Profile of miRNAs in small extracellular vesicles released from glioblastoma cells treated by boron neutron capture therapy.

PURPOSE: Boron neutron capture therapy (BNCT) is a tumor cell-selective particle-radiation therapy. In BNCT, administered p-boronophenylalanine (BPA) is selectively taken up by tumor cells, and the tumor is irradiated with thermal neutrons. High-LET α-particles and recoil 7Li, which have a path length of 5-9 µm, are generated by the capture reaction between 10B and thermal neutrons and selectively kill tumor cells that have uptaken 10B. Although BNCT has prolonged the survival time of malignant glioma patients, recurrences are still to be resolved. miRNAs, that are encapsulated in small extracellular vesicles (sEVs) in body fluids and exist stably may serve critical role in recurrence. In this study, we comprehensively investigated microRNAs (miRNAs) in sEVs released from post-BNCT glioblastoma cells. METHOD: Glioblastoma U87 MG cells were treated with 25 ppm of BPA in the culture media and irradiated with thermal neutrons. After irradiation, they were plated into dishes and cultured for 3 days in the 5% CO2 incubator. Then, sEVs released into the medium were collected by column chromatography, and miRNAs in sEVs were comprehensively investigated using microarrays. RESULT: An increase in 20 individual miRNAs (ratio > 2) and a decrease in 2 individual miRNAs (ratio < 0.5) were detected in BNCT cells compared with non-irradiated cells. Among detected miRNAs, 20 miRNAs were associated with worse prognosis of glioma in Kaplan Meier Survival Analysis of overall survival in TCGA. CONCLUSION: These miRNA after BNCT may proceed tumors, modulate radiation resistance, or inhibit invasion and affect the prognosis of glioma.

Observation of morphological abnormalities in silkworm pupae after feeding 137CsCl-supplemented diet to evaluate the effects of low dose-rate exposure.

Since the Fukushima Dai-ichi Nuclear Power Plant (FDNPP) accident, morphological abnormalities in lepidopteran insects, such as shrinkage and/or aberration of wings, have been reported. Butterflies experimentally exposed to radiocesium also show such abnormalities. However, because of a lack of data on absorbed dose and dose-effect relationship, it is unclear whether these abnormalities are caused directly by radiation. We conducted a low dose-rate exposure experiment in silkworms reared from egg to fully developed larvae on a 137CsCl-supplemented artificial diet and estimated the absorbed dose to evaluate morphological abnormalities in pupal wings. We used 137CsCl at 1.3 × 103 Bq/g fresh weight to simulate 137Cs contamination around the FDNPP. Absorbed doses were estimated using a glass rod dosimeter and Monte Carlo particle transport simulation code PHITS. Average external absorbed doses were approximately 0.24 (on diet) and 0.016 mGy/day (near diet); the average internal absorbed dose was approximately 0.82 mGy/day. Pupal wing structure is sensitive to radiation exposure. However, no significant differences were observed in the wing-to-whole body ratio of pupae between the 137CsCl-exposure and control groups. These results suggest that silkworms are insensitive to low dose-rate exposure due to chronic ingestion of high 137Cs at a high concentration.

Age-related isomerization of Asp in human immunoglobulin G kappa chain.

Isomerization of aspartate (Asp) is a common non-enzymatic posttranslational modification. Isomerized residues accumulate in proteins associated with age-related human disorders such as cataract and are well known to affect protein structure and function. We previously detected d-Asp-containing peptides in human serum. In this study, we investigated whether isomerized Asp residues are present in human immunoglobulin G (IgG) kappa chain by a qualitative d-amino acid analysis based on diastereomer formation and liquid chromatography tandem mass spectrometry (LC-MS/MS). We also investigated the d/l ratio of Asp residues in the IgG kappa chain in serum from donors aged 25, 37, 41, 54 and 67 years. As a result, two isomerized Asp residues, Asp151 and Asp170, were detected in the IgG kappa chain, and the d/l ratio of these residues was found to increase with aging. To assess the effects of this isomerization, we synthesized four isomeric peptides of IgG kappa chain containing lα-, lß-, dα-, or dß-Asp at position 170, and compared their secondary structures by CD spectroscopy. Peptide containing normal lα-Asp170 showed type II ß-turn structure, while the other isomeric peptides showed random structure, clearly indicating that substitution of a single Asp isomer alters the secondary structure of the peptide. Because IgG is a main component of humoral immunity, Asp isomerization in IgG may reflect changes of structure and decrease in immune function. Proteome research on serum from the standpoint of racemization might enable us to develop new kinds of biomarker and new directions to study the aging process.

Identification of á´ -amino acid-containing peptides in human serum.

Biologically uncommon d-aspartate (d-Asp) residues have been shown to accumulate in proteins associated with age-related human disorders, such as cataract and Alzheimer disease. Such d-Asp-containing proteins are unlikely to be broken down completely because metabolic enzymes recognize only proteins or peptides composed exclusively of l-amino acids. Therefore, undigested d-Asp-containing peptides may exist in blood and, if detectable, may be a useful biomarker for associated diseases. In this study, we investigated d-amino acid-containing peptides in adult human serum by a qualitative d-amino acid analysis based on a diastereomer method and LC-MS/MS method. As a result, two d-Asp-containing peptides were detected in serum, both derived from the fibrinogen ß-chain, a glycoprotein that helps in the formation of blood clots. One of the peptides was fibrinopeptide B, which prevents fibrinogen from forming polymers of fibrin, and the other was same peptide with C-terminal Arginine missing. To our knowledge, this is the first report of the presence of d-amino acid-containing peptides in serum and the approach described will provide a new direction on the serum proteome and fragmentome.

Isomeric Replacement of a Single Aspartic Acid Induces a Marked Change in Protein Function: The Example of Ribonuclease A.

lα-Aspartic acid (Asp) residues in proteins are nonenzymatically isomerized to abnormal lß-, dα-, and dß-Asp isomers under physiological conditions. Such an isomerization of Asp residues is considered to be a trigger of protein denaturation because it either elongates the main chain or induces a different orientation of the side chain within the protein structure or both. However, previous studies have found no direct evidence of the effects of Asp isomers on protein function. Therefore, the production of Asp-isomer-containing proteins is required to verify the effects of Asp isomerization. Here, we describe the production of an Asp-isomer-containing protein using the expressed protein ligation. As a model protein, bovine pancreatic ribonuclease A (RNase A, EC 3.1.27.5), which catalyzes the cleavage of phosphodiester bonds in RNA, was used. In this study, lα-Asp at position 121 in RNase A was replaced by lß-, dα-, and dß-Asp. The objective aspartic acid at position 121 is located near the active site and related to RNA cleavage. The RNase A with lα-Asp at position 121 showed a normal activity. By contrast, the catalytic activity of lß-, dα-, and dß-Asp-containing RNase A was markedly decreased. This study represents the first synthesis and analysis of a protein containing four different Asp isomers.

Substrate stereoselectivity of mammalian D-aspartyl endopeptidase.

The formation and accumulation of D-aspartate residue (D-Asp) in proteins caused by oxidative stress leads to dysfunction and/or denaturation of proteins, and is consequently responsible for aging-related misfolding diseases such as cataracts, prion disease, and Alzheimer's disease. We sought to identify that an unknown protease selectively degrades the noxious D-Asp-containing protein, namely D-aspartyl endopeptidase (DAEP), and finally purified it from the inner mitochondrial membrane of mouse liver. In order to analyze the substrate stereoselectivity of DAEP, we synthesized a peptide corresponding to 55-65 (Thr-Val-Leu-Asp-Ser-Gly-Ile-Ser-Glu-Val-Arg) of human αA-crystallin and its corresponding diastereoisomers in which L-α-Asp was replaced with L-ß-, D-α- or D-ß-Asp residue at position 58. Following incubation of that peptide with purified DAEP, it was only degraded at D-α-Asp(58), independent of ATP or NAD. This result indicates that DAEP stereoselectively recognizes and degrades its substrate at the internal D-α-Asp residue. DAEP therefore seems to physiologically serve as the quality control system against the noxious D-Asp-containing protein in the long life span of mammals.

UV B-irradiation enhances the racemization and isomerizaiton of aspartyl residues and production of Nε-carboxymethyl lysine (CML) in keratin of skin.

UV-B irradiation is one of the risk factors in age-related diseases. We have reported that biologically uncommon D-ß-Asp residues accumulate in proteins from sun-exposed elderly human skin. A previous study also reported that carboxymethyl lysine (CML; one of the advanced glycation end products (AGEs)) which is produced by the oxidation of glucose and peroxidation of lipid, also increases upon UV B irradiation. The formation of D-ß-Asp and CML were reported as the alteration of proteins in UV B irradiated skin, independently. In this study, in order to clarify the relationship between the formation of D-ß-Asp and CML, immunohistochemical analysis using anti-D-ß-Asp containing peptide antibodies and anti-CML antibodies was performed in UV B irradiated mice. Immunohistochemical analyses clearly indicated that an anti-D-ß-Asp containing peptide antibody and anti-CML antibody reacted at a common area in UV B irradiated skin. Western blot analyses of the proteins isolated from UV B irradiated skin demonstrated that proteins of 50-70 kDa were immunoreactive towards antibodies for both D-ß-Asp containing peptide and CML. These proteins were identified by proteomic analysis as members of the keratin families including keratin-1, keratin-6B, keratin-10, and keratin-14.

Collapse of homochirality of amino acids in proteins from various tissues during aging.

Prior to the emergence of life, it is believed that only L-amino acids were selected for formation of proteins, and that D-amino acids were eliminated on the primitive Earth. Whilst homochirality is essential for life, recently the occurrence of proteins containing D-beta-aspartyl (Asp) residues from various tissues of elderly subjects has been reported. Here, we discuss the presence of D-beta-Asp-containing proteins in the lens, ciliary body, drusen, and sclera of the eye, skin, cardiac muscle, blood vessels of the lung, chief cells of the stomach, longitudinal and circular muscles of the stomach, and small and large intestines. Since the D-beta-Asp residue occurs through a succinimide intermediate, this isomer may potentially be generated in proteins more easily than initially thought. UV Rays and oxidative stress can accelerate the formation of the D-beta-Asp residue in proteins.

Influence of oxidative stress on D-aspartyl endopeptidase activity.

It is strongly suggested that D-aspartic acid (D-Asp)-containing proteins are spontaneously generated by oxidative stress and would cause many aging-related misfolding diseases, such as cataracts, prion disease, and Alzheimer's disease. We have identified a D-Asp-containing protein-specific protease, D-aspartyl endopeptidase (DAEP), from mammalian mitochondria, serving as a scavenger against the noxious D-Asp-containing protein. Recently, it has been shown that the activity of Lon, an ATP-dependent protease degrading oxidatively damaged proteins in mitochondria, decreases with aging by oxidative stress. However, an obvious relation between DAEP activity and oxidative stress with aging remains to be demonstrated. In the present study, we showed that there was a remarkable decrease in DAEP activity in superoxide dismutase-deficient mice, which formed excess reactive oxygen species (ROS). Our result suggests that a decrease in DAEP activity by oxidative stress may cause the accumulation of D-Asp-containing protein, leading to mitochondria-associated diseases.

Structural consideration of mammalian D-aspartyl endopeptidase.

D-aspartyl endopeptidase (DAEP) is a specific protease for D-aspartic acid (D-Asp)-containing protein, which has been implicated in the pathogenesis of age-related and misfolding diseases such as Alzheimer's disease. Therefore, DAEP would serve as a defensive system against the noxious D-Asp-containing protein. However, it is unclear how DAEP exerts its unique enzymatic function, since its higher-order structure remains quite unsolved. In this study, we analyzed the conformation of purified DAEP from the mitochondrial membrane of mouse by atomic force microscopy the advantage of which is its ability to study biological macromolecules and even living organisms in an ambient air environment. DAEP formed a ring-like structure with a diameter of ca. 40 nm. Our data suggest that DAEP topologically belongs to the AAA+ protease family such as proteasome, Lon, and mitochondrial membrane-bound i-/m-AAA protease.

Influence of Lß-, Dα- and Dß-Asp isomers of the Asp-76 residue on the properties of αA-crystallin 70-88 peptide.

