Lysosome-associated membrane protein-1 and -2 (LAMP-1 and LAMP-2, respectively) are type I transmembrane proteins. LAMP-2 comprises three splice isoforms (LAMP-2A, -B and-C) with different cytoplasmic tails (CTs). These three CTs possess different tyrosine-based motifs (GYXXΦ, where Φ is a bulky hydrophobic amino acid) at their C-termini. Interactions between tyrosine-based motifs and μ-subunits of four tetrameric adaptor protein (AP) complexes are necessary for their vesicular transport to lysosomes. Little is known about how the interaction strengths of these tyrosine motifs with μ-subunits affect the localization of isoforms to lysosomes. The interactions were first investigated using a yeast two-hybrid system to address this question. LAMP-2A-CT interacted with all four μ-subunits (μ1, μ2, μ3A and μ4 of AP-1, AP-2, AP-3 and AP-4, respectively). The interaction with μ3A was more robust than that with other μ-subunits. LAMP-2B-CT interacted exclusively and moderately with μ3A. LAMP-2C-CT did not detectably interact with any of the four μ-subunits. Immunofluorescence microscopy showed that all isoforms were localized in late endosomes and lysosomes. LAMP-2C was present in the plasma membrane and early endosomes; however, LAMP-2A and -2B were barely detectable in these organelles. In cell fractionation, LAMP-2A was the most abundant in the dense lysosomes, whereas LAMP-2C was significantly present in the low-density fraction containing the plasma membrane and early endosomes, in addition to the dense lysosomes. LAMP-2B considerably existed in the low-density late endosomal fraction. These data strongly suggest that the LAMP-2 isoforms are distributed differently in endocytic organelles depending on their interaction strengths with AP-3.

Lysosome-associated membrane protein-1 (LAMP-1) is a type I membrane glycoprotein consisting of a large luminal domain, a membrane-anchoring domain, and a short cytoplasmic tail (CT). The tyrosine-based motif (G378Y379QTI382) in its CT exclusively binds to adapter protein complex-3 (AP-3), which may facilitate the incorporation of LAMP-1 into transport vesicles to late endosomes and lysosomes. Of this sequence, Y379 is critical, and hydrophobic I382 is optimal for the AP-3 binding and the efficient delivery to lysosomes. However, it is not clear how important G378 is in the AP-3 binding and lysosome transport. To clarify its importance, four mutants in which G378 was replaced with alanine, aspartic acid, glutamic acid, and asparagine (designated as G378A, G378D, G378E, and G378N, respectively) were prepared and their interaction strengths with AP-3 and lysosomal abundance were compared with those of wild-type (WT)-LAMP-1. A yeast two-hybrid system was applied to measure the interaction strengths of WT-, G378A-, G378D-, G378E-, and G378N-CTs with the medium subunit of AP-3 (μ3A). The G378A-, G378D-, and G378E-CTs as strongly interacted with μ3A as WT-CT, but the G378N-CT exhibited a very weak interaction with it. In the cell fractionation analyses, the lysosomal levels of G378A, G378D, and G378E were almost the same as that of WT whereas a lesser amount of G378N existed in the lysosomes. Taken together, it is considered that Y379 and I382 are essential for AP-3-mediated vesicular transport of LAMP-1 while the Y379-preceding amino acid is not restricted to glycine but asparagine at this position is less suitable for that.

Lysosomal integral membrane protein-2 (LIMP-2) is a type III transmembrane protein that is highly glycosylated and mainly localized to the lysosomal membrane. The diverse functions of LIMP-2 are currently being uncovered; however, its participation in macroautophagy, usually described as autophagy, has not yet been well-investigated. To determine the possible involvement of LIMP-2 in autophagic activity, we examined the intracellular amount of microtubule-associated protein 1 light chain 3 (LC3)-II, which is well-correlated with autophagosome levels, in exogenous rat LIMP-2-expressing COS7 and HEK293 cells. Transient or stable expression of LIMP-2-myc significantly increased the levels of LC3-II. Conversely, knockdown of LIMP-2 decreased the LC3-II levels in NIH3T3 cells. Furthermore, approaches using lysosomal protease inhibitors and mCherry-GFP-LC3 fluorescence suggested that exogenous expression of LIMP-2 increased the biogenesis of autophagosomes rather than decreased the lysosomal turnover of LC3-II. Considering the results of the biochemical assay and the quantitative fluorescence assay together, it is suggested that LIMP-2 has a possible involvement in autophagic activity, especially autophagosome biogenesis.

