Inefficient SRP interaction with a nascent chain triggers a mRNA quality control pathway.

Misfolded proteins are often cytotoxic, unless cellular systems prevent their accumulation. Data presented here uncover a mechanism by which defects in secretory proteins lead to a dramatic reduction in their mRNAs and protein expression. When mutant signal sequences fail to bind to the signal recognition particle (SRP) at the ribosome exit site, the nascent chain instead contacts Argonaute2 (Ago2), and the mutant mRNAs are specifically degraded. Severity of signal sequence mutations correlated with increased proximity of Ago2 to nascent chain and mRNA degradation. Ago2 knockdown inhibited degradation of the mutant mRNA, while overexpression of Ago2 or knockdown of SRP54 promoted degradation of secretory protein mRNA. The results reveal a previously unappreciated general mechanism of translational quality control, in which specific mRNA degradation preemptively regulates aberrant protein production (RAPP).

Spc1 regulates the signal peptidase-mediated processing of membrane proteins.

Signal peptidase (SPase) cleaves the signal sequences (SSs) of secretory precursors. It contains an evolutionarily conserved membrane protein subunit, Spc1, that is dispensable for the catalytic activity of SPase and whose role remains unknown. In this study, we investigated the function of yeast Spc1. First, we set up an in vivo SPase cleavage assay using variants of the secretory protein carboxypeptidase Y (CPY) with SSs modified in the N-terminal and hydrophobic core regions. When comparing the SS cleavage efficiencies of these variants in cells with or without Spc1, we found that signal-anchored sequences became more susceptible to cleavage by SPase without Spc1. Furthermore, SPase-mediated processing of model membrane proteins was enhanced in the absence of Spc1 and was reduced upon overexpression of Spc1. Spc1 co-immunoprecipitated with proteins carrying uncleaved signal-anchored or transmembrane (TM) segments. Taken together, these results suggest that Spc1 protects TM segments from SPase action, thereby sharpening SPase substrate selection and acting as a negative regulator of the SPase-mediated processing of membrane proteins.

Diagnostic criteria for temporomandibular disorders-INfORM recommendations: Comprehensive and short-form adaptations for children.

BACKGROUND: The Diagnostic Criteria for Temporomandibular Disorders (DC/TMD) are used worldwide in adults. Until now, no adaptation for use in children has been proposed. OBJECTIVE: The aim of this study was to present comprehensive and short-form adaptations of Axis I and Axis II of the DC/TMD for adults that are appropriate for use with children in clinical and research settings. METHODS: Global Delphi studies with experts in TMDs and in pain psychology identified ways of adapting the DC/TMD for children. RESULTS: The proposed adaptation is suitable for children aged 6-9 years. Proposed changes in Axis I include (i) adapting the language of the Demographics and the Symptom Questionnaires to be developmentally appropriate for children, (ii) adding a general health questionnaire for children and one for their parents, (iii) replacing the TMD Pain Screener with the 3Q/TMD questionnaire and (iv) modifying the clinical examination protocol. Proposed changes in Axis II include (i) for the Graded Chronic Pain Scale, to be developmentally appropriate for children, (ii) adding anxiety and depression assessments that have been validated in children and (iii) adding three constructs (stress, catastrophising and sleep disorders) to assess psychosocial functioning in children. CONCLUSION: The recommended DC/TMD, including Axis I and Axis II, for children aged 6-9 years, is appropriate for use in clinical and research settings. This adapted the first version for children includes changes in Axis I and Axis II changes requiring reliability and validity testing in international settings. Official translations to different languages according to INfORM requirements will enable a worldwide dissemination and implementation.

Diagnostic criteria for temporomandibular disorders-INfORM recommendations: Comprehensive and short-form adaptations for adolescents.

