Turnover of EDEM1, an ERAD-enhancing factor, is mediated by multiple degradation routes.

Quality-based protein production and degradation in the endoplasmic reticulum (ER) are essential for eukaryotic cell survival. During protein maturation in the ER, misfolded or unassembled proteins are destined for disposal through a process known as ER-associated degradation (ERAD). EDEM1 is an ERAD-accelerating factor whose gene expression is upregulated by the accumulation of aberrant proteins in the ER, known as ER stress. Although the role of EDEM1 in ERAD has been studied in detail, the turnover of EDEM1 by intracellular degradation machinery, including the proteasome and autophagy, is not well understood. To clarify EDEM1 regulation in the protein level, degradation mechanism of EDEM1 was examined. Our results indicate that both ERAD and autophagy degrade EDEM1 alike misfolded degradation substrates, although each degradation machinery targets EDEM1 in different folded states of proteins. We also found that ERAD factors, including the SEL1L/Hrd1 complex, YOD1, XTP3B, ERdj3, VIMP, BAG6, and JB12, but not OS9, are involved in EDEM1 degradation in a mannose-trimming-dependent and -independent manner. Our results suggest that the ERAD accelerating factor, EDEM1, is turned over by the ERAD itself, similar to ERAD clients.

Identification of EGF Receptor and Thrombospondin-1 as Endogenous Targets of ER-Associated Degradation Enhancer EDEM1 in HeLa Cells.

Secretory and membrane proteins are vital for cell activities, including intra- and intercellular communication. Therefore, protein quality control in the endoplasmic reticulum (ER) is an essential and crucial process for eukaryotic cells. Endoplasmic reticulum-associated degradation (ERAD) targets misfolded proteins during the protein maturation process in the ER and leads to their disposal. This process maintains the ER productive function and prevents misfolded protein stress (i.e., ER stress). The ERAD-stimulating factor ER degradation-enhancing α mannosidase-like 1 protein (EDEM1) acts on misfolded proteins to accelerate ERAD, thereby maintaining the productivity of the ER. However, the detail mechanism underlying the function of EDEM1 in ERAD is not completely understood due to a lack of established physiological substrate proteins. In this study, we attempted to identify substrate proteins for EDEM1 using siRNA. The matrix component thrombospondin-1 (TSP1) and epidermal growth factor receptor (EGFR) were identified as candidate targets of EDEM1. Their protein maturation status and cellular localization were markedly affected by knockdown of EDEM1. We also showed that EDEM1 physically associates with EGFR and enhances EGFR degradation via ERAD. Our data highlight the physiological role of EDEM1 in maintaining specific target proteins and provide a potential approach to the regulation of expression of clinically important proteins.

Endoplasmic Reticulum Associated Degradation of Spinocerebellar Ataxia-Related CD10 Cysteine Mutant.

Spinocerebellar ataxia (SCA) is one of the most severe neurodegenerative diseases and is often associated with misfolded protein aggregates derived from the genetic mutation of related genes. Recently, mutations in CD10 such as C143Y have been identified as SCA type 43. CD10, also known as neprilysin or neuroendopeptidase, digests functional neuropeptides, such as amyloid beta, in the extracellular region. In this study, we explored the cellular behavior of CD10 C143Y to gain an insight into the functional relationship of the mutation and SCA pathology. We found that wild-type CD10 is expressed on the plasma membrane and exhibits endopeptidase activity in a cultured cell line. CD10 C143Y, however, forms a disulfide bond-mediated oligomer that does not appear by the wild-type CD10. Furthermore, the CD10 C143Y mutant was retained in the endoplasmic reticulum (ER) by the molecular chaperone BiP and was degraded through the ER-associated degradation (ERAD) process, in which representative ERAD factors including EDEM1, SEL1L, and Hrd1 participate in the degradation. Suppression of CD10 C143Y ERAD recovers intracellular transport but not enzymatic activity. Our results indicate that the C143Y mutation in CD10 negatively affects protein maturation and results in ER retention and following ERAD. These findings provide beneficial insight into SCA type 43 pathology.

The prevalence and antimicrobial susceptibility of Streptococcus pneumoniae isolated from patients at Jikei University Hospitals after the implementation of the pneumococcal vaccination program in Japan.