Lysosome-associated membrane protein-1 (LAMP-1) is a type I membrane glycoprotein with a COOH-terminal cytoplasmic tail (CT) containing a lysosome-targeting signal of GYQTI382-COOH. This sequence is categorized as a tyrosine-based motif of GYXXΦ where Φ is a bulky hydrophobic amino acid residue. Lysosomal localization of LAMP-1 varies by changing the COOH-terminal amino acid residues. Adaptor protein (AP) complexes are reported to recognize the tyrosine-based signal peptide for efficient lysosomal transport of LAMP-1. In order to better understand the role of APs in lysosomal transport of LAMP-1, we have studied interactions of wild-type (WT) and mutated CTs of LAMP-1 with medium (μ) subunits of the four APs by a yeast two-hybrid (Y2H) system and subsequent computer-based molecular modeling. Among the μ subunits of AP-1, AP-2, AP-3 and AP-4, called μ1, μ2, μ3A, and μ4, respectively, the WT-CT significantly interacted with μ3Α in the Y2H system. The degree of interaction of the WT and mutated CTs with μ3A from the Y2H analyses correlated with that of their dissociation constants determined by computer-based molecular modeling, and also with that of the late endosomal and lysosomal amount of WT and the similar mutants of LAMP-1. These results reinforce the notion that AP-3 makes a major contribution to the vesicular transport of LAMP-1 to late endosomes and lysosomes via a direct intracellular route.

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.

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.

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 down-regulation of MPD transcription in the liver both play a role in the development of hypocholesterolemia in SHRSP.

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.

Lysosome-associated membrane protein-1 (LAMP-1) is a type I transmembrane protein with a short cytoplasmic tail that possesses a lysosome-targeting signal of GYQTI(382)-COOH. Wild-type (WT)-LAMP-1 was exclusively localized in high density lysosomes, and efficiency of LAMP-1's transport to lysosomes depends on its COOH-terminal amino acid residue. Among many different COOH-terminal amino acid substitution mutants of LAMP-1, a leucine-substituted mutant (I382L) displays the most efficient targeting to late endosomes and lysosomes [Akasaki et al. (2010) J. Biochem. 148: , 669-679]. In this study, we generated two human hepatoma cell lines (HepG2 cell lines) that stably express WT-LAMP-1 and I382L, and compared their intracellular distributions. The subcellular fractionation study using Percoll density gradient centrifugation revealed that WT-LAMP-1 had preferential localization in the high density secondary lysosomes where endogenous human LAMP-1 was enriched. In contrast, a major portion of I382L was located in a low density fraction. The low density fraction also contained approximately 80% of endogenous human LAMP-1 and significant amounts of endogenous β-glucuronidase and LAMP-2, which probably represents occurrence of low density lysosomes in the I382L-expressing cells. Double immunofluorescence microscopic analyses distinguished I382L-containing intracellular vesicles from endogenous LAMP-1-containing lysosomes and early endosomes. Altogether, constitutive expression of I382L causes its aberrant intracellular localization and generation of low density lysosomes, indicating that the COOH-terminal isoleucine is critical for normal localization of LAMP-1 in the dense lysosomes.

The spontaneously hypertensive rat (SHR)/NDmcr-cp (SHR-cp), which is a metabolic syndrome model rat, was reported to show hypercholesteremia, as compared with lean littermates. The serum total cholesterol level in SHR-cp at 18 weeks of age is higher than that of normotensive Wistar Kyoto rat (WKY), but that in SHR-cp at 10 weeks of age is the same. The objective of this study is to clarify whether there are differences in the system regulating serum cholesterol levels between SHR-cp and WKY at 10 weeks of age. Total serum cholesterol levels, and cholesterol levels of high density lipoprotein (HDL), low density lipoprotein (LDL), and very low density lipoprotein (VLDL) were similar in the two strains. However, the cholesterol levels in the liver of SHR-cp were lower than those of WKY. Next, mRNA levels of receptors (scavenger receptor class B type 1 [SRB1], LDL receptor [LDLR]) involved in uptake from serum to liver or enzymes of cholesterol catabolism (CYP7A1 and CYP8B1) and biosynthesis (mevalonate pyrophosphate decarboxylases [MPD]) in liver were compared between SHR-cp and WKY. High levels of MPD and LDLR and low levels of SRB1 were shown in SHR-cp, as compared with WKY. CYP7A1 and CYP8B1 levels were similar between SHR-cp and WKY. These results suggest that the serum cholesterol level in SHR-cp by the balance or regulation between the rise in cholesterol uptake and reduction in cholesterol biosynthesis in the liver is the same as that in WKY.

