Ubiquitin ligase CHIP suppresses cancer stem cell properties in a population of breast cancer cells

Cancer stem cells (CSCs) have several distinctive characteristics, including high metastatic potential, tumor-initiating potential, and properties that resemble normal stem cells such as self-renewal, differentiation, and drug efflux. Because of these characteristics, CSC is regarded to be responsible for cancer progression and patient prognosis. In our previous study, we showed that a ubiquitin E3 ligase carboxyl terminus of Hsc70-interacting protein (CHIP) suppressed breast cancer malignancy. Moreover, a recent clinical study reported that CHIP expression levels were associated with favorable prognostic parameters of patients with breast cancer. Here we show that CHIP suppresses CSC properties in a population of breast cancer cells. CHIP depletion resulted in an increased proportion of CSCs among breast cancers when using several assays to assess CSC properties. From our results, we propose that inhibition of CSC properties may be one of the functions of CHIP as a suppressor of cancer progression.     

Development of an Ultra-Rapid Diagnostic Method Based on Heart-Type Fatty Acid Binding Protein Levels in the CSF of CJD Patients

Creutzfeldt-Jakob disease (CJD) is a transmissible, fatal, neurodegenerative disease in humans. Recently, various drugs have been reported to be useful in the treatment of CJD; however, for such treatments to be useful it is essential to rapidly and accurately diagnose CJD. 124 CJD patients and 87 with other diseases causing rapid progressive dementia were examined. Cerebral spinal fluid (CSF) from CJD patients was analyzed by 2D-PAGE and the protein expression pattern was compared with that from healthy subjects. One of three CJD-specific spots was found to be fatty acid binding protein (FABP), and heart-type FABP (H-FABP) was analyzed as a new biochemical marker for CJD. H-FABP ELISA results were compared between CJD patients and patients with other diseases (n = 211). Visual readout accuracy of the Rapicheck^® H-FABP test panel for CSF was analyzed using an independent measure of CSF H-FABP concentration. The distribution of H-FABP in the brains of CJD patients was examined by immunohistochemistry. ELISA sensitivity and specificity were 90.3% and 92.9%, respectively, and Rapicheck^® H-FABP sensitivity and specificity were 87.9% and 96.0%, respectively. ELISA and Rapicheck^® H-FABP assays provided comparable results for 14-3-3 protein and total tau protein. Elevated H-FABP levels were associated with an accumulation of abnormal prion protein, astrocytic gliosis, and neuronal loss in the cerebral cortices of CJD patients. In conclusion, Rapicheck^® H-FABP of CSF specimens enabled quick and frequent diagnosis of CJD. H-FABP represents a new biomarker for CJD distinct from 14-3-3 protein and total tau protein.     

Lectin-like ERAD players in ER and cytosol

Protein quality control in the endoplasmic reticulum (ER) is an elaborate process conserved from yeast to mammals, ensuring that only newly synthesized proteins with correct conformations in the ER are sorted further into the secretory pathway. It is well known that high-mannose type N-glycans are involved in protein-folding events. In the quality control process, proteins that fail to achieve proper folding or proper assembly are degraded in a process known as ER-associated degradation (ERAD). The ERAD pathway comprises multiple steps including substrate recognition and targeting to the retro-translocation machinery, retrotranslocation from the ER into the cytosol, and proteasomal degradation through ubiquitination. Recent studies have documented the important roles of sugar-recognition (lectin-type) molecules for trimmed high-mannose type N-glycans and glycosidases in the ERAD pathways in both ER and cytosol. In this review, we discuss a fundamental system that monitors glycoprotein folding in the ER and the unique roles of the sugar-recognizing ubiquitin ligase and peptide:N-glycanase (PNGase) in the cytosolic ERAD pathway.     