Studies have shown that pneumococcal vaccination reduces the incidence of Streptococcus pneumoniae infections but does not change the prevalence of S. pneumoniae nasopharyngeal colonization. To comprehensively and longitudinally assess the epidemiology of S. pneumoniae after the introduction of pneumococcal vaccination, we monitored the prevalence and antimicrobial susceptibility of S. pneumoniae, irrespective of its serotypes or pathogenicity, by analyzing specimens collected from a large number of patients at Jikei University Hospitals from 2009 to 2017. A total of 5763 S. pneumoniae isolates were identified out of 375,435 specimens from various sources of patients in different age groups. The prevalence of S. pneumoniae isolated only from patients <5 years old was significantly reduced with the widespread use of pneumococcal vaccines, although this reduction differed by areas where patients resided. The incidence of pneumococcal infections, including bacteremia and otitis media, clearly decreased among patients <5 years old after the introduction of pneumococcal vaccination, while the prevalence of S. pneumoniae isolated from blood specimens of patients 15-64 years old increased, suggesting the involvement of non-vaccine serotypes in the incidence of invasive pneumococcal infections. The antimicrobial susceptibility of S. pneumoniae improved after the introduction of pneumococcal vaccination. Our results show that pneumococcal vaccination has a suppressive effect on the prevalence of S. pneumoniae and the incidence of pneumococcal infections, at least for children <5 years old, in association with an improvement in the antimicrobial susceptibility of S. pneumoniae. However, further measures will be needed to control invasive pneumococcal infections caused by non-vaccine serotypes.

Decreased Prevalence of qacA-Positive Methicillin-Resistant Staphylococcus aureus in Hospitalized Patients in Tokyo, Japan.

Presence of methicillin-resistant Staphylococcus aureus (MRSA) strains carrying plasmid-borne multidrug efflux pump-encoding gene, qacA/B, is a serious issue for infection control in hospitals, because they can survive hand hygiene. The qacA/B genes are divided into five subtypes: qacA, qacBI, qacBII, qacBIII, and qacBIV. The aim of this study was to investigate the prevalence and risk factors of hospitalized patients infected by respective qacA/B-positive MRSA strains between 2010 and 2016 in Tokyo, Japan. Of the 600 total MRSA strains observed, the qacA/B-positive strains constituted 19.8% (199 isolates), of which 56.8% (113 isolates), 28.6% (57 isolates), and 14.6% (29 isolates) were classified as qacA, qacBIII, and qacBII-positive strains, respectively. The prevalence of qacA-positive MRSA strains significantly decreased from July 2010 to June 2011 (34.0%) to July 2015 to May 2016 (5.3%) (p < 0.05). When staphylococcal cassette chromosome (SCC)mec types of the respective qacA/B-positive strains were determined, 81.4% of the qacA-positive strains were classified into SCCmec type II, which has recently been decreasing in hospital-acquired MRSA in Japan. Risk factor analysis showed that there were no specific clinical departments associated with the presence of qacA-positive strains. Our findings suggest that change in the MRSA clonal lineages impact to decrease the prevalence of qacA-positive strains in Japanese hospitals.

EDEM1's mannosidase-like domain binds ERAD client proteins in a redox-sensitive manner and possesses catalytic activity.

Endoplasmic reticulum (ER) degradation-enhancing α-mannosidase-like 1 protein (EDEM1) is a protein quality control factor that was initially proposed to recognize N-linked glycans on misfolded proteins through its mannosidase-like domain (MLD). However, recent studies have demonstrated that EDEM1 binds to some misfolded proteins in a glycan-independent manner, suggesting a more complex binding landscape for EDEM1. In this study, we have identified a thiol-dependent substrate interaction between EDEM1 and the α1-antitrypsin ER-associated protein degradation (ERAD) clients Z and NHK, specifically through the single Cys residue on Z/NHK (Cys256), required for binding under stringent detergent conditions. In addition to the thiol-dependent interaction, the presence of weaker protein-protein interactions was confirmed, suggestive of bipartite client-binding properties. About four reactive thiols on EDEM1 were identified and were not directly responsible for the observed redox-sensitive binding by EDEM1. Moreover, a protein construct comprising the EDEM1 MLD had thiol-dependent binding properties along with its active glycan-trimming activities. Lastly, we identified an additional intrinsically disordered region (IDR) located at the C terminus of EDEM1 in addition to its previously identified N-terminal IDR. We also determined that both IDRs are required for binding to the ERAD component ERdj5 as an interaction with ERdj5 was not observed with the MLD alone. Together, our findings indicate that EDEM1 employs different binding modalities to interact with ERAD clients and ER quality control (ERQC) machinery partners and that some of these properties are shared with its homologues EDEM2 and EDEM3.

Change in genotype of methicillin-resistant Staphylococcus aureus (MRSA) affects the antibiogram of hospital-acquired MRSA.

