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Photocross-linking of nascent chains to the STT3 subunit of the oligosaccharyltransferase complex

Wed, 11/19/2014 - 9:17am

In eukaryotic cells, polypeptides are N glycosylated after passing through the membrane of the ER into the ER lumen. This modification is effected cotranslationally by the multimeric oligosaccharyltransferase (OST) enzyme. Here, we report the first cross-linking of an OST subunit to a nascent chain that is undergoing translocation through, or integration into, the ER membrane. A photoreactive probe was incorporated into a nascent chain using a modified Lys-tRNA and was positioned in a cryptic glycosylation site (-Q-K-T- instead of -N-K-T-) in the nascent chain. When translocation intermediates with nascent chains of increasing length were irradiated, nascent chain photocross-linking to translocon components, Sec61alpha and TRAM, was replaced by efficient photocross-linking solely to a protein identified by immunoprecipitation as the STT3 subunit of the OST. No cross-linking was observed in the absence of a cryptic sequence or in the presence of a competitive peptide substrate of the OST. As no significant nascent chain photocross-linking to other OST subunits was detected in these fully assembled translocation and integration intermediates, our results strongly indicate that the nascent chain portion of the OST active site is located in STT3.

Oligosaccharyltransferase isoforms that contain different catalytic STT3 subunits have distinct enzymatic properties

Wed, 11/19/2014 - 9:17am

Oligosaccharyltransferase (OST) is an integral membrane protein that catalyzes N-linked glycosylation of nascent proteins in the lumen of the endoplasmic reticulum. Although the yeast OST is an octamer assembled from nonhomologous subunits (Ost1p, Ost2p, Ost3p/Ost6p, Ost4p, Ost5p, Wbp1p, Swp1p, and Stt3p), the composition of the vertebrate OST was less well defined. The roles of specific OST subunits remained enigmatic. Here we show that genomes of most multicellular eukaryotes encode two homologs of Stt3p and mammals express two homologs of Ost3p. The Stt3p and Ost3p homologs are assembled together with the previously described mammalian OST subunits (ribophorins I and II, OST48, and DAD1) into complexes that differ significantly in enzymatic activity. Tissue and cell type-specific differences in expression of the Stt3p homologs suggest that the enzymatic properties of oligosaccharyltransferase are regulated in eukaryotes to respond to alterations in glycoprotein flux through the secretory pathway and may contribute to tissue-specific glycan heterogeneity.

The diversity of dolichol-linked precursors to Asn-linked glycans likely results from secondary loss of sets of glycosyltransferases

Wed, 11/19/2014 - 9:17am

The vast majority of eukaryotes (fungi, plants, animals, slime mold, and euglena) synthesize Asn-linked glycans (Alg) by means of a lipid-linked precursor dolichol-PP-GlcNAc2Man9Glc3. Knowledge of this pathway is important because defects in the glycosyltransferases (Alg1-Alg12 and others not yet identified), which make dolichol-PP-glycans, lead to numerous congenital disorders of glycosylation. Here we used bioinformatic and experimental methods to characterize Alg glycosyltransferases and dolichol-PP-glycans of diverse protists, including many human pathogens, with the following major conclusions. First, it is demonstrated that common ancestry is a useful method of predicting the Alg glycosyltransferase inventory of each eukaryote. Second, in the vast majority of cases, this inventory accurately predicts the dolichol-PP-glycans observed. Third, Alg glycosyltransferases are missing in sets from each organism (e.g., all of the glycosyltransferases that add glucose and mannose are absent from Giardia and Plasmodium). Fourth, dolichol-PP-GlcNAc2Man5 (present in Entamoeba and Trichomonas) and dolichol-PP- and N-linked GlcNAc2 (present in Giardia) have not been identified previously in wild-type organisms. Finally, the present diversity of protist and fungal dolichol-PP-linked glycans appears to result from secondary loss of glycosyltransferases from a common ancestor that contained the complete set of Alg glycosyltransferases.

Mapping the interaction of the STT3 subunit of the oligosaccharyl transferase complex with nascent polypeptide chains

Wed, 11/19/2014 - 9:17am

Many secretory and membrane proteins are N-glycosylated by the oligosaccharyl transferase complex during their translocation across the endoplasmic reticulum membrane. Several experimental observations suggest that the highly conserved STT3 subunit contains the active site of the oligosaccharyl transferase. Here, we report a detailed study of the interaction between the active site of the STT3 protein and nascent polypeptide chains using an in vitro photocrosslinking technique. Our results show that the addition of a glycan moiety in a stretch of approximately 15 residues surrounding a QK(*)T cross-linking site impairs the interaction between the nascent chain and STT3.