BACKGROUND: The Diagnostic Criteria for Temporomandibular Disorders (DC/TMD) for use in adults is in use worldwide. Until now, no version of this instrument for use in adolescents has been proposed. OBJECTIVE: To present comprehensive and short-form adaptations of the adult version of DC/TMD that are appropriate for use with adolescents in clinical and research settings. METHODS: International experts in TMDs and experts in pain psychology participated in a Delphi process to identify ways of adapting the DC/TMD protocol for physical and psychosocial assessment of adolescents. RESULTS: The proposed adaptation defines adolescence as ages 10-19 years. Changes in the physical diagnosis (Axis I) include (i) adapting the language of the Demographics and the Symptom Questionnaires to be developmentally appropriate for adolescents, (ii) adding two general health questionnaires, one for the adolescent patient and one for their caregivers and (iii) replacing the TMD Pain Screener with the 3Q/TMD questionnaire. Changes in the psychosocial assessment (Axis II) include (i) adapting the language of the Graded Chronic Pain Scale to be developmentally appropriate for adolescents, (ii) adding anxiety and depression assessment that have been validated for adolescents and (iii) adding three constructs (stress, catastrophizing and sleep disorders) to assess psychosocial functioning in adolescents. CONCLUSION: The recommended DC/TMD, including Axis I and Axis II for adolescents, is appropriate to use in clinical and research settings. This adapted first version for adolescents includes changes in Axis I and Axis II requiring reliability and validity testing in international settings. Official translations of the comprehensive and short-form to different languages according to INfORM requirements will enable a worldwide dissemination and implementation.

Diagnostic criteria for temporomandibular disorders in children and adolescents: An international Delphi study-Part 2-Development of Axis II.

BACKGROUND: Unlike the psychosocial assessment established for adults in the Diagnostic Criteria for Temporomandibular Disorders (DC/TMD), a standardised psychosocial assessment for children and adolescents with TMD complaints has not yet been established. OBJECTIVES: To develop a new standardised instrument set to assess the psychosocial functioning in children and adolescents by adapting the psychosocial status and pain-related disability (Axis II) of the adult DC/TMD and by including new instruments. METHODS: A modified Delphi method was used to survey 23 international TMD experts and four international experts in pain-related psychological factors for consensus regarding assessment tools for psychosocial functioning and pain-related disability in children and adolescents. The TMD experts reviewed 29 Axis II statements at round 1, 13 at round 2 and 2 at round 3. Agreement was set at 80% for first-round consensus level and 70% for each of the second and third rounds. The psychological experts completed a complementary Delphi survey to reach a consensus on tools to use to assess more complex psychological domains in children and adolescents. For the psychological experts, the first round included 10 open-ended questions on preferred screening tools for depression, anxiety, catastrophising, sleep problems and stress in children (ages 6-9 years old) and adolescents (ages 10-19 years old) as well as on other domains suggested for investigation. In the second round, the psychological experts received a 9-item questionnaire to prioritise the suggested instruments from most to least recommended. RESULTS: The TMD experts, after three Delphi rounds, reached consensus on the changes of DC/TMD to create a form to evaluate Axis II in children and adolescents with TMD complaints. The psychological experts added tools to assess depression and anxiety, sleep disorders, catastrophising, stress and resilience. CONCLUSION: Through international expert consensus, this study adapted Axis II of the adult DC/TMD to assess psychosocial functioning and pain-related disability in children and adolescents. The adapted Axis II protocols will be validated in the target populations.

Internet-based treatment for adolescents with symptomatic temporomandibular joint disc displacement with reduction. A randomized controlled clinical trial.

OBJECTIVE: To evaluate treatment outcome of a jaw exercise (JE) intervention program combined with an information/counselling program (IC) vs. information/counselling alone. MATERIALS AND METHODS: A clinical sample of 83 adolescents, experiencing painful clicking or catching/locking of the jaw, and diagnosed with symptomatic disc displacement with reduction according to RDC/TMD, were randomly assigned to JE/IC or IC program. Both programs were internet-delivered. The adolescents were examined clinically at baseline, at a 2-month mid-evaluation, and at 4months posttreatment by examiners blinded to which programs the adolescents were assigned to. RESULTS: The JE/IC group showed significantly more improvements of painful catching/locking (p = .017), eating ability (p = .006) and of their jaw function limitation (p = .026) compared to the IC group. Significantly more adolescents in the JE/IC group also reported a ≥50% improvement of the catching/locking of the jaw with pain (p = .024) and for eating ability (p = .034) based on a severity index. Treatment method credibility and satisfaction were also significantly higher in the JE/IC group. CONCLUSION: The internet-delivered JE/IC program showed a better outcome compared to IC alone. The former is thus a feasible and cost-effective treatment for adolescents with symptomatic disc displacement with reduction.

Diagnostic criteria for temporomandibular disorders (DC/TMD) for children and adolescents: An international Delphi study-Part 1-Development of Axis I.

BACKGROUND: Since in children and adolescence prevalence is assessed mainly on self-reported or proxy-reported signs and symptoms; there is a need to develop a more comprehensive standardised process for the collection of clinical information and the diagnosis of TMD in these populations. OBJECTIVE: To develop new instruments and to adapt the diagnostic criteria for temporomandibular disorders (DC/TMD) for the evaluation of TMD in children and adolescents. METHOD: A modified Delphi method was used to seek international consensus among TMD experts. Fourteen clinicians and researchers in the field of oro-facial pain and TMD worldwide were invited to participate in a workshop initiated by the International Network for Orofacial Pain and Related Disorders Methodology (INfORM scientific network) at the General Session of the International Association for Dental Research (IADR, London 2018), as the first step in the Delphi process. Participants discussed the protocols required to make physical diagnoses included in the Axis I of the DC/TMD. Thereafter, nine experts in the field were added, and the first Delphi round was created. This survey included 60 statements for Axis I, and the experts were asked to respond to each statement on a five-item Likert scale ranging from 'Strongly disagree' to 'Strongly agree'. Consensus level was set at 80% agreement for the first round, and at 70% for the next. RESULTS: After three rounds of the Delphi process, a consensus among TMD experts was achieved and two adapted DC/TMD protocols for Axis I physical diagnoses for children and adolescents were developed. CONCLUSION: Through international consensus among TMD experts, this study adapted the Axis I of the DC/TMD for use in evaluating TMD in children and adolescents.

Murine astrotactins 1 and 2 have a similar membrane topology and mature via endoproteolytic cleavage catalyzed by a signal peptidase.

Astrotactin 1 (Astn1) and Astn2 are membrane proteins that function in glial-guided migration, receptor trafficking, and synaptic plasticity in the brain as well as in planar polarity pathways in the skin. Here we used glycosylation mapping and protease protection approaches to map the topologies of mouse Astn1 and Astn2 in rough microsomal membranes and found that Astn2 has a cleaved N-terminal signal peptide, an N-terminal domain located in the lumen of the rough microsomal membranes (topologically equivalent to the extracellular surface in cells), two transmembrane helices, and a large C-terminal lumenal domain. We also found that Astn1 has the same topology as Astn2, but we did not observe any evidence of signal peptide cleavage in Astn1. Both Astn1 and Astn2 mature through endoproteolytic cleavage in the second transmembrane helix; importantly, we identified the endoprotease responsible for the maturation of Astn1 and Astn2 as the endoplasmic reticulum signal peptidase. Differences in the degree of Astn1 and Astn2 maturation possibly contribute to the higher levels of the C-terminal domain of Astn1 detected on neuronal membranes of the central nervous system. These differences may also explain the distinct cellular functions of Astn1 and Astn2, such as in membrane adhesion, receptor trafficking, and planar polarity signaling.

Orientational preferences of neighboring helices can drive ER insertion of a marginally hydrophobic transmembrane helix.

α-helical integral membrane proteins critically depend on the correct insertion of their transmembrane α helices into the lipid bilayer for proper folding, yet a surprisingly large fraction of the transmembrane α helices in multispanning integral membrane proteins are not sufficiently hydrophobic to insert into the target membrane by themselves. How can such marginally hydrophobic segments nevertheless form transmembrane helices in the folded structure? Here, we show that a transmembrane helix with a strong orientational preference (N(cyt)-C(lum) or N(lum)-C(cyt)) can both increase and decrease the hydrophobicity threshold for membrane insertion of a neighboring, marginally hydrophobic helix. This effect helps explain the "missing hydrophobicity" in polytopic membrane proteins.

Does adolescent self-reported TMD pain persist into early adulthood? A longitudinal study.

Objective: To follow up 2209 individuals in a longitudinal study and assess self-reported TMD pain, painful and non-painful comorbid conditions, and pain-related disability.Material and methods: During 2012-2014, questionnaires were sent to 2209 eligible individuals who had been screened for TMD pain each year during 2000-2003. The two screening questions were (1) Do you have pain in the temple, face, jaw joint, or jaws once a week or more often? and (2) Do you have pain when you open your mouth wide or chew once a week or more often? If the patient answered 'yes' to one or both of the questions, TMD pain was recorded. Non-respondents received reminders; telephone interviews were offered a randomised group. The questionnaire queried TMD pain, and painful and non-painful comorbid conditions.Results: The overall response rate was 36.5%. Individuals were placed into one of four pain groups defined by their pain experience at baseline and at the follow-up: no TMD pain (69.0%), new TMD pain (13.0%), previous TMD pain (9.9%), and persistent TMD pain (8.1%). Based on the self-report surveys, significantly more responders with TMD pain at follow-up had had pain as adolescents than not. Of adolescents with TMD pain, 45.1% had pain at follow-up as young adults, while 15.8% had pain at follow-up without a previous history of TMD pain. Individuals with persistent TMD pain had high frequencies of comorbid pains (p < .001), 45.2% reported moderate-severe depression scores (p < .001), and 13.0% had moderate pain-related disability (GCPS).Conclusions: Based on self-report surveys, TMD pain in adolescence appears to triple the risk of TMD pain in young adulthood, and persistent pain increased comorbid pain and psychosocial distress.

Temporomandibular pain in adolescents with a history of preterm birth.

AIM: To evaluate the frequency of temporomandibular disorder (TMD) pain among adolescents with a history of preterm birth compared to a matched control group. METHODS: A group of 192 preterm-born adolescents was followed up at the age of 17-19 years and compared to matched controls. Self-report questionnaires included screening questions about TMD pain, chronic diseases, general health, depression, anxiety, anger, antisocial behaviour and self-concept. TMD pain was defined as answering "yes" to one or both of the following questions: "Do you have pain in the temple, face, temporomandibular joint or jaws once a week or more?" and "Do you have pain when you open your mouth wide or chew once a week or more often?" Data analysis was performed using chi-square test and logistic regression model with likelihood ratio test. RESULTS: A TMD pain frequency of 23% of preterm-born adolescents and 26% among the controls was found, with no significant differences between the groups. Neither were there differences regarding anxiety, depression, anger or self-confidence. Within the preterm group, adolescents with TMD pain registered tension and pain in the body, trouble sleeping, stomach pain and feelings of hopelessness about the future. The controls with TMD pain, more reported having a bad life, feeling like a failure and having bodily pain. Among tested background variables, only TMJ locking or intermittent locking once a week or more was found to explain TMD pain in adolescents. CONCLUSION: A high frequency of TMD pain was found in both groups, one possible explanation could be TMJ dysfunction.

Refined topology model of the STT3/Stt3 protein subunit of the oligosaccharyltransferase complex.