Recently, the dissemination of community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) into hospitals has frequently been reported worldwide. Hospital-acquired MRSA (HA-MRSA) strains exhibit high-level resistance to multiple antimicrobial agents, whereas CA-MRSA strains are usually susceptible to non-ß-lactams. Thus, it is predicted that the antibiogram of the HA-MRSA population would change along with the change in genotype of MRSA. Here, we investigated the changes in the MRSA population along with the MRSA antibiogram in a hospital between 2010 and 2016. Staphylococcal cassette chromosome (SCC) mec typing showed that the predominant HA-MRSA strains in the hospital dramatically changed from SCCmec type II, which is the major type of HA-MRSA, to SCCmec type IV, which is the major type of CA-MRSA. Multilocus sequence typing revealed that the predominant SCCmec type IV strain was a clonal complex (CC) 8 clone, which is mainly found among CA-MRSA. Furthermore, the CC1-SCCmec type IV (CC1-IV) clone significantly increased. Both the CC8-IV and CC1-IV clones exhibited high antimicrobial susceptibility. The antibiogram change of the HA-MRSA population was consistent with the antimicrobial susceptibilities and increased prevalence of the CC8-IV and CC1-IV clones. Our data reveal that the change in the genotypes of MRSA strains could impact the antibiogram of HA-MRSA population.

HtrA1 Is Specifically Up-Regulated in Active Keloid Lesions and Stimulates Keloid Development.

Keloids occur after failure of the wound healing process; inflammation persists, and various treatments are ineffective. Keloid pathogenesis is still unclear. We have previously analysed the gene expression profiles in keloid tissue and found that HtrA1 was markedly up-regulated in the keloid lesions. HtrA1 is a serine protease suggested to play a role in the pathogenesis of various diseases, including age-related macular degeneration and osteoarthritis, by modulating extracellular matrix or cell surface proteins. We analysed HtrA1 localization and its role in keloid pathogenesis. Thirty keloid patients and twelve unrelated patients were enrolled for in situ hybridization, immunohistochemical, western blot, and cell proliferation analyses. Fibroblast-like cells expressed more HtrA1 in active keloid lesions than in surrounding lesions. The proportion of HtrA1-positive cells in keloids was significantly higher than that in normal skin, and HtrA1 protein was up-regulated relative to normal skin. Silencing HtrA1 gene expression significantly suppressed cell proliferation. HtrA1 was highly expressed in keloid tissues, and the suppression of the HtrA1 gene inhibited the proliferation of keloid-derived fibroblasts. HtrA1 may promote keloid development by accelerating cell proliferation and remodelling keloid-specific extracellular matrix or cell surface molecules. HtrA1 is suggested to have an important role in keloid pathogenesis.

Reglucosylation by UDP-glucose:glycoprotein glucosyltransferase 1 delays glycoprotein secretion but not degradation.

UDP-glucose:glycoprotein glucosyltransferase 1 (UGT1) is a central quality control gatekeeper in the mammalian endoplasmic reticulum (ER). The reglucosylation of glycoproteins supports their rebinding to the carbohydrate-binding ER molecular chaperones calnexin and calreticulin. A cell-based reglucosylation assay was used to investigate the role of UGT1 in ER protein surveillance or the quality control process. UGT1 was found to modify wild-type proteins or proteins that are expected to eventually traffic out of the ER through the secretory pathway. Trapping of reglucosylated wild-type substrates in their monoglucosylated state delayed their secretion. Whereas terminally misfolded substrates or off-pathway proteins were most efficiently reglucosylated by UGT1, the trapping of these mutant substrates in their reglucosylated or monoglucosylated state did not delay their degradation by the ER-associated degradation pathway. This indicated that monoglucosylated mutant proteins were actively extracted from the calnexin/calreticulin binding-reglucosylation cycle for degradation. Therefore trapping proteins in their monoglucosylated state was sufficient to delay their exit to the Golgi but had no effect on their rate of degradation, suggesting that the degradation selection process progressed in a dominant manner that was independent of reglucosylation and the glucose-containing A-branch on the substrate glycans.

Stepwise assembly of fibrinogen is assisted by the endoplasmic reticulum lectin-chaperone system in HepG2 cells.

The endoplasmic reticulum (ER) plays essential roles in protein folding and assembly of secretory proteins. ER-resident molecular chaperones and related enzymes assist in protein maturation by co-operated interactions and modifications. However, the folding/assembly of multimeric proteins is not well understood. Here, we show that the maturation of fibrinogen, a hexameric secretory protein (two trimers from α, ß and γ subunits), occurs in a stepwise manner. The αγ complex, a precursor for the trimer, is retained in the ER by lectin-like chaperones, and the ß subunit is incorporated into the αγ complex immediately after translation. ERp57, a protein disulfide isomerase homologue, is involved in the hexamer formation from two trimers. Our results indicate that the fibrinogen hexamer is formed sequentially, rather than simultaneously, using kinetic pause by lectin chaperones. This study provides a novel insight into the assembly of most abundant multi-subunit secretory proteins.

A case of NDM-1-producing Acinetobacter baumannii transferred from India to Japan.

A 52-year-old male Japanese businessman with massive cerebral bleeding was transferred from India to Japan and was admitted to our hospital. Multidrug-resistant Acinetobacter baumannii was isolated from his sputum. The minimum inhibitory concentrations for this strain were as follows: imipenem, 64 µg/ml; meropenem, 32 µg/ml; ciprofloxacin, 16 µg/ml; amikacin, 16 µg/ml; aztreonam, 16 µg/ml; colistin, <1 µg/ml. This A. baumannii strain had both bla NDM-1 and bla OXA-23 by polymerase chain reaction analysis. In Japan, NDM-1-producing bacteria are extremely rare in clinical specimens. To date, three NDM-1-positive cases have been detected in Japan, and this is the first case of A. baumannii-producing NDM-1 in Japan. Our case suggests that NDM-1-producing bacteria could be introduced into our country easily. There is concern that various resistant bacteria may be transferred from epidemic countries as a result of international medical care.

Characterization of early EDEM1 protein maturation events and their functional implications.

The endoplasmic reticulum (ER) quality control factor EDEM1 associates with a number of ER proteins and ER-associated degradation (ERAD) substrates; however, an understanding of its role in ERAD is unclear. The early maturation events for EDEM1 including signal sequence cleavage and glycosylation were analyzed, and their relationship to the function of EDEM1 was determined. EDEM1 has five N-linked glycosylation sites with the most C-terminal site recognized poorly cotranslationally, resulting in the accumulation of EDEM1 containing four or five glycans. The fifth site was modified post-translationally when bypassed cotranslationally. Signal sequence cleavage of EDEM1 was found to be a slow and inefficient process. Signal sequence cleavage produced a soluble form of EDEM1 that efficiently associated with the oxidoreductase ERdj5 and most effectively accelerated the turnover of a soluble ERAD substrate. In contrast, a type-II membrane form of EDEM1 was generated when the signal sequence was uncleaved, creating an N-terminal transmembrane segment. The membrane form of EDEM1 efficiently associated with the ER membrane protein SEL1L and accelerated the turnover of a membrane-associated ERAD substrate. Together, these results demonstrated that signal sequence cleavage functionally regulated the association of EDEM1-soluble and membrane-integrated isoforms with distinct ERAD machinery and substrates.

Calsperin is a testis-specific chaperone required for sperm fertility.

Calnexin (CANX) and calreticulin (CALR) are homologous lectin chaperones located in the endoplasmic reticulum and cooperate to mediate nascent glycoprotein folding. In the testis, calmegin (CLGN) and calsperin (CALR3) are expressed as germ cell-specific counterparts of CANX and CALR, respectively. Here, we show that Calr3(-/-) males produced apparently normal sperm but were infertile because of defective sperm migration from the uterus into the oviduct and defective binding to the zona pellucida. Whereas CLGN was required for ADAM1A/ADAM2 dimerization and subsequent maturation of ADAM3, a sperm membrane protein required for fertilization, we show that CALR3 is a lectin-deficient chaperone directly required for ADAM3 maturation. Our results establish the client specificity of CALR3 and demonstrate that the germ cell-specific CALR-like endoplasmic reticulum chaperones have contrasting functions in the development of male fertility. The identification and understanding of the maturation mechanisms of key sperm proteins will pave the way toward novel approaches for both contraception and treatment of unexplained male infertility.

Sorting things out through endoplasmic reticulum quality control.

The endoplasmic reticulum (ER) is a highly organized and specialized organelle optimized for the production of proteins. It is comprised of a highly interconnected network of tubules that contain a large set of resident proteins dedicated to the maturation and processing of proteins that traverse the eukaryotic secretory pathway. As protein maturation is an imperfect process, frequently resulting in misfolding and/or the formation of aggregates, proteins are subjected to a series of evaluation processes within the ER. Proteins deemed native are sorted for anterograde trafficking, while immature or non-native proteins are initially retained in the ER in an attempt to rescue the aberrant products. Terminally misfolded substrates are eventually targeted for turnover through the ER-associated degradation or ERAD pathway to protect the cell from the release of a defective product. A clearer picture of the identity of the machinery involved in these quality control evaluation processes and their mechanisms of actions has emerged over the past decade.