Proteins have been considered to consist exclusively of L-amino acids in living tissues. However, our previous studies showed that two specific aspartyl (Asp) residues in αA- and αB-crystallins from human eye lenses invert to the D-isomers to a high degree during aging. The reaction is also accompanied by isomerization into a form containing ß-Asp (isoaspartate) residues. The appearance of D- and ß-Asp in a protein potentially induces large changes to the higher order structure of the protein as well as to its function. However, it remains unclear whether the formation of the Asp isomer is the direct trigger of the change to the higher order structure and function. In this study, in order to clarify the effect of the inversion to D-isomers in a protein, we synthesized peptides corresponding to the 70-88 (KFVIFLDVKHFSPEDLTVK) fragment of human αA-crystallin and its corresponding diastereoisomers in which Lα-Asp was replaced with Lß-Asp, Dα-Asp, and Dß-Asp at position 76 and compared their biochemical properties with that of normal peptide. The peptides containing abnormal isomers (Lß-Asp, Dα-Asp, and Dß-Asp residues, respectively) were more hydrophilic than the normal peptide (containing Lα-Asp), lost ß-sheet structure and changed to random structures. The normal peptide promoted the aggregation of insulin while the other three isomers suppressed the aggregation of insulin. This is the first evidence that a single substitution of an Asp isomer in a peptide induces a large change to the properties of the peptide.

Localization of D-ß-aspartyl residue-containing proteins in various tissues.

Prior to the emergence of life, it is believed that only l-amino acids were selected for formation of protein and that d-amino acids were eliminated on the primitive Earth. Whilst homochirality is essential for life, the occurrence of proteins containing d-beta-aspartyl (Asp) residues in various tissues from elderly subjects has been reported recently. Here, we demonstrate the presence of a d-beta-Asp-containing protein in the cardiac muscle of heart, blood vessels of the lung, chief cells of the stomach, longitudinal and circular muscle of the stomach, small intestine and large intestine. Since the d-beta-Asp residue occurs through a succinimide intermediate, this isomer may potentially be generated in proteins more easily than initially thought. Formation of the d-beta-Asp residue in proteins may be related to stress.

Screening system for D-Asp-containing proteins using D-aspartyl endopeptidase and two-dimensional gel electrophoresis.

D-Asp-containing proteins have been implicated in many aging-related diseases. To clarify the role of D-Asp-containing proteins in such diseases, we developed a screening system for these proteins using a D-aspartyl endopeptidase that specifically cleaves the proteins at the C-terminus. The digested proteins were detected by means of two-dimensional gel electrophoresis and identified using nano-liquid chromatography/tandem mass spectrometry. We were able to detect myelin basic protein, a known D-Asp-containing protein, in the brain tissues of mice; this indicates that our system is effective for screening D-Asp-containing proteins.

Differential rate constants of racemization of aspartyl and asparaginyl residues in human alpha A-crystallin mutants.

Asp58 and Asp151 in alpha A-crystallin of human eye lenses become highly inverted and isomerized to d-beta-Asp residues with age. Racemization was previously shown to proceed rapidly when the residue on the carboxyl side of the Asp residue is small. Asn was also demonstrated to be more susceptible to racemization than Asp in protein. In this study, the changes of rate constants for racemization at Asp58 and Asp151 and at Asn58 and Asn151 were investigated using D58N, S59T, D151N and A152V mutants obtained through site-directed mutagenesis. The rate constant of racemization at Asn151 in D151N was found to be 1.5 times more rapid than Asp151 in the wild-type. For A152V, the rate constant at Asp151 was 1/4 that of the wild-type. There were no significant differences in the rate constants of racemization for both Asp58 and Asn58 residues. The aggregate size of D58N, S59T and D151N mutants increased or increased in polydispersity and their chaperone activities decreased. The size and chaperone activity of A152V was unchanged. These results suggest that structures close to Asp58 and Asp151 residues in the protein affect the rate constant of Asp racemization and the size and chaperone function of alpha A-crystallin.

Distribution of CESP-1 protein in the corneal endothelium and other tissues.