In the present study, we investigated the dose-dependent effect of delta-tocotrienol long term (48, 72 h) on the melanin content of cells treated with delta-tocotrienol, and whether cells treated with delta-tocotrienol for long a time show cytotoxicity. We also examined whether other enzymes responsible for melanin biosynthesis, tyrosinase-related protein-1 (TRP-1) and -2 (TRP-2), are involved in the decrease in melanin levels. Protein levels in cells treated with 25 or 50 microM delta-tocotrienol for 48 h or 72 h were similar to those in control cells. Melanin content decreased by 44 (25 microM delta-tocotrienol) to 50% (50 microM) at 48 h, and by 14 to 21% at 72 h, compared to control levels. Tyrosinase activity, amounts of tyrosinase and TRP-1 decreased dependent on dose : by 50 (25 microM delta-tocotrienol) to 75% (50 microM), 20 to 45% and 42 to 82% at 48 h, and by 25 to 50%, 75 to 80% and 78 to 77% at 72 h, respectively. Although the amount of TRP-2 increased by 20% on treatment with 25 microM delta-tocotrienol for 48 h, it decreased by 52% on treatment with 50 microM delta-tocotrienol for 48 h. The amount of TRP-2 dose-dependently decreased by 55% and 75% on 72 h by treatment with 25 and 50 microM delta-tocotrienol, respectively. From these findings, delta-tocotrienol at up to 50 microM dose-dependently caused a reduction in melanin content by the decrease of TRP-1 and TRP-2 as well as tyrosinase, and no cytotoxicity.

We previously reported that a decrease in the melanin content of mouse melanoma cells (B16 cells) treated with delta-tocotrienol was the result of a decrease in the level of tyrosinase activity and protein. Use of delta-tocotrienol as a whitening agent, may therefore have side effects. In the present study, we examined whether delta-tocotrienol caused side effects (the release of lysosomes from and a decrease in the cholesterol content of cells). We also examined the release of melanosomes (lysosome-related organella). Neither of lysosomes nor melanosomes were released from cells treated with delta-tocotrienol, since beta-glucuronidase (melanosomal and lysosomal enzyme) activity, melanin content (melanosomal marker), and tyrosinase (melanosomal enzyme) activity did not increase in the cell culture medium. Although mevalonate pyrophosphate decarboxylase (MPD; an enzyme of cholesterol biosynthesis) was significantly reduced in the cells treated with delta-tocotrienol, cholesterol content was not. Thus, delta-tocotrienol might be useful as a therapeutic or preventive drug for hyperpigmentation and as a component of whitening and/or lightening cosmetics not causing severe side effect (reduction of cholesterol content and release of lysosomes/melanosomes), although delta-tocotrienol cause a decrease of MPD.

In the present study, we examined levels of oxidative stress in the serum, brain and kidneys of normotensive Wistar Kyoto rats (WKY) and stroke prone spontaneously hypertensive rats (SHRSP) at 10 weeks of age. Levels of advanced oxidation protein products (AOPP), oxidized albumin and oxidized proteins, markers of oxidative stress, were significantly decreased in serum among SHRSP as compared with WKY. Levels of oxidized proteins determined by immunoblotting were significantly increased in the brain, but not kidney, of SHRSP. The mRNA level of super oxide dismutase (SOD) determined by real time polymerase chain reaction (PCR) and the protein level of catalase assessed by immunoblotting were significantly increased in the brain of SHRSP. From these results, it was suggested that levels of oxidative stress were higher in the brain than serum or kidneys of SHRSP at 10 weeks of age, but are not caused by decreases in the expression of SOD and catalase.

The effect of high and low molecular weight chitosans (HMC; 1000 kDa, LMC; 30 kDa) on oxidative stress and hypercholesterolemia was investigated using male 6-week-old Wistar Kyoto rats as a normal model (Normal-rats) and spontaneously hypertensive rat/ND mcr-cp (SHP/ND) as a metabolic syndrome model (MS-rats), respectively. In Normal-rats, the ingestion of both chitosans over a 4 week period resulted in a significant decrease in total body weight (BW), glucose (Gl), triglyceride (TG), low density lipoprotein (LDL) and serum creatinine (Cre) levels. The ingestion of both chitosans also resulted in a lowered ratio of oxidized to reduced albumin and an increase in total plasma antioxidant activity. In addition to similar results in Normal-rats, the ingestion of only HMC over a 4 week period resulted in a significant decrease in total cholesterol levels in MS-rats. Further, the ingestion of LMC resulted in a significantly higher antioxidant activity than was observed for HMC in both rat models. In in vitro studies, LMC caused a significantly higher reduction in the levels of two stable radicals, compared to HMC, and the effect was both dose- and time-dependent. The findings also show that LDL showed strong binding in the case of HMC. These results suggest that LMC has a high antioxidant activity as well as antilipidemic effects, while HMC results in a significant reduction in the levels of pro-oxidants such as LDL in the gastrointestinal tract, thereby inhibiting the subsequent development of oxidative stress in the systemic circulation in metabolic model rats.

