Cleavage of amyloid-beta precursor protein (APP) by membrane-type matrix metalloproteinases

Amyloid-beta precursor protein (APP) was identified on expression cloning from a human placenta cDNA library as a gene product that modulates the activity of membrane-type matrix metalloproteinase-1 (MT1-MMP). Co-expression of MT1-MMP with APP in HEK293T cells induced cleavage and shedding of the APP ectodomain when co-expressed with APP adaptor protein Fe65. Among the MT-MMPs tested, MT3-MMP and MT5-MMP also caused efficient APP shedding. The recombinant APP protein was cleaved by MT3-MMP in vitro at the A463-M464, N579-M580, H622-S623, and H685-Q686 peptide bonds, which included a cleavage site within the amyloid beta peptide region known to produce a C-terminal fragment. The Swedish-type mutant of APP, which produces a high level of amyloid beta peptide, was more effectively cleaved by MT3-MMP than wild-type APP in both the presence and absence of Fe65; however, amyloid beta peptide production was not affected by MT3-MMP expression. Expression of MT3-MMP enhanced Fe65-dependent transactivation by APP fused to the Gal4 DNA-binding and transactivation domains. These results suggest that MT1-MMP, MT3-MMP and MT5-MMP should play an important role in the regulation of APP functions in tissues including the central nervous system.     

A protein recycling system for nuclear magnetic resonance-based screening of drug candidates

A sample-treating system for nuclear magnetic resonance (NMR)-based interaction screening between drug candidates (small molecules) and a protein of interest was developed by applying high-performance liquid chromatography (HPLC) technology. The system prepares a test solution by mixing a (15)N-labeled protein solution and a solution of each candidate compound, loads it to a flow cell-type NMR probe, and recycles the protein after the data acquisition. The system was designed to behave differently according to the information obtained in NMR measurements. In a test operation with a 100-compound library, the system could single out known interacting substances properly. Recovery values of the protein and one representative compound were 75 and 71%, respectively, and the recovered protein was found intact as intended.     

Enzyme-responsive release of encapsulated proteins from biodegradable hollow capsules

Biodegradable hollow capsules encapsulating proteins were prepared via layer-by-layer assembly of chitosan and dextran sulfate on protein-entrapping mesoporous silica particles and the subsequent removal of the silica. The enzymatic degradation of the capsules in the presence of chitosanase was explored by scanning electron microscopy (SEM). With increasing time, the chitosan component was degraded by chitosanase, and the capsules began to deform and were finally destroyed. Sustained release of the encapsulated proteins was attained by using the enzymatic degradation of the hollow capsules. The release behavior was successfully manipulated by altering the charge of capsule surface.     

Spread of X-chromosome inactivation into chromosome 15 is associated with Prader–Willi syndrome phenotype in a boy with a t(X;15)(p21.1;q11.2) translocation

X-chromosome inactivation (XCI) is an essential mechanism in females that compensates for the genome imbalance between females and males. It is known that XCI can spread into an autosome of patients with X;autosome translocations. The subject was a 5-year-old boy with Prader?CWilli syndrome (PWS)-like features including hypotonia, hypo-genitalism, hypo-pigmentation, and developmental delay. G-banding, fluorescent in situ hybridization, BrdU-incorporated replication, human androgen receptor gene locus assay, SNP microarrays, ChIP-on-chip assay, bisulfite sequencing, and real-time RT-PCR were performed. Cytogenetic analyses revealed that the karyotype was 46,XY,der(X)t(X;15)(p21.1;q11.2),?15. In the derivative chromosome, the X and half of the chromosome 15 segments showed late replication. The X segment was maternal, and the chromosome 15 region was paternal, indicating its post-zygotic origin. The two chromosome 15s had a biparental origin. The DNA methylation level was relatively high in the region proximal from the breakpoint, and the level decreased toward the middle of the chromosome 15 region; however, scattered areas of hypermethylation were found in the distal region. The promoter regions of the imprinted SNRPN and the non-imprinted OCA2 genes were completely and half methylated, respectively. However, no methylation was found in the adjacent imprinted gene UBE3A, which contained a lower density of LINE1 repeats. Our findings suggest that XCI spread into the paternal chromosome 15 led to the aberrant hypermethylation of SNRPN and OCA2 and their decreased expression, which contributes to the PWS-like features and hypo-pigmentation of the patient. To our knowledge, this is the first chromosome-wide methylation study in which the DNA methylation level is demonstrated in an autosome subject to XCI.     

