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-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.

Expectations and Experiences of Internet-Based Therapy for Adolescents with TMD Pain.

AIMS: To investigate expectations and experiences of internet-based therapy (IBT) in adolescents with temporomandibular disorder (TMD) pain. METHODS: Seven adolescents were strategically selected for this study. All patients had received IBT for their TMD pain in a previous randomized controlled trial. One-on-one interviews were conducted in a nonclinical setting. The interviews were semi-structured, following an interview guide with six domains. The recorded interviews were transcribed, and a qualitative inductive content analysis was then performed. RESULTS: Content analysis indicated that the expectations of the adolescents and their experiences of IBT as a treatment for TMD pain can be understood in light of three main categories: (1) To become better; (2) An ambivalent experience; and (3) A personal challenge. The adolescents expressed expectations of less TMD pain after treatment, but also of improvement in general well-being and everyday life. Although their experiences of IBT varied, adolescents described having mixed feelings about treatment and feeling that it was personally challenging. CONCLUSION: Gained understanding of expectations and experiences is a necessary basis for revising the IBT program to meet the demands of adolescents and to improve treatment adherence. Furthermore, the content of the three categories clarifies the values of adolescents, and this understanding can in turn contribute to the development of new patient-centered treatment programs.

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.

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 (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.

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.

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.

Membrane integration and topology of RIFIN and STEVOR proteins of the Plasmodium falciparum parasite.

The malarial parasite Plasmodium exports its own proteins to the cell surfaces of red blood cells (RBCs) during infection. Examples of exported proteins include members of the repetitive interspersed family (RIFIN) and subtelomeric variable open reading frame (STEVOR) family of proteins from Plasmodium falciparum. The presence of these parasite-derived proteins on surfaces of infected RBCs triggers the adhesion of infected cells to uninfected cells (rosetting) and to the vascular endothelium potentially obstructing blood flow. While there is a fair amount of information on the localization of these proteins on the cell surfaces of RBCs, less is known about how they can be exported to the membrane and the topologies they can adopt during the process. The first step of export is plausibly the cotranslational insertion of proteins into the endoplasmic reticulum (ER) of the parasite, and here, we investigate the insertion of three RIFIN and two STEVOR proteins into the ER membrane. We employ a well-established experimental system that uses N-linked glycosylation of sites within the protein as a measure to assess the extent of membrane insertion and the topology it assumes when inserted into the ER membrane. Our results indicate that for all the proteins tested, transmembranes (TMs) 1 and 3 integrate into the membrane, so that the protein assumes an overall topology of Ncyt-Ccyt. We also show that the segment predicted to be TM2 for each of the proteins likely does not reside in the membrane, but is translocated to the lumen.

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.

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.

Transmembrane but not soluble helices fold inside the ribosome tunnel.

Integral membrane proteins are assembled into the ER membrane via a continuous ribosome-translocon channel. The hydrophobicity and thickness of the core of the membrane bilayer leads to the expectation that transmembrane (TM) segments minimize the cost of harbouring polar polypeptide backbones by adopting a regular pattern of hydrogen bonds to form α-helices before integration. Co-translational folding of nascent chains into an α-helical conformation in the ribosomal tunnel has been demonstrated previously, but the features governing this folding are not well understood. In particular, little is known about what features influence the propensity to acquire α-helical structure in the ribosome. Using in vitro translation of truncated nascent chains trapped within the ribosome tunnel and molecular dynamics simulations, we show that folding in the ribosome is attained for TM helices but not for soluble helices, presumably facilitating SRP (signal recognition particle) recognition and/or a favourable conformation for membrane integration upon translocon entry.

Direct Detection of Membrane-Inserting Fragments Defines the Translocation Pores of a Family of Pathogenic Toxins.