The relationship between protein and mRNA levels of mevalonate pyrophosphate decarboxylase (MPD) in rat tissues remains to be clarified. In this study, we examined the distribution of the mRNA in Wistar rat tissues by real-time PCR. When the relative expression of MPD in 1 mg of tissue was quantified using glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) as an internal control, the mRNA level was found to be markedly higher in the spleen and liver than in other organs. The correlation coefficient between protein and mRNA levels of MPD was 0.847, indicating that the protein level of MPD in Wistar rat is distributed in the tissues almost entirely dependent on the mRNA level of MPD. We previously reported that the protein levels of MPD in the liver and brain of spontaneously hypertensive rats, stroke-prone (SHRSP) are reduced. Thus, we compared mRNA levels of MPD in the liver and brain between Wistar Kyoto rats (WKY) and SHRSP. The levels in liver of SHRSP were significantly decreased, but not in brain, as compared with WKY. Also, mRNA levels of sterol regulatory element binding protein-2, which is transcription factor of cholesterol, in liver of SHRSP was similar to those of WKY. These findings indicate that the reduced protein level of MPD in the liver of SHRSP is caused by a decrease in mRNA level of MPD, and that in the brain of SHRSP is caused by increased degradation of the MPD protein.

We previously reported that mevalonate pyrophosphate decarboxylase (MPD) is located in the cytosol and that MPD level in the liver is higher than in other rat tissues. In the present study, we further investigated the tissue distribution of MPD in guinea pigs by immunoblotting using anti-rat MPD antiserum. When immunoblot analysis was carried out using guinea pig brain, the antiserum reacted with 46-kDa protein as well as a substance with the same molecular weight of MPD in mice. Protein of 46-kDa detected in guinea pig liver treated with 0.1% pravastatin, a 3-hydroxy-3-methylglutaryl-CoA reductase inhibitor indicating a liver-specific effect, was increased 3-fold as compared with nontreated guinea pigs; however, 46-kDa protein in the brain treated with pravastatin was similar to that treated without pravastatin. When the subcellular distribution of MPD in the brain, liver, kidney, and testis, was examined by cell fractionation, MPD was mostly detected in the cytosol fraction of all tissues. From these data, the 46-kDa protein was identified as MPD. Next, when the tissue distribution of MPD was examined, MPD in the liver was higher than in other tissues. The relative amount of MPD in guinea pig kidney was higher than in rats and similar to in mice, as MPD in the liver of the same species was taken as 1. Furthermore, the correlation coefficient between guinea pigs and rats or mice in the tissue distribution of MPD was 0.69 or 0.72, respectively. These data indicate a relationship in tissue distribution between guinea pigs and rats or mice, although the tissue-specific regulator of MPD between species somewhat differed.

Chitosan, a cationic polysaccharide, is widely employed as dietary supplement and in pharmacological and biomedical applications. Although numerous studies have focused on its applications as pharmaceutical excipients or bioactive reagents, relationships between molecular weight (Mr) and biological properties remain unclear. The focus of this study was on the antioxidant properties of several Mr chitosans. We measured the ability of seven Mr chitosans (CT1; 2.8 kDa, CT2; 17.0 kDa, CT3; 33.5 kDa, CT4; 62.6 kDa, CT5; 87.7 kDa, CT6; 604 kDa, CT7; 931 kDa) to protect plasma protein from oxidation by peroxyl radicals derived from 2,2'-azobis (2-amidinopropane) dihydrochloride (AAPH). A comparison of the antioxidant action of high Mr chitosans (CT6-CT7) with that of low Mr chitosans (CT1-CT5) showed that low Mr chitosans (CT1-CT5) were more effective in preventing the formation of carbonyl groups in plasma protein exposed to peroxyl radicals. AAPH substantially increases plasma protein carbonyl content via the oxidation of human serum albumin (HSA). We also measured the ability of these chitosans to protect HSA against oxidation by AAPH. Low Mr chitosans (CT1-CT5) were found to effectively prevent the formation of carbonyl groups in HSA, when exposed to peroxyl radicals. Low Mr chitosans were also good scavengers of N-centered radicals, but high Mr chitosans were much less effective. We also found a strong correlation between antioxidant activity and the Mr of chitosans in vitro. These activities were also determined by using the 'TPAC' test. These results suggest that low Mr chitosans (CT1-CT3) may be absorbed well from the gastrointestinal tract and inhibit neutrophil activation and oxidation of serum albumin that is frequently observed in patients plasma undergoing hemodialysis, resulting in a reduction in oxidative stress associated with uremia.