PURPOSE: The gene expression profile of human corneal endothelium (CE) was established with the gene signature system. A novel gene, GS3582, was abundantly transcribed in the CE compared with other tissues according to a human gene expression database. This protein was designated corneal endothelium-specific protein (CESP)-1. The tissue distribution and subcellular localization of CESP-1 was assessed in humans and mice, to investigate its physiological function. METHODS: Rabbit and mouse CESP-1 cDNAs were cloned, and a polyclonal anti-human CESP-1 antibody (Ab) and anti-mouse N- or C-terminal ovary-specific acidic protein (OSAP)-1 Ab were produced. CESP-1 expression was investigated in human and mouse corneas by Western blot and/or immunohistochemical analysis. The distribution of CESP-1 in human tissues was also examined by Western blot analysis. To identify the subcellular localization of CESP-1, cultured human CE was colabeled with anti-human CESP-1 Ab and anti-cytochrome c monoclonal Ab or anti-GRP78 monoclonal Ab for confocal microscopy. RESULTS: The rabbit and mouse CESP-1 cDNA sequences contained an open reading frame coding 242 and 283 amino acids, respectively. Mouse CESP-1 was entirely consistent with mouse OSAP. Western blot analysis showed that CESP-1 was expressed in the human corneal epithelium, CE, cultured CE, brain, testis, and ovary. Mouse CESP-1 was also expressed in mouse corneal epithelium and CE with anti-mouse C- but not N-terminal OSAP Ab according to immunohistochemical analysis. Subcellular localization of CESP-1 to the mitochondria was demonstrated in cultured human CE. The N-terminal of CESP-1, possessing a mitochondrial targeting sequence, may be processed after the protein is imported into the mitochondria. CONCLUSIONS: CESP-1 was distributed in the corneal epithelium, the CE and cultured human CE, as well as the brain, testis, and ovary. CESP-1 was localized in the mitochondria of cultured human CE. These findings may provide some clues about the physiological function of CESP-1.

Mammalian D-aspartyl endopeptidase: a scavenger for noxious racemized proteins in aging.

The accumulation of D-isomers of aspartic acid (D-Asp) in proteins during aging has been implicated in the pathogenesis of Alzheimer's disease, cataracts, and arteriosclerosis. Here, we identified a specific lactacystin-sensitive endopeptidase that cleaves the D-Asp-containing protein and named it D-aspartyl endopeptidase (DAEP). DAEP has a multi-complex structure (MW: 600kDa) and is localized in the inner mitochondrial membrane of mouse and rabbit, but DAEP activity was not detected in Escherichia coli, Saccharomyces cerevisiae, and Caenorhabditis elegans. A specific inhibitor for DAEP was newly synthesized, and inhibited DAEP activity (IC(50), 3microM), a factor of 10 greater than lactacystin on DAEP. On the other hand, the inhibitor did not inhibit either the 20S or 26S proteasome.

Construction of human corneal endothelial cDNA library and identification of novel active genes.

PURPOSE: To describe genes expressed in human corneal endothelial cells and identify novel genes. METHODS: Sixteen human donor corneas that had no history of corneal disease, infection, or intraocular surgery were used within 7 days of death. Total RNA was extracted from corneal endothelial cells with attached Descemet membranes. A 3'-directed cDNA library was constructed from mRNA by using a pUC19-based primer. These sequences were compared with each other to determine their frequency and were searched against GenBank for identification. To identify novel specific and abundant transcript genes in corneal endothelial cells, the novel genes were compared with an expressed sequence tag database, the expected sequence extended, and 5' rapid amplification of cDNA ends-polymerase chain reaction cloning performed. RESULTS: The human corneal endothelial cDNA library showed that the most abundant transcript was prostaglandin D2 synthase. The remaining transcript genes that were present in abundance consisted of lactate dehydrogenase-A, gene signature (GS) 3582, which is a novel gene without a known function, and matrix Gla protein. The full-length sequence of GS3582 showed similarity to genes obtained in ovary and TESTIS. CONCLUSIONS: A human corneal endothelial cDNA library was constructed. An expression profile of corneal endothelium provides probes to monitor physiologic and pathologic conditions of this tissue in terms of gene expression.