Large clostridial toxins (LCTs) are a family of homologous proteins toxins that are directly responsible for the symptoms associated with a number of clostridial infections that cause disease in humans and in other animals. LCTs damage tissues by delivering a glucosyltransferase domain, which inactivates small GTPases, across the endosomal membrane and into the cytosol of target cells. Elucidating the mechanism of translocation for LCTs has been hampered by difficulties associated with identifying marginally hydrophobic segments that insert into the bounding membrane to form the translocation pore. Here, we directly measured the membrane-insertion partitioning propensity for segments spanning the putative pore-forming region using a translocon-mediated insertion assay and synthetic peptides. We identified membrane-inserting segments, as well as a conserved and functionally important negatively charged residue that requires protonation for efficient membrane insertion. We provide a model of the LCT pore, which provides insights into translocation for this enigmatic family of α-helical translocases.

Structure and topology around the cleavage site regulate post-translational cleavage of the HIV-1 gp160 signal peptide.

Like all other secretory proteins, the HIV-1 envelope glycoprotein gp160 is targeted to the endoplasmic reticulum (ER) by its signal peptide during synthesis. Proper gp160 folding in the ER requires core glycosylation, disulfide-bond formation and proline isomerization. Signal-peptide cleavage occurs only late after gp160 chain termination and is dependent on folding of the soluble subunit gp120 to a near-native conformation. We here detail the mechanism by which co-translational signal-peptide cleavage is prevented. Conserved residues from the signal peptide and residues downstream of the canonical cleavage site form an extended alpha-helix in the ER membrane, which covers the cleavage site, thus preventing cleavage. A point mutation in the signal peptide breaks the alpha helix allowing co-translational cleavage. We demonstrate that postponed cleavage of gp160 enhances functional folding of the molecule. The change to early cleavage results in decreased viral fitness compared to wild-type HIV.

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.

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.

Epitopes of anti-RIFIN antibodies and characterization of rif-expressing Plasmodium falciparum parasites by RNA sequencing.

Variable surface antigens of Plasmodium falciparum have been a major research focus since they facilitate parasite sequestration and give rise to deadly malaria complications. Coupled with its potential use as a vaccine candidate, the recent suggestion that the repetitive interspersed families of polypeptides (RIFINs) mediate blood group A rosetting and influence blood group distribution has raised the research profile of these adhesins. Nevertheless, detailed investigations into the functions of this highly diverse multigene family remain hampered by the limited number of validated reagents. In this study, we assess the specificities of three promising polyclonal anti-RIFIN antibodies that were IgG-purified from sera of immunized animals. Their epitope regions were mapped using a 175,000-peptide microarray holding overlapping peptides of the P. falciparum variable surface antigens. Through immunoblotting and immunofluorescence imaging, we show that different antibodies give varying results in different applications/assays. Finally, we authenticate the antibody-based detection of RIFINs in two previously uncharacterized non-rosetting parasite lines by identifying the dominant rif transcripts using RNA sequencing.

Treatment Seeking and Self-Constructed Explanations of Pain and Pain Management Strategies Among Adolescents with Temporomandibular Disorder Pain.

AIMS: To explore adolescents' explanations of their temporomandibular disorder (TMD) pain, their pain management strategies for TMD pain, and their treatment-seeking behavior. METHODS: One-on-one interviews were conducted with 21 adolescents aged 15 to 19 years who had TMD pain and followed a semistructured interview guide. Subjects were strategically selected from patients referred to an orofacial pain clinic. All participants had been examined and received a pain diagnosis based on the Research Diagnostic Criteria for TMD. The interviews focused on the adolescents' experiences of TMD pain, their strategies for handling pain, and how they seek care. The interviews were recorded, transcribed verbatim, and analyzed using qualitative manifest content analysis. RESULTS: Qualitative manifest content analysis revealed two categories: (1) self-constructed explanations, with three subcategories (situation-based explanatory model, physical/biologic model, and psychological explanatory model); and (2) pain management strategies, with four subcategories (social support, treatment, relaxation/rest, and psychological strategies). Adolescents used physical activities and psychological and pharmacologic treatment to manage pain. Reasons for seeking treatment were to be cured, to obtain an explanation for their pain, and because their symptoms bother others. CONCLUSION: Adolescents living with TMD pain develop self-constructed explanations and pain management strategies. With access to these descriptions, dentists can be better prepared to have a dialogue with their adolescent patients about their own explanations of pain, the nature of pain, and in which situations the pain appears. Dentists can also explore adolescent patients' pain management strategies and perhaