Kenji Akasaki

Chromatoid bodies: aggresome-like characteristics and degradation sites for organelles of spermiogenic cells.

We investigated the localization of several markers for lysosomes and aggresomes in the chromatoid bodies (CBs) by immunoelectron microscopy. We found so-called aggresomal markers such as Hsp70 and ubiquitin in the core of the CBs and vimentin and proteasome subunit around the CBs. Ubiquitin-conjugating enzyme (E2) was also found in the CBs. In tubulovesicular structures surrounding the CBs, lysosomal markers were detected but an endoplasmic reticulum retention signal (KDEL) was not. Moreover, proteins located in each subcellular compartment, including the cytosol, mitochondria, and nucleus, were detected in the CBs. Signals for cytochrome oxidase I (COXI) coded on mitochondrial DNA were also found in the CBs. Quantitative analysis of labeling density showed that all proteins examined were concentrated in the CBs to some extent. These results show that the CBs have some aggresomal features, suggesting that they are not a synthetic site as proposed previously but a degradation site where unnecessary DNA, RNA, and proteins are digested.

Dendritic Cell-Lysosomal-Associated Membrane Protein (LAMP) and LAMP-1-HIV-1 Gag Chimeras Have Distinct Cellular Trafficking Pathways and Prime T and B Cell Responses to a Diverse Repertoire of Epitopes

Ag processing is a critical step in defining the repertoire of epitope-specific immune responses. In the present study, HIV-1 p55Gag Ag was synthesized as a DNA plasmid with either lysosomal-associated membrane protein-1 (LAMP/gag) or human dendritic cell-LAMP (DC-LAMP/gag) and used to immunize mice. Analysis of the cellular trafficking of these two chimeras demonstrated that both molecules colocalized with MHC class II molecules but differed in their overall trafficking to endosomal/lysosomal compartments. Following DNA immunization, both chimeras elicited potent Gag-specific T and B cell immune responses in mice but differ markedly in their IL-4 and IgG1/IgG2a responses. The DC-LAMP chimera induced a stronger Th type 1 response. ELISPOT analysis of T cell responses to 122 individual peptides encompassing the entire p55gag sequence (15-aa peptides overlapping by 11 residues) showed that DNA immunization with native gag, LAMP/gag, or DC-LAMP/gag induced responses to identical immunodominant CD4+ and CD8+ peptides. However, LAMP/gag and DC-LAMP/gag plasmids also elicited significant responses to 23 additional cryptic epitopes that were not recognized after immunization with native gag DNA. The three plasmids induced T cell responses to a total of 39 distinct peptide sequences, 13 of which were induced by all three DNA constructs. Individually, DC-LAMP/gag elicited the most diverse response, with a specific T cell response against 35 peptides. In addition, immunization with LAMP/gag and DC-LAMP/gag chimeras also promoted Ab secretion to an increased number of epitopes. These data indicate that LAMP-1 and DC-LAMP Ag chimeras follow different trafficking pathways, induce distinct modulatory immune responses, and are able to present cryptic epitopes.

Immunocytochemical study of the surrounding envelope of autophagic vacuoles in cultured rat hepatocytes

By the use of electron immunoperoxidase cytochemistry at the ultrastructural level, the relationship of the surrounding sac of the autophagic vacuoles to the different cytomembranes was studied. When the endoplasmic reticulum was completely stained for microsomal carboxyesterase E1, the enzyme was not found to be labeled in the developed envelopes forming autophagic vacuoles. The autophagic envelope at the formative stages was also devoid of albumin which intensely stained Golgi cisternae. However, although it was rare, the endoplasmic reticulum showed an electron-lucent region like an early autophagic envelope in its cisternae which was lacking in carboxyesterase E1. In addition, deeply curving swelled cisternae where carboxyesterase E1 was found at the edges were occasionally encountered. These observations suggest that the segregating membranes arise from an endoplasmic reticulum and the structural characteristics of the endoplasmic membranes change at very early stages of formation of autophagic vacuoles. Acid phosphatase, a lysosomal marker enzyme, began to be localized on sections of the double membranes of newly created autophagic vacuoles. The enzyme spread all along the limiting membranes of the autophagic vacuoles, while, at the same time, the double membranes were converted into a single membrane. A lysosomal membrane glycoprotein (LGP107) was also localized on the surrounding envelope of autophagic vacuoles in a fashion similar to that of acid phosphatase. Lysosomal hydrolases seem to play some role in the conversion of double limiting membranes into a single limiting membrane.