The oligosaccharyltransferase complex, localized in the endoplasmic reticulum (ER) of eukaryotic cells, is responsible for the N-linked glycosylation of numerous protein substrates. The membrane protein STT3 is a highly conserved part of the oligosaccharyltransferase and likely contains the active site of the complex. However, understanding the catalytic determinants of this system has been challenging, in part because of a discrepancy in the structural topology of the bacterial versus eukaryotic proteins and incomplete information about the mechanism of membrane integration. Here, we use a glycosylation mapping approach to investigate these questions. We measured the membrane integration efficiency of the mouse STT3-A and yeast Stt3p transmembrane domains (TMDs) and report a refined topology of the N-terminal half of the mouse STT3-A. Our results show that most of the STT3 TMDs are well inserted into the ER membrane on their own or in the presence of the natural flanking residues. However, for the mouse STT3-A hydrophobic domains 4 and 6 and yeast Stt3p domains 2, 3a, 3c, and 6 we measured reduced insertion efficiency into the ER membrane. Furthermore, we mapped the first half of the STT3-A protein, finding two extra hydrophobic domains between the third and the fourth TMD. This result indicates that the eukaryotic STT3 has 13 transmembrane domains, consistent with the structure of the bacterial homolog of STT3 and setting the stage for future combined efforts to interrogate this fascinating system.

Gene Duplication Leads to Altered Membrane Topology of a Cytochrome P450 Enzyme in Seed Plants.

Evolution of the phenolic metabolism was critical for the transition of plants from water to land. A cytochrome P450, CYP73, with cinnamate 4-hydroxylase (C4H) activity, catalyzes the first plant-specific and rate-limiting step in this pathway. The CYP73 gene is absent from green algae, and first detected in bryophytes. A CYP73 duplication occurred in the ancestor of seed plants and was retained in Taxaceae and most angiosperms. In spite of a clear divergence in primary sequence, both paralogs can fulfill comparable cinnamate hydroxylase roles both in vitro and in vivo. One of them seems dedicated to the biosynthesis of lignin precursors. Its N-terminus forms a single membrane spanning helix and its properties and length are highly constrained. The second is characterized by an elongated and variable N-terminus, reminiscent of ancestral CYP73s. Using as proxies the Brachypodium distachyon proteins, we show that the elongation of the N-terminus does not result in an altered subcellular localization, but in a distinct membrane topology. Insertion in the membrane of endoplasmic reticulum via a double-spanning open hairpin structure allows reorientation to the lumen of the catalytic domain of the protein. In agreement with participation to a different functional unit and supramolecular organization, the protein displays modified heme proximal surface. These data suggest the evolution of divergent C4H enzymes feeding different branches of the phenolic network in seed plants. It shows that specialization required for retention of gene duplicates may result from altered protein topology rather than change in enzyme activity.

Folding and Intramembraneous BRICHOS Binding of the Prosurfactant Protein C Transmembrane Segment.

Surfactant protein C (SP-C) is a novel amyloid protein found in the lung tissue of patients suffering from interstitial lung disease (ILD) due to mutations in the gene of the precursor protein pro-SP-C. SP-C is a small α-helical hydrophobic protein with an unusually high content of valine residues. SP-C is prone to convert into ß-sheet aggregates, forming amyloid fibrils. Nature's way of solving this folding problem is to include a BRICHOS domain in pro-SP-C, which functions as a chaperone for SP-C during biosynthesis. Mutations in the pro-SP-C BRICHOS domain or linker region lead to amyloid formation of the SP-C protein and ILD. In this study, we used an in vitro transcription/translation system to study translocon-mediated folding of the WT pro-SP-C poly-Val and a designed poly-Leu transmembrane (TM) segment in the endoplasmic reticulum (ER) membrane. Furthermore, to understand how the pro-SP-C BRICHOS domain present in the ER lumen can interact with the TM segment of pro-SP-C, we studied the membrane insertion properties of the recombinant form of the pro-SP-C BRICHOS domain and two ILD-associated mutants. The results show that the co-translational folding of the WT pro-SP-C TM segment is inefficient, that the BRICHOS domain inserts into superficial parts of fluid membranes, and that BRICHOS membrane insertion is promoted by poly-Val peptides present in the membrane. In contrast, one BRICHOS and one non-BRICHOS ILD-associated mutant could not insert into membranes. These findings support a chaperone function of the BRICHOS domain, possibly together with the linker region, during pro-SP-C biosynthesis in the ER.