Safety evaluation of probiotic bifidobacteria by analysis of mucin degradation activity and translocation ability

Although probiotic-containing nutrient formulas for infants and toddlers have become very popular, some adverse effects related to translocation of probiotic strains have been reported. We assessed the safety of probiotic bifidobacteria that have been used in clinical investigations and proven to have beneficial effects, by analyzing mucin degradation activity and translocation ability. Mucin degradation activities of three probiotic bifidobacteria strains; Bifidobacterium longum BB536, Bifidobacterium breve M-16V and Bifidobacterium infantis M-63, were evaluated by three in vitro tests comprising growth in liquid medium, SDS-PAGE analysis of degraded mucin residues, and degradation assay in Petri dish. All test strains and control type strains failed to grow in the liquid medium containing mucin as the only carbon source, although good growth was obtained from fecal sample. In the SDS-PAGE analyses of mucin residues and observation of mucinolytic zone in agar plate, the three test strains also showed no mucin degradation activity as the type strains, although fecal sample yielded positive results. In another study, a high dose of B. longum BB536 was administered orally to conventional mice to examine the translocation ability. No translocation into blood, liver, spleen, kidney and mesenteric lymph nodes was observed and no disturbance of epithelial cells and mucosal layer in the ileum, cecum and colon was detected, indicating that the test strain had no translocation ability and induced no damage to intestinal surface. These results resolve the concern about bacterial translocation when using bifidobacteria strains as probiotics, which have been tested in various clinical trials, supporting the continuous use of these probiotic strains without anxiety.     

Uncovering the roles of PINK1 and parkin in mitophagy

Parkinson disease (PD) is the second most prevalent neurodegenerative disorder, and thus elucidation of the pathogenic mechanism and establishment of a fundamental cure is essential in terms of public welfare. Fortunately, our understanding of the pathogenesis of two types of recessive familial PDs—early-onset familial PD caused by dysfunction of the PTEN-induced putative kinase 1 (PINK1) gene and autosomal recessive juvenile Parkinsonism (ARJP) caused by a mutation in the Parkin gene—has evolved and continues to expand.Key words: PINK1, parkin, ubiquitin, mitochondria, autophagy, mitophagy, membrane potential, quality controlSince the cloning of PINK1 and Parkin, numerous papers have been published about the corresponding gene products, but the mechanism by which dysfunction of PINK1 and/or Parkin causes PD remain unclear. Parkin encodes a ubiquitin ligase E3, a substrate recognition member of the ubiquitination pathway, whereas PINK1 encodes a mitochondria-targeted serine-threonine kinase that contributes to the maintenance of mitochondrial integrity. Based on their molecular functions, it is clear that Parkin-mediated ubiquitination and PINK1 phosphorylation are key events in disease pathogenesis. The underlying mechanism, however, is not as well defined and claims of pathogenicity, until recently, remained controversial. Although Parkin''s E3 activity was clearly demonstrated in vitro, we were unable to show a clear E3 activity of Parkin in cell/in vivo. In addition, despite a predicted mitochondrial localization signal for PINK1, we were unable to detect PINK1 on mitochondria by either immunoblotting or immunocytochemistry. More confusingly, overexpression of nontagged PINK1 mainly localized to the cytoplasm under steady state conditions.Work by Dr. Youle''s group at the National Institutes of Health in 2008, however, offered new insights. They reported that Parkin associated with depolarized mitochondria and that Parkin-marked mitochondria were subsequently cleared by autophagy. Soon after their publication, we also examined the function of Parkin and PINK1 following a decrease in mitochondrial membrane potential. Our findings, described below (Fig. 1), have contributed to the development of a mechanism explaining pathogenicity.Open in a separate windowFigure 1Model of mitochondrial quality control mediated by PINK1 and Parkin. Under steady-state conditions, the mature 60 kDa PINK1 is constantly cleaved by an unknown protease to a 50 kDa intermediate form that is subsequently degraded, presumably by the proteasome (upper part). The protein, however, is stabilized on depolarized mitochondria because the initial processing event is inhibited by a decrease in mitochondrial membrane potential (lower part). Accumulated PINK1 recruits cytosolic Parkin onto depolarized mitochondria resulting in activation of its E3 activity. Parkin then ubiquitinates a mitochondrial substrate(s). As a consequence, damaged mitochondria are degraded via mitophagy. Ub, ubiquitin.(1) We sought to determine the subcellular localization of endogenous PINK1, and realized that endogenous PINK1 is barely detectable under steady-state conditions. However, a decrease in mitochondrial membrane-potential following treatment with the mitochondrial uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP) results in the gradual accumulation of endogenous PINK1 on mitochondria. Importantly, when CCCP is washed out, the accumulated endogenous PINK1 rapidly disappears (within 30 min) both in the presence and absence of cycloheximide. These results support the hypothesis that PINK1 is constantly transported to the mitochondria, but is rapidly degraded in a membrane potential-dependent manner (see below for details). We speculate that PINK1 is stabilized by a decrease in mitochondrial membrane potential and as a result accumulates on depolarized mitochondria.(2) We examined the potential role of PINK1 in the mitochondrial recruitment of Parkin. In control MEFs (PINK1^+/+), Parkin is selectively recruited to the mitochondria following CCCP treatment, and subsequently results in the selective disappearance of the mitochondria via autophagy (called mitophagy). In sharp contrast, Parkin is not translocated to the mitochondria in PINK1 knockout (PINK1^−/−) MEFs following CCCP treatment, and subsequent mitochondrial degradation is also completely impeded. These results suggest that PINK1 is “a Parkin-recruitment factor” that recruits Parkin from the cytoplasm to damaged mitochondria in a membrane potential-dependent manner for mitophagy.(3) We monitored the E3 activity of Parkin using an artificial pseudo-substrate fused to Parkin in cells. Parkin''s E3 activity was repressed under steady-state conditions; however, we find that Parkin ubiquitinates the pseudo-substrate when it is retrieved to the depolarized mitochondria, suggesting that activation of the latent Parkin E3 activity is likewise dependent on a decrease in mitochondrial membrane potential.(4) PINK1 normally exists as either a long (approximately 60 kDa) or a short (approximately 50 kDa) protein. Because the canonical mitochondrial targeting signal (matrix targeting signal) is cleaved after import into the mitochondria, the long form has been designated as the precursor and the short form as the mature PINK1. However, our subcellular localization study of endogenous PINK1 following CCCP treatment shows that the long form is recovered in the mitochondrial fraction, suggesting that it is not the pre-import precursor form. Moreover, by monitoring the degradation process of PINK1 following recovery of membrane potential, we realized that the short form of PINK1 transiently appears soon after CCCP is washed out and then later disappears, suggesting that the processed form of PINK1 is an intermediate in membrane-potential-dependent degradation. In conclusion, these results imply that PINK1 cleavage does not reflect a canonical maturation process accompanying mitochondrial import as initially thought, but rather represents constitutive degradation in healthy mitochondria by a two-step mechanism; i.e., first limited processing and subsequent complete degradation probably via the proteasome.(5) PINK1 accumulation by decrease of membrane potential and subsequent recruitment of Parkin onto mitochondria are presumably etiologically important because they are impeded for the most part by disease-linked mutations of PINK1 or Parkin.These results, together with reports by other groups, strongly suggest that recessive familial PD is caused by dysfunction of quality control for depolarized mitochondria.At present, we do not know whether the aforementioned pathogenic mechanism of recessive familial PD can be generalized to prevalent sporadic PD. However, the clinical symptoms of recessive familial PD caused by dysfunction of PINK1 or Parkin resembles that of idiopathic PD except early-onset pathogenesis, and thus it is plausible that there is a common pathogenic mechanism. We accordingly believe that our results provide solid insight into the molecular mechanisms of PD pathogenesis, not only for familial forms caused by Parkin and PINK1 mutations, but also the major sporadic form of PD.To fully understand the molecular mechanism of PINK1-Parkin-mediated mitophagy, further details need to be addressed including: identifying the protease(s) that processes PINK1 in a mitochondrial membrane-potential dependent manner and that presumably monitors mitochondrial integrity; identifying a physiological substrate(s) of PINK1; determining the molecular mechanism underlying Parkin activation; and identifying the protein(s) linking Parkin-mediated ubiquitination to mitophagy. A detailed mechanism of the aforementioned events will be the focus of future research, however, we feel our conclusion that PINK1 and Parkin function in the removal of depolarized mitochondria is evident and hope that our studies will provide a solid foundation for further studies.     