Identification and characterization of two distinct kyotorphin-hydrolyzing enzymes in rat brain

We identified and characterized two kyotorphin-hydrolyzing peptidases (KTPases) in a soluble fraction of rat brain. When the soluble fraction was chromatographed with DEAE-Sephacel, the enzyme activity was resolved into two peaks, which were designated as KTPases I and II in their order of elution. KTPases I and II accounted for 95% and 5% of the KTPase activity in the soluble fraction, respectively. KTPases I and II hydrolyzed kyotorphin with Km values of 22 microM and 110 microM, respectively. By gel filtration, Mr values of KTPases I and II were determined to be 55,000 and 98,000, respectively. Immunological analyses of KTPase II with an anti-enkephalin aminopeptidase antibody indicated that KTPase II was identical to an enkephalin aminopeptidase with Mr = 98,000. However, KTPase I was a novel peptidase responsible for the major kyotorphin-degrading activity in the soluble fraction of rat brain.

Ile (476), a constituent of di-leucine-based motif of a major lysosomal membrane protein, LGP85/LIMP II, is important for its proper distribution in late endosomes and lysosomes

Lysosomal membrane glycoprotein termed LGP85 or LIMP II extends a COOH-terminal cytoplasmic tail of R459GQGSMDEGTADERAPLIRT478, in which an L475 I476 sequence lies as a di-leucine-based motif for lysosomal targeting. In the present study, we explored the role of the I476 residue in the localization of LGP85 to the endocytic organelles using two substitution mutants called I476A and I476L in which alanine and leucine are replaced at I476, respectively, and I476R477T478-deleted LGP85 called Delta 476-478. Immunofluorescence analyses showed that I476A and I476L are largely colocalized in intracellular organelles with an endogenous late endosomal and lysosomal marker, LAMP-1, but there were some granules in which staining for the LGP85 mutants was prominent, while Delta 476-478 is detected in LAMP-1-positive and LAMP-1-negative intracellular organelles, and on the cell surface. The subcellular fractionation studies revealed that I476A, I476L, and Delta 476-478 are different from wild-type LGP85 in the distribution of early endosomes, late endosomes, and lysosomes. I476A and I476L are present more in late endosomes than in the densest lysosomes, whereas wild-type LGP85 is mainly lysosomal. Substitution of I476 for A and L differentially modified the ratios of late endosomal to lysosomal LGP85. A major portion of Delta 476-478 resided in the light buoyant density fraction containing plasma membrane and early endosomes. Taken together, these results indicate that the existence of the 476th amino acid residue is essential for localization of LGP85 to late endocytic compartments. The fact that isoleucine but not leucine is in the 476th position is especially of importance in the proper distribution of LGP85 in late endosomes and lysosomes.

Purification and characterization of a soluble form of lysosome‐associated membrane glycoprotein‐2 (lamp‐2) from rat liver lysosomal contents

Lysosomal membrane of rat liver contains a highly glycosylated protein referred to as lamp‐2. Lamp‐2 occurs to a significant extent in a soluble fraction of rat liver lysosomes. The soluble form of lamp‐2 (SF‐lamp‐2) was purified to electrophoretic homogeneity. An apparent molecular weight (Mr) of SF‐lamp‐2 on sodium dodecy sulfate‐polyacrylamide gel electrophoresis was determined to be 91,000 which is 5,000 less than that of the membranous form of lamp‐2 (MF‐lamp‐2). SF‐ and MF‐lamp‐2 were very similar to each other in terms of sialic acid content, NH2‐terminal amino acid sequence and isoelectric point. Gel filtration data indicated that native SF‐lamp‐2 has an Mr=360,000. Taken together, SF‐lamp‐2 forms a tetrameric structure consisiting of a homogenous polypeptide lacking a membrane‐spanning domain and a cytoplasmic tail near the COOH‐terminus.

COOH-terminal isoleucine of lysosome-associated membrane protein-1 is optimal for its efficient targeting to dense secondary lysosomes

Lysosome-associated membrane protein-1 (LAMP-1) consists of a highly glycosylated luminal domain, a single-transmembrane domain and a short cytoplasmic tail that possesses a lysosome-targeting signal (GYQTI(382)) at the COOH terminus. It is hypothesized that the COOH-terminal isoleucine, I(382), could be substituted with any other bulky hydrophobic amino acid residue for LAMP-1 to exclusively localize in lysosomes. In order to test this hypothesis, we compared subcellular distribution of four substitution mutants with phenylalanine, leucine, methionine and valine at the COOH-terminus (termed I382F, I382L, I382M and I382V, respectively) with that of wild-type (WT)-LAMP-1. Double-labelled immunofluorescence analyses showed that these substitution mutants were localized as significantly to late endocytic organelles as WT-LAMP-1. However, the quantitative subcellular fractionation study revealed different distribution of WT-LAMP-1 and these four COOH-terminal mutants in late endosomes and dense secondary lysosomes. WT-LAMP-1 was accumulated three to six times more in the dense lysosomal fraction than the four mutants. The level of WT-LAMP-1 in late endosomal fraction was comparable to those of I382F, I382M and I382V. Conversely, I382L in the late endosomal fraction was approximately three times more abundant than WT-LAMP-1. These findings define the presence of isoleucine residue at the COOH-terminus of LAMP-1 as critical in governing its efficient delivery to secondary lysosomes and its ratio of lysosomes to late endosomes.