The evolution of N-glycan-dependent endoplasmic reticulum quality control factors for glycoprotein folding and degradation

Wed, 11/19/2014 - 9:17am

Asn-linked glycans (N-glycans) play important roles in the quality control (QC) of glycoprotein folding in the endoplasmic reticulum (ER) lumen and in ER-associated degradation (ERAD) of proteins by cytosolic proteasomes. A UDP-Glc:glycoprotein glucosyltransferase glucosylates N-glycans of misfolded proteins, which are then bound and refolded by calreticulin and/or calnexin in association with a protein disulfide isomerase. Alternatively, an alpha-1,2-mannosidase (Mns1) and mannosidase-like proteins (ER degradation-enhancing alpha-mannosidase-like proteins 1, 2, and 3) are part of a process that results in the dislocation of misfolded glycoproteins into the cytosol, where proteins are degraded in the proteasome. Recently we found that numerous protists and fungi contain 0-11 sugars in their N-glycan precursors versus 14 sugars in those of animals, plants, fungi, and Dictyostelium. Our goal here was to determine what effect N-glycan precursor diversity has on N-glycan-dependent QC systems of glycoprotein folding and ERAD. N-glycan-dependent QC of folding (UDP-Glc:glycoprotein glucosyltransferase, calreticulin, and/or calnexin) was present and active in some but not all protists containing at least five mannose residues in their N-glycans and was absent in protists lacking Man. In contrast, N-glycan-dependent ERAD appeared to be absent from the majority of protists. However, Trypanosoma and Trichomonas genomes predicted ER degradation-enhancing alpha-mannosidase-like protein and Mns1 orthologs, respectively, each of which had alpha-mannosidase activity in vitro. Phylogenetic analyses suggested that the diversity of N-glycan-dependent QC of glycoprotein folding (and possibly that of ERAD) was best explained by secondary loss. We conclude that N-glycan precursor length has profound effects on N-glycan-dependent QC of glycoprotein folding and ERAD.

Unique Asn-linked oligosaccharides of the human pathogen Entamoeba histolytica

Wed, 11/19/2014 - 9:17am

N-Glycans of Entamoeba histolytica, the protist that causes amebic dysentery and liver abscess, are of great interest for multiple reasons. E. histolytica makes an unusual truncated N-glycan precursor (Man(5)GlcNAc(2)), has few nucleotide sugar transporters, and has a surface that is capped by the lectin concanavalin A. Here, biochemical and mass spectrometric methods were used to examine N-glycan biosynthesis and the final N-glycans of E. histolytica with the following conclusions. Unprocessed Man(5)GlcNAc(2), which is the most abundant E. histolytica N-glycan, is aggregated into caps on the surface of E. histolytica by the N-glycan-specific, anti-retroviral lectin cyanovirin-N. Glc(1)Man(5)GlcNAc(2), which is made by a UDP-Glc: glycoprotein glucosyltransferase that is part of a conserved N-glycan-dependent endoplasmic reticulum quality control system for protein folding, is also present in mature N-glycans. A swainsonine-sensitive alpha-mannosidase trims some N-glycans to biantennary Man(3)GlcNAc(2). Complex N-glycans of E. histolytica are made by the addition of alpha1,2-linked Gal to both arms of small oligomannose glycans, and Gal residues are capped by one or more Glc. In summary, E. histolytica N-glycans include unprocessed Man(5)GlcNAc(2), which is a target for cyanovirin-N, as well as unique, complex N-glycans containing Gal and Glc.

Cotranslational and posttranslational N-glycosylation of polypeptides by distinct mammalian OST isoforms

Wed, 11/19/2014 - 9:17am

Asparagine-linked glycosylation of polypeptides in the lumen of the endoplasmic reticulum is catalyzed by the hetero-oligomeric oligosaccharyltransferase (OST). OST isoforms with different catalytic subunits (STT3A versus STT3B) and distinct enzymatic properties are coexpressed in mammalian cells. Using siRNA to achieve isoform-specific knockdowns, we show that the OST isoforms cooperate and act sequentially to mediate protein N-glycosylation. The STT3A OST isoform is primarily responsible for cotranslational glycosylation of the nascent polypeptide as it enters the lumen of the endoplasmic reticulum. The STT3B isoform is required for efficient cotranslational glycosylation of an acceptor site adjacent to the N-terminal signal sequence of a secreted protein. Unlike STT3A, STT3B efficiently mediates posttranslational glycosylation of a carboxyl-terminal glycosylation site in an unfolded protein. These distinct and complementary roles for the OST isoforms allow sequential scanning of polypeptides for acceptor sites to insure the maximal efficiency of N-glycosylation.

Meaning in life: an important factor for the psychological well-being of chronically ill patients?

Fri, 11/14/2014 - 12:01pm

PURPOSE: This study aimed to investigate 2 dimensions of meaning in life--Presence of Meaning (i.e., the perception of your life as significant, purposeful, and valuable) and Search for Meaning (i.e., the strength, intensity, and activity of people's efforts to establish or increase their understanding of the meaning in their lives)--and their role for the well-being of chronically ill patients.

RESEARCH DESIGN: A sample of 481 chronically ill patients (M = 50 years, SD = 7.26) completed measures on meaning in life, life satisfaction, optimism, and acceptance. We hypothesized that Presence of Meaning and Search for Meaning will have specific relations with all 3 aspects of well-being.

RESULTS: Cluster analysis was used to examine meaning in life profiles. Results supported 4 distinguishable profiles (High Presence High Search, Low Presence High Search, High Presence Low Search, and Low Presence Low Search) with specific patterns in relation to well-being and acceptance. Specifically, the 2 profiles in which meaning is present showed higher levels of well-being and acceptance, whereas the profiles in which meaning is absent are characterized by lower levels. Furthermore, the results provided some clarification on the nature of the Search for Meaning process by distinguishing between adaptive (the High Presence High Search cluster) and maladaptive (the Low Presence High Search cluster) searching for meaning in life.

CONCLUSIONS: The present study provides an initial glimpse in how meaning in life may be related to the well-being of chronically ill patients and the acceptance of their condition. Clinical implications are discussed.

Differences in Access to Care and Healthcare Utilization Among Sexual Minorities: A Master's Thesis

Fri, 11/14/2014 - 10:54am

BACKGROUND: The barriers in accessing healthcare for gay, lesbian and bisexuals individuals are not well explored. These challenges as well as a lack of knowledge concerning this understudied group has prompted the Institute of Medicine to create a research agenda to build a foundational understanding of gay, lesbian and bisexual health and the barriers they encounter.1 the primary aim of this study will be to compare the differences in health care access and utilization between gay/lesbian, bisexual and heterosexual individuals using a large, nationally representative dataset of the U.S. population.

METHODS: Data from 2001 to 2012 from the National Health and Nutrition Examination Survey was pooled. Using logistic regression, we calculated the unadjusted and adjusted odds ratios of having health insurance, having a routine place and seeing a provider at least one in the past year.

RESULTS: We found that gay men were more likely to have health insurance coverage (ORadj:2.13 95%CI: 1.15,3.92), while bisexual men were at a small disadvantage in having health insurance coverage (ORadj:0.82 95%CI: 0.46,1.46). Bisexual men were more likely to have received health care in the past 12 months (ORadj:3.11 95%CI: 1.74,5.55). Lesbian women were less likely to have health insurance coverage (ORadj-lesbian:0.58 95%CI: 0.34,0.97).

CONCLUSION: This study contributed to the limited knowledge on understanding the health care access and utilization among gay, lesbian and bisexual individuals, which was classified as a high priority by the Institute of Medicine. Expanding health insurance coverage through the Affordable Care Act and Universal Partnership Coverage may reduce the disparities among gay, lesbian and bisexual individuals.

A Feedback Loop Couples Musashi-1 Activity to Omega-9 Fatty Acid Biosynthesis: A PhD Dissertation

Fri, 11/14/2014 - 10:54am

All living creatures change their gene expression program in response to nutrient availability and metabolic demands. Nutrients and metabolites can directly control transcription and activate second-­‐messenger systems. In bacteria, metabolites also affect post-­‐transcriptional regulatory mechanisms, but there are only a few isolated examples of this regulation in eukaryotes. Here, I present evidence that RNA-­‐binding by the stem cell translation regulator Musashi-­‐1 (MSI1) is allosterically inhibited by 18-­‐22 carbon ω-­‐9 monounsaturated fatty acids. The fatty acid binds to the N-­‐terminal RNA Recognition Motif (RRM) and induces a conformational change that prevents RNA association. Musashi proteins are critical for development of the brain, blood, and epithelium. I identify stearoyl-­‐CoA desaturase-­‐1 as a MSI1 target, revealing a feedback loop between ω-­‐9 fatty acid biosynthesis and MSI1 activity. To my knowledge, this is the first example of an RNA-­‐binding protein directly regulated by fatty acid. This finding may represent one of the first examples of a potentially broad network connecting metabolism with post-­‐transcriptional regulation.

Impact of COPD on the Mortality and Treatment of Patients Hospitalized with Acute Decompensated Heart Failure (The Worcester Heart Failure Study): A Masters Thesis

Fri, 11/14/2014 - 10:54am

Objective: Chronic obstructive pulmonary disease (COPD) is a common comorbidity in patients with heart failure, yet little is known about the impact of this condition in patients with acute decompensated heart failure (ADHF), especially from a more generalizable, community-based perspective. The primary objective of this study was to describe the in-hospital and post discharge mortality and treatment of patients hospitalized with ADHF according to COPD status.