Distribution of In Vitro Fermentation Ability of Lacto-N-Biose I,a Major Building Block of Human Milk Oligosaccharides,in Bifidobacterial Strains

This study investigated the potential utilization of lacto-N-biose I (LNB) by individual strains of bifidobacteria. LNB is a building block for the human milk oligosaccharides, which have been suggested to be a factor for selective growth of bifidobacteria. A total of 208 strains comprising 10 species and 4 subspecies were analyzed for the presence of the galacto-N-biose/lacto-N-biose I phosphorylase (GLNBP) gene (lnpA) and examined for growth when LNB was used as the sole carbohydrate source. While all strains of Bifidobacterium longum subsp. longum, B. longum subsp. infantis, B. breve, and B. bifidum were able to grow on LNB, none of the strains of B. adolescentis, B. catenulatum, B. dentium, B. angulatum, B. animalis subsp. lactis, and B. thermophilum showed any growth. In addition, some strains of B. pseudocatenulatum, B. animalis subsp. animalis, and B. pseudolongum exhibited the ability to utilize LNB. With the exception for B. pseudocatenulatum, the presence of lnpA coincided with LNB utilization in almost all strains. These results indicate that bifidobacterial species, which are the predominant species found in infant intestines, are potential utilizers of LNB. These findings support the hypothesis that GLNBP plays a key role in the colonization of bifidobacteria in the infant intestine.Bifidobacteria are gram-positive anaerobic bacteria that naturally colonize the human intestinal tract and are believed to be beneficial to human health (21, 30). Breastfeeding has been shown to be associated with an infant fecal microbiota dominated by bifidobacteria, whereas the fecal microbiota of infants who are consuming alternative diets has been described as being mixed and adult-like (12, 21). It has been suggested that the selective growth of bifidobacteria observed in breast-fed newborns is related to the oligosaccharides and other factors that are contained in human milk (human milk oligosaccharides [HMOs]) (3, 4, 10, 11, 16, 17, 34). Kitaoka et al. (15) have recently found that bifidobacteria possess a unique metabolic pathway that is specific for lacto-N-biose I (LNB; Galβ1-3GlcNAc) and galacto-N-biose (GNB; Galβ1-3GalNAc). LNB is a building block for the type 1 HMOs [such as lacto-N-tetraose (Galβ1-3GlcNAcβ1-3Galβ1-4Glc), lacto-N-fucopentaose I (Fucα1-2Galβ1-3GlcNAcβ1-3Galβ1-4Glc), and lacto-N-difucohexaose I (Fucα1-2Galβ1-3[Fucα1-4]GlcNAcβ1-3Galβ1-4Glc)], and GNB is a core structure of the mucin sugar that is present in the human intestine and milk (18, 27). The GNB/LNB pathway, as previously illustrated by Wada et al. (33), involves proteins/enzymes that are required for the uptake and degradation of disaccharides such as the GNB/LNB transporter (29, 32), galacto-N-biose/lacto-N-biose I phosphorylase (GLNBP; LnpA) (15, 24) (renamed from lacto-N-biose phosphorylase after the finding of phosphorylases specific to GNB [23] and LNB [22]), N-acetylhexosamine 1-kinase (NahK) (25), UDP-glucose-hexose 1-phosphate uridylyltransferase (GalT), and UDP-galactose epimerase (GalE). Some bifidobacteria have been demonstrated to be enzymatically equipped to release LNB from HMOs that have a type 1 structure (lacto-N biosidase; LnbB) (33) or GNB from the core 1-type O-glycans in mucin glycoproteins (endo-α-N-acetylgalatosaminidase) (6, 13, 14). It has been suggested that the presence of the LnbB and GNB/LNB pathways in some bifidobacterial strains could provide a nutritional advantage for these organisms, thereby increasing their populations within the ecosystem of these breast-fed newborns (33).The species that predominantly colonize the infant intestine are the bifidobacterial species B. breve, B. longum subsp. infantis, B. longum subsp. longum, and B. bifidum (21, 28). On the other hand, strains of B. adolescentis, B. catenulatum, B. pseudocatenulatum, and B. longum subsp. longum are frequently isolated from the adult intestine (19), and strains of B. animalis subsp. animalis, B. animalis subsp. lactis, B. thermophilum and B. pseudolongum have been shown to naturally colonize the guts of animals (1, 2, 7, 8). However, it is unclear whether there is a relationship between the differential colonization of the bifidobacterial species and the presence of the GNB/LNB pathway. In the present study, we investigated the ability of individual bifidobacterial strains in the in vitro fermentation of LNB and in addition, we also tried to determine whether or not the GLNBP gene (lnpA), which is a key enzyme of the GNB/LNB pathway, was present.     