Thermodynamics of Membrane Insertion and Refolding of the Diphtheria Toxin T-Domain.

The diphtheria toxin translocation (T) domain inserts into the endosomal membrane in response to the endosomal acidification and enables the delivery of the catalytic domain into the cell. The insertion pathway consists of a series of conformational changes that occur in solution and in the membrane and leads to the conversion of a water-soluble state into a transmembrane state. In this work, we utilize various biophysical techniques to characterize the insertion pathway from the thermodynamic perspective. Thermal and chemical unfolding measured by differential scanning calorimetry, circular dichroism, and tryptophan fluorescence reveal that the free energy of unfolding of the T-domain at neutral and mildly acidic pH differ by 3-5 kcal/mol, depending on the experimental conditions. Fluorescence correlation spectroscopy measurements show that the free energy change from the membrane-competent state to the interfacial state is approximately -8 kcal/mol and is pH-independent, while that from the membrane-competent state to the transmembrane state ranges between -9.5 and -12 kcal/mol, depending on the membrane lipid composition and pH. Finally, the thermodynamics of transmembrane insertion of individual helices was tested using an in vitro assay that measures the translocon-assisted integration of test sequences into the microsomal membrane. These experiments suggest that even the most hydrophobic helix TH8 has only a small favorable free energy of insertion. The free energy for the insertion of the consensus insertion unit TH8-TH9 is slightly more favorable, yet less favorable than that measured for the entire protein, suggesting a cooperative effect for the membrane insertion of the helices of the T-domain.

Live-cell topology assessment of URG7, MRP6102 and SP-C using glycosylatable green fluorescent protein in mammalian cells.

Experimental tools to determine membrane topology of a protein are rather limited in higher eukaryotic organisms. Here, we report the use of glycosylatable GFP (gGFP) as a sensitive and versatile membrane topology reporter in mammalian cells. gGFP selectively loses its fluorescence upon N-linked glycosylation in the ER lumen. Thus, positive fluorescence signal assigns location of gGFP to the cytosol whereas no fluorescence signal and a glycosylated status of gGFP map the location of gGFP to the ER lumen. By using mammalian gGFP, the membrane topology of disease-associated membrane proteins, URG7, MRP6102, SP-C(Val) and SP-C(Leu) was confirmed. URG7 is partially targeted to the ER, and inserted in Cin form. MRP6102 and SP-C(Leu/Val) are inserted into the membrane in Cout form. A minor population of untargeted SP-C is removed by proteasome dependent quality control system.

Apolar surface area determines the efficiency of translocon-mediated membrane-protein integration into the endoplasmic reticulum.

Integral membrane proteins are integrated cotranslationally into the membrane of the endoplasmic reticulum in a process mediated by the Sec61 translocon. Transmembrane α-helices in a translocating polypeptide chain gain access to the surrounding membrane through a lateral gate in the wall of the translocon channel [van den Berg B, et al. (2004) Nature 427:36-44; Zimmer J, et al. (2008) Nature 455:936-943; Egea PF, Stroud RM (2010) Proc Natl Acad Sci USA 107:17182-17187]. To clarify the nature of the membrane-integration process, we have measured the insertion efficiency into the endoplasmic reticulum membrane of model hydrophobic segments containing nonproteinogenic aliphatic and aromatic amino acids. We find that an amino acid's contribution to the apparent free energy of membrane-insertion is directly proportional to the nonpolar accessible surface area of its side chain, as expected for thermodynamic partitioning between aqueous and nonpolar phases. But unlike bulk-phase partitioning, characterized by a nonpolar solvation parameter of 23 cal/(mol · Å(2)), the solvation parameter for transfer from translocon to bilayer is 6-10 cal/(mol · Å(2)), pointing to important differences between translocon-guided partitioning and simple water-to-membrane partitioning. Our results provide compelling evidence for a thermodynamic partitioning model and insights into the physical properties of the translocon.

Validity and reliability of OIDP and OHIP-14: a survey of Chinese high school students.

BACKGROUND: To determine the impact of oral diseases on everyday life, measures of oral quality of life are needed. In complementing traditional disease-based measures, they assess the need for oral care to evaluate oral health care programs and management of treatment. To assess the reliability and validity of the Oral Impact of Daily Performance (OIDP) and the short-form Oral Health Impact Profile (OHIP-14) among high school students in Xi'an, the capital of Shanxi province, China. METHODS: Cross-sectional one-stage stratified random cluster sample using high schools as the primary sampling unit. Students completed self-administered questionnaires at school. The survey included the OHIP-14 and OIDP inventories, translated and culturally adapted for China, and global oral health and socio-behavioral measures. RESULTS: A total of 5,608 students participated in the study, with a 93% response rate (mean age 17.2, SD 0.8, 52% females, 45.3% urban residents).The proportion experiencing at least one impact (at any frequency) during the previous six months was 62.9% for the OHIP-14 and 45.8% for the OIDP. Cronbach's alpha measured internal consistency at 0.85 for OHIP-14 and 0.75 for OIDP while Cohen's kappa varied between 0.27 and 0.58 for OHIP-14 items and between 0.23 and 0.65 for OIDP items. Kappa scores for the OHIP-14 and OIDP additive scores were 0.52 and 0.66, respectively. Both measures varied systematically and in the expected direction, with global oral health measures showing criterion validity. The correlation between OIDP and OHIP-14 was rs +0.65. That both measures varied systematically with socio-behavioral factors indicates construct validity. CONCLUSION: Both the OIDP and OHIP-14 inventories had reasonable reliability and construct validity in relation to subjective global oral health indicators among adolescents attending high schools in China and thus appear to be useful oral health -related quality of life measures in this context. Overall, the OHIP-14 and OIDP performed equally well, although OHIP-14 had superior content validity due to its sensitivity towards less severe impacts.

EpCAM associates with endoplasmic reticulum aminopeptidase 2 (ERAP2) in breast cancer cells.

Epithelial cell adhesion molecule (EpCAM) is an epithelial and cancer cell "marker" and there is a cumulative and growing evidence of its signaling role. Its importance has been recognized as part of the breast cancer stem cell phenotype, the tumorigenic breast cancer stem cell is EpCAM(+). In spite of its complex functions in normal cell development and cancer, relatively little is known about EpCAM-interacting proteins. We used breast cancer cell lines and performed EpCAM co-immunoprecipitation followed by mass spectrometry in search for novel potentially interacting proteins. The endoplasmic reticulum aminopeptidase 2 (ERAP2) was found to co-precipitate with EpCAM and to co-localize in the cytoplasm/ER and the plasma membrane. ERAP2 is a proteolytic enzyme set in the endoplasmic reticulum (ER) where it plays a central role in the trimming of peptides for presentation by MHC class I molecules. Expression of EpCAM and ERAP2 in vitro in the presence of dog pancreas rough microsomes (ER vesicles) confirmed N-linked glycosylation, processing in ER and the size of EpCAM. The association between ERAP2 and EpCAM is a unique and novel finding that provides new ideas on EpCAM processing and on how antigen presentation may be regulated in cancer.

Molecular code for transmembrane-helix recognition by the Sec61 translocon.

Transmembrane alpha-helices in integral membrane proteins are recognized co-translationally and inserted into the membrane of the endoplasmic reticulum by the Sec61 translocon. A full quantitative description of this phenomenon, linking amino acid sequence to membrane insertion efficiency, is still lacking. Here, using in vitro translation of a model protein in the presence of dog pancreas rough microsomes to analyse a large number of systematically designed hydrophobic segments, we present a quantitative analysis of the position-dependent contribution of all 20 amino acids to membrane insertion efficiency, as well as of the effects of transmembrane segment length and flanking amino acids. The emerging picture of translocon-mediated transmembrane helix assembly is simple, with the critical sequence characteristics mirroring the physical properties of the lipid bilayer.