Involvement of microRNA214 and transcriptional regulation in reductions in mevalonate pyrophosphate decarboxylase mRNA levels in stroke-prone spontaneously hypertensive rat livers

Hypocholesterolemia has been epidemiologically identified as one of the causes of stroke (cerebral hemorrhage). We previously reported that lower protein levels of mevalonate pyrophosphate decarboxylase (MPD), which is responsible for reducing serum cholesterol levels in stroke-prone spontaneously hypertensive rats (SHRSP), in the liver were caused by a reduction in mRNA levels. However, the mechanism responsible for reducing MPD expression levels in the SHRSP liver remains unclear. Thus, we compared microRNA (miR)-214 combined with the 3′-untranslated region of MPD mRNA and heterogeneous nuclear RNA (hnRNA) between SHRSP and normotensive Wistar Kyoto rats (WKY). miR-214 levels in the liver were markedly higher in SHRSP than in WKY, whereas hnRNA levels were significantly lower. These results indicate that the upregulation of miR-214 and downregulation of MPD transcription in the liver both play a role in the development of hypocholesterolemia in SHRSP. Graphical abstract The upregulation of miR-214 and downregulation of MPD transcription in the liver both play a role in the development of hypocholesterolemia in SHRSP.

β‐Taxilin participates in differentiation of C2C12 myoblasts into myotubes

Myogenesis is required for the development of skeletal muscle. Accumulating evidence indicates that the expression of several genes are upregulated during myogenesis and these genes play pivotal roles in myogenesis. However, the molecular mechanism underlying myogenesis is not fully understood. In this study, we found that β-taxilin, which is specifically expressed in the skeletal muscle and heart tissues, was progressively expressed during differentiation of C2C12 myoblasts into myotubes, prompting us to investigate the role of β-taxilin in myogenesis. In C2C12 cells, knockdown of β-taxilin impaired the fusion of myoblasts into myotubes, and decreased the diameter of myotubes. We also found that β-taxilin interacted with dysbindin, a coiled-coil-containing protein. Knockdown of dysbindin conversely promoted the fusion of myoblasts into myotubes and increased the diameter of myotubes in C2C12 cells. Furthermore, knockdown of dysbindin attenuated the inhibitory effect of β-taxilin depletion on myotube formation of C2C12 cells. These results demonstrate that β-taxilin participates in myogenesis through suppressing the function of dysbindin to inhibit the differentiation of C2C12 myoblasts into myotubes.

The Major Lysosomal Membrane Proteins LAMP-1 and LAMP-2 Participate in Differentiation of C2C12 Myoblasts

Lysosomes are organelles that play a crucial role in the degradation of endocytosed molecules, phagocytosed macromolecules and autophagic substrates. The membrane of lysosomes contains several highly glycosylated membrane proteins, and lysosome-associated membrane protein (LAMP)-1 and LAMP-2 account for a major portion of the lysosomal membrane glycoproteins. Although it is well known that LAMP-2 deficiency causes Danon disease, which is characterized by cardiomyopathy, myopathy and mental retardation, the roles of lysosomal membrane proteins including LAMP-1 and LAMP-2 in myogenesis are not fully understood. In this study, to understand the role of LAMP proteins in the course of differentiation of myoblasts into myotubes, we used C2C12 myoblasts and found that the protein and mRNA levels of LAMP-1 and LAMP-2 were increased in the course of differentiation of C2C12 myoblasts into myotubes. Then, we investigated the effects of LAMP-1 or LAMP-2 knockdown on C2C12 myotube formation, and found that LAMP-1 or LAMP-2 depletion impaired the differentiation of C2C12 myoblasts and reduced the diameter of C2C12 myotubes. LAMP-2 knockdown more severely impaired C2C12 myotube formation compared with LAMP-1 knockdown, and knockdown of LAMP-1 did not exacerbate the suppressive effects of LAMP-2 knockdown on C2C12 myotube formation. In addition, knockdown of LAMP-1 or LAMP-2 decreased the expression levels of myogenic regulatory factors, MyoD and myogenin. These results demonstrate that both LAMP-1 and LAMP-2 are involved in C2C12 myotube formation and LAMP-2 may contribute dominantly to it.