Ischemia-induced angiogenesis is impaired in aminopeptidase A deficient mice via down-regulation of HIF-1α

Aminopeptidase A (APA; EC 3.4.11.7) is a transmembrane metalloprotease with several functions in tumor angiogenesis. To investigate the role of APA in the process of ischemia-induced angiogenesis, we evaluated the cellular angiogenic responses under hypoxic conditions and the process of perfusion recovery in the hindlimb ischemia model of APA-deficient (APA-KO; C57Bl6/J strain) mice.Western blotting of endothelial cells (ECs) isolated from the aorta of APA-KO mice revealed that the accumulation of hypoxia-inducible factor-1α (HIF-1α) protein in response to hypoxic challenge was blunted. Regarding the proteasomal ubiquitination, a proteasome inhibitor MG-132 restored the reduced accumulation of HIF-1α in ECs from APA-KO mice similar to control mice under hypoxic conditions. These were associated with decreased growth factor secretion and capillary formation in APA-KO mice. In the hindlimb ischemia model, perfusion recovery in APA-KO mice was decreased in accordance with a significantly lower capillary density at 2 weeks. Regarding vasculogenesis, no differences were observed in cell populations and distribution patterns between wild type and APA-KO mice in relation to endothelial progenitor cells.Our results suggested that Ischemia-induced angiogenesis is impaired in APA-KO mice partly through decreased HIF-1α stability by proteasomal degradation and subsequent suppression of HIF-1α-driven target protein expression such as growth factors. APA is a functional target for ischemia-induced angiogenesis.     

Genetic immunization based on the ubiquitin-fusion degradation pathway against Trypanosoma cruzi

Cytotoxic CD8⁺ T cells are particularly important to the development of protective immunity against the intracellular protozoan parasite, Trypanosoma cruzi, the etiological agent of Chagas disease. We have developed a new effective strategy of genetic immunization by activating CD8⁺ T cells through the ubiquitin-fusion degradation (UFD) pathway. We constructed expression plasmids encoding the amastigote surface protein-2 (ASP-2) of T. cruzi. To induce the UFD pathway, a chimeric gene encoding ubiquitin fused to ASP-2 (pUB-ASP-2) was constructed. Mice immunized with pUB-ASP-2 presented lower parasitemia and longer survival period, compared with mice immunized with pASP-2 alone. Depletion of CD8⁺ T cells abolished protection against T. cruzi in mice immunized with pUB-ASP-2 while depletion of CD4⁺ T cells did not influence the effective immunity. Mice deficient in LMP2 or LMP7, subunits of immunoproteasomes, were not able to develop protective immunity induced. These results suggest that ubiquitin-fused antigens expressed in antigen-presenting cells were effectively degraded via the UFD pathway, and subsequently activated CD8⁺ T cells. Consequently, immunization with pUB-ASP-2 was able to induce potent protective immunity against infection of T. cruzi.     

Organophosphorus compound detection on a cell chip with yeast coexpressing hydrolase and eGFP

Organophosphorus compounds (OPs) such as pesticides, fungicides, and herbicides are highly toxic but are nevertheless extensively used worldwide. To detect OPs, we constructed a yeast strain that co-displays organophosphorus hydrolase (OPH) and enhanced green fluorescent protein (EGFP) on the cell surface using a Flo1p anchor system. OP degradation releases protons and causes a change in pH. This pH change results in structural deformation of EGFP, which triggers quenching of its fluorescence, thereby making this cell useful for visual detection of OPs. Fluorescence microscopy confirmed the high-intensity fluorescence displayed by EGFP on the cell surface. The yeast strain possessed sufficient OPH hydrolytic activities for degrading OPs, as measured by incubation with 1 mM paraoxon for 24 h at 30°C. In addition, with 20 mM paraoxon at 30°C, fluorescence quenching of EGFP on the single yeast cell was observed within 40 s in a microchamber chip. These observations suggest that engineered yeast cells are suitable for simultaneous degradation and visual detection of OPs.