Methods: The study population consisted of patients hospitalized with ADHF at all 11 medical centers in central Massachusetts during 4 study years: 1995, 2000, 2002, and 2004.

Results: Of the 9,748 patients hospitalized with ADHF during the years under study, 35.9% had a history of COPD. The average age of this population was 76.1 years, 43.9% were men, and 93.3% were white. At the time of hospital discharge, patients with COPD were less likely to have received evidence-based heart failure medications, including beta-blockers and ACE inhibitors/angiotensin receptor blockers, than patients without COPD. Multivariable adjusted in-hospital death rates were similar for patients with and without COPD. However, among patients who survived to hospital discharge, patients with COPD had a significantly higher risk of dying at 1 (adjusted RR 1.10; 95% CI 1.06, 1.14) and 5-years (adjusted RR 1.40; 95% CI 1.28, 1.42) after hospital discharge than patients who were not previously diagnosed with COPD.

Conclusions: COPD is a common co-morbidity in patients hospitalized with ADHF and is associated with a worse long-term prognosis. Further research is required to understand the complex interactions of these diseases and to ensure that patients with ADHF and COPD receive optimal treatment modalities.

Melanoma in Hong Kong between 1983 and 2002: a decreasing trend in incidence observed in a complex socio-political and economic setting

Thu, 11/13/2014 - 2:36pm

The aim of this study was to examine the incidence and mortality of cutaneous melanoma (CM) in Hong Kong. The epidemiology, clinical, and pathological features of melanoma in Asians are different from those in the European population, yet there is little in the literature that describes about melanoma in Asians. Data from the Hong Kong Cancer Registry from 1983 to 2002 were collected and reviewed. Population-based data were analyzed, focusing on the mortality and incidence rates over this 20-year period. The mean Hong Kong CM incidence rate between 1983 and 2002 was 0.8/100 000 for men and 0.6/100 000 for women. There was an overall decrease in the incidence of CM in Hong Kong between 1983 and 2002 (P

Pruritus ani as a manifestation of systemic contact dermatitis: resolution with dietary nickel restriction

Thu, 11/13/2014 - 2:36pm

Pruritus ani is a common distressing problem with numerous possible causes. When locally applied agents trigger irritation or allergic response, skin changes of dermatitis usually accompany the itch. Focal pruritus in the absence of dermatitis is not generally considered to be a manifestation of contact allergy. Furthermore, focal pruritus is not listed among the possible diverse presentations of the systemic delivery of a proven contact allergen. We report a case of a gentleman with a 1.5-year history of treatment-resistant pruritus ani. When patch testing revealed a positive reaction to nickel sulfate, he admitted to daily peanut butter consumption. His symptoms resolved with dietary nickel restriction. Patch testing may be useful in patients with pruritus of the anogenital region, not only to elucidate potential contact exposures contributing to the symptom but also to suggest possible dietary precipitants.

A mouse model of vitiligo with focused epidermal depigmentation requires IFN-gamma for autoreactive CD8(+) T-cell accumulation in the skin

Thu, 11/13/2014 - 2:35pm

Vitiligo is an autoimmune disease of the skin causing disfiguring patchy depigmentation of the epidermis and, less commonly, hair. Therapeutic options for vitiligo are limited, reflecting in part limited knowledge of disease pathogenesis. Existing mouse models of vitiligo consist of hair depigmentation but lack prominent epidermal involvement, which is the hallmark of human disease. They are thus unable to provide a platform to fully investigate disease mechanisms and treatment. CD8(+) T cells have been implicated in the pathogenesis of vitiligo, and expression of IFN-gamma is increased in the lesional skin of patients, however, it is currently unknown what role IFN-gamma has in disease. Here, we have developed an adoptive transfer mouse model of vitiligo using melanocyte-specific CD8(+) T cells, which recapitulates the human condition by inducing epidermal depigmentation while sparing the hair. Like active lesions in human vitiligo, histology of depigmenting skin reveals a patchy mononuclear infiltrate and single-cell infiltration of the epidermis. Depigmentation is accompanied by accumulation of autoreactive CD8(+) T cells in the skin, quantifiable loss of tyrosinase transcript, and local IFN-gamma production. Neutralization of IFN-gamma with antibody prevents CD8(+) T-cell accumulation and depigmentation, suggesting a therapeutic potential for this approach.

Eruption of bullae within psoriatic plaques: a rare adverse effect of narrow-band ultraviolet B (NB-UVB) phototherapy

Thu, 11/13/2014 - 2:35pm

The sudden eruption of bullae within psoriatic plaques is an uncommon adverse effect of narrow-band UVB phototherapy (TL-01 radiation). We report the case of a 49-year-old man who developed a bullous eruption after several NB-UVB treatments and will review important aspects of this unusual phototherapy complication.