Structural and biochemical characterization of glycoside hydrolase family 79 β-glucuronidase from Acidobacterium capsulatum

We present the first structure of a glycoside hydrolase family 79 β-glucuronidase from Acidobacterium capsulatum, both as a product complex with β-D-glucuronic acid (GlcA) and as its trapped covalent 2-fluoroglucuronyl intermediate. This enzyme consists of a catalytic (β/α)(8)-barrel domain and a β-domain with irregular Greek key motifs that is of unknown function. The enzyme showed β-glucuronidase activity and trace levels of β-glucosidase and β-xylosidase activities. In conjunction with mutagenesis studies, these structures identify the catalytic residues as Glu(173) (acid base) and Glu(287) (nucleophile), consistent with the retaining mechanism demonstrated by (1)H NMR analysis. Glu(45), Tyr(243), Tyr(292)-Gly(294), and Tyr(334) form the catalytic pocket and provide substrate discrimination. Consistent with this, the Y292A mutation, which affects the interaction between the main chains of Gln(293) and Gly(294) and the GlcA carboxyl group, resulted in significant loss of β-glucuronidase activity while retaining the side activities at wild-type levels. Likewise, although the β-glucuronidase activity of the Y334F mutant is ~200-fold lower (k(cat)/K(m)) than that of the wild-type enzyme, the β-glucosidase activity is actually 3 times higher and the β-xylosidase activity is only 2.5-fold lower than the equivalent parameters for wild type, consistent with a role for Tyr(334) in recognition of the C6 position of GlcA. The involvement of Glu(45) in discriminating against binding of the O-methyl group at the C4 position of GlcA is revealed in the fact that the E45D mutant hydrolyzes PNP-β-GlcA approximately 300-fold slower (k(cat)/K(m)) than does the wild-type enzyme, whereas 4-O-methyl-GlcA-containing oligosaccharides are hydrolyzed only 7-fold slower.     

Intravitreal injection or topical eye-drop application of a μ-calpain C2L domain peptide protects against photoreceptor cell death in Royal College of Surgeons' rats, a model of retinitis pigmentosa

Mitochondrial μ-calpain initiates apoptosis-inducing factor (AIF)-dependent apoptosis in retinal photoreceptor degeneration. Mitochondrial μ-calpain inhibitors may represent therapeutic targets for the disease. Therefore, we sought to identify inhibitors of mitochondrial calpains and determine their effects in Royal College of Surgeons' (RCS) rats, an animal model of retinitis pigmentosa (RP). We synthesized 20-mer peptides of the C2-like (C2L) domain of μ-calpain. Two μ-calpain peptides N2 and N9 inhibited mitochondrial μ-calpain activity (IC(50); 892 and 498nM, respectively), but not other proteases. Western blotting showed that 50μM of both μ-calpain peptides caused specific degradation of mitochondrial μ-calpain. Three-dimensional structure of calpains suggested that the peptides N2 and N9 corresponded to the regions forming salt bridges between the protease core domain 2 and the C2L domain. We determined the inhibitory regions of μ-calpain peptides N2 and N9 using 10-mers, and one peptide, N2-10-2, inhibited the activity of mitochondrial μ-calpain (IC(50); 112nM). We next conjugated the peptide N2-10-2 to the C-terminal of HIV-1 tat (HIV), a cell-penetrating peptide. Using isolated rat liver mitochondria, 50μM HIV-conjugated μ-calpain N2-10-2 peptide (HIV-Nμ, IC(50); 285nM) significantly inhibited AIF truncation. The intravitreal injection of 20mM HIV-Nμ also prevented retinal photoreceptor apoptosis determined by TUNEL staining, and preserved retinal function assessed by electroretinography in RCS rats. Topical application of 40mM HIV-Nμ also prevented apoptosis of retinal photoreceptors in RCS rats. Our results demonstrate that HIV-Nμ, a peptide inhibitor of mitochondrial μ-calpain, offers a new modality for treating RP.     

Formation of nanofilms on cell surfaces to improve the insertion efficiency of a nanoneedle into cells

A nanoneedle, an atomic force microscope (AFM) tip etched to 200 nm in diameter and 10 μm in length, can be inserted into cells with the aid of an AFM and has been used to introduce functional molecules into cells and to analyze intracellular information with minimal cell damage. However, some cell lines have shown low insertion efficiency of the nanoneedle. Improvement in the insertion efficiency of a nanoneedle into such cells is a significant issue for nanoneedle-based cell manipulation and analysis. Here, we have formed nanofilms composed of extracellular matrix molecules on cell surfaces and found that the formation of the nanofilms improved insertion efficiency of a nanoneedle into fibroblast and neural cells. The nanofilms were shown to improve insertion efficiency even in cells in which the formation of actin stress fibers was inhibited by the ROCK inhibitor Y27632, suggesting that the nanofilms with the mesh structure directly contributed to the improved insertion efficiency of a nanoneedle.