgG) dependent activities are important in host defense and autoimmune diseases. Various cell types including macrophages and neutrophils contribute to pathogen destruction and tissue damage through binding of IgG to Fcγ receptors (FcγR). One member of this family, FcγRIIA, is a transmembrane glycoprotein known to mediate binding and internalization of IgG-containing targets. FcγRIIA has been observed to translocate into lipids rafts upon binding IgG-containing targets. We hypothesize that lipid rafts participate to different extents in binding and internalizing targets of different sizes. We demonstrate that disruption of lipid rafts with 8 mM methyl-β-cyclodextrin (MβCD) nearly abolishes binding (91% reduction) and phagocytosis (60% reduction) of large IgG-coated targets. Conversely, binding and internalization of small IgG-complexes is less dependent on lipid rafts (49% and 17% inhibition at 8 mM MβCD, respectively). These observations suggest that differences between phagocytosis and endocytosis may arise as early as the initial stages of ligand recognition.     

The expression and function of β-1,4-galactosyltransferase-I in dendritic cells

β-1,4-Galactosyltransferase-I (GalTI) is unusual among the galactosyltransferase family, which has two isoforms that differ only in the length of their cytoplasmic domains [1]. In this study, we found that both the long and short isoforms of GalTI were expressed in human monocyte-derived dendritic cells (MoDCs), and localized in the cytoplasm near nucleus and cytomembrane. The expression level of GalTI and cellular adhesion ability was increased when DCs continued to mature. We also demonstrated that the cellular adhesion ability of DCs was inhibited by α-lactalbumin (α-LA) via interference with cell surface GalTI function, suggesting that the adhesion ability was positively correlated with the expression of cell surface long GalTI. α-LA also could inhibit DC-T clustering and CD4⁺ T cell proliferation. Collectively, the data suggests that GalTI might act as a key adhesion molecular participating in T cells-DCs contacts.     

Loss of βarrestin1 and βarrestin2 contributes to pulmonary hypoplasia and neonatal lethality in mice

Less is known about the connection between the malfunction of βarrestins and developmental defects as the mice with either of two βarrestin isoforms knockout appear normal. In order to address the biological function of βarrestins during developmental process, we generate βarrestin1/2 double knockout mice. We found that βarrestin1/2 dual-null mice developed respiratory distress and atelectasis that subsequently caused neonatal death. Morphological examination revealed type II pneumocyte immaturity. Our results indicate that not only βarrestin1/2 double knockout lung tissue show disturbances in cell proliferation but βarrestin1 and βarrestin2 contribute to pulmonary surfactant complex generation during pulmonary maturation. Intra-amniotic delivery of recombinant adenovirus expressing βarrestin1 or βarrestin2 enhances surfactant-associated proteins synthesis in vivo. Our mRNA microarray data further reveal that βarrestin1/2 double knockout results in downregulation of a significant proportion of genes involved in several lung morphogenesis processes. Together, our study demonstrates that βarrestin1 and βarrestin2 collaborate in embryonic development processes for epithelial pneumocyte differentiation and lung maturation.