The role of UDP-Glc:glycoprotein glucosyltransferase 1 in the maturation of an obligate substrate prosaposin.

An endoplasmic reticulum (ER) quality control system assists in efficient folding and disposal of misfolded proteins. N-linked glycans are critical in these events because their composition dictates interactions with molecular chaperones. UDP-glucose:glycoprotein glucosyltransferase 1 (UGT1) is a key quality control factor of the ER. It adds glucoses to N-linked glycans of nonglucosylated substrates that fail a quality control test, supporting additional rounds of chaperone binding and ER retention. How UGT1 functions in its native environment is poorly understood. The role of UGT1 in the maturation of glycoproteins at basal expression levels was analyzed. Prosaposin was identified as a prominent endogenous UGT1 substrate. A dramatic decrease in the secretion of prosaposin was observed in ugt1(-/-) cells with prosaposin localized to large juxtanuclear aggresome-like inclusions, which is indicative of its misfolding and the essential role that UGT1 plays in its proper maturation. A model is proposed that explains how UGT1 may aid in the folding of sequential domain-containing proteins such as prosaposin.

Isolation and identification of ATP-secreting bacteria from mice and humans.

In a recent report, ATP, which was possibly secreted by some intestinal bacteria, was shown to cause colitis in mice via Th17 cell differentiation. However, the ATP-secreting bacteria have not been isolated and identified. In the present study, we report that Enterococcus gallinarum, which is a vancomycin-resistant Gram-positive coccus isolated from mice and humans, secretes ATP.

Sec22b is a negative regulator of phagocytosis in macrophages.

The endoplasmic reticulum (ER) is proposed to be a membrane donor for phagosome formation. In support of this, we have previously shown that the expression level of syntaxin 18, an ER-localized SNARE protein, correlates with phagocytosis activity. To obtain further insights into the involvement of the ER in phagocytosis we focused on Sec22b, another ER-localized SNARE protein that is also found on phagosomal membranes. In marked contrast to the effects of syntaxin 18, we report here that phagocytosis was nearly abolished in J774 macrophages stably expressing mVenus-tagged Sec22b, without affecting the cell surface expression of the Fc receptor or other membrane proteins related to phagocytosis. Conversely, the capacity of the parental J774 cells for phagocytosis was increased when endogenous Sec22b expression was suppressed. Domain analyses of Sec22b revealed that the R-SNARE motif, a selective domain for forming a SNARE complex with syntaxin18 and/or D12, was responsible for the inhibition of phagocytosis. These results strongly support the ER-mediated phagocytosis model and indicate that Sec22b is a negative regulator of phagocytosis in macrophages, most likely by regulating the level of free syntaxin 18 and/or D12 at the site of phagocytosis.

EDEM1 recognition and delivery of misfolded proteins to the SEL1L-containing ERAD complex.

Terminally misfolded or unassembled secretory proteins are retained in the endoplasmic reticulum (ER) and subsequently cleared by the ER-associated degradation (ERAD) pathway. The degradation of ERAD substrates involves mannose trimming of N-linked glycans; however, the mechanisms of substrate recognition and sorting to the ERAD pathway are poorly defined. EDEM1 (ER degradation-enhancing alpha-mannosidase-like 1 protein) has been proposed to play a role in ERAD substrate signaling or recognition. We show that EDEM1 specifically binds nonnative proteins in a glycan-independent manner. Inhibition of mannosidase activity with kifunensine or disruption of the EDEM1 mannosidase-like domain by mutation had no effect on EDEM1 substrate binding but diminished its association with the ER membrane adaptor protein SEL1L. These results support a model whereby EDEM1 binds nonnative proteins and uses its mannosidase-like domain to target aberrant proteins to the ER membrane dislocation and ubiquitination complex containing SEL1L.

Sweet bays of ERAD.

Proteins that improperly mature in the endoplasmic reticulum (ER) are dislocated to the cytoplasm for proteasome-mediated destruction. A recent study provides insight into the incompletely understood processes for selection and targeting of aberrant proteins for ER-associated protein degradation. The identification of the ER chaperones GRP94 and BiP as binding partners for the mannose-binding proteins OS-9 and XTP3-B, indicates that these protein complexes bind to aberrant proteins and direct them to the Hrd1 dislocation and ubiquitylation complex in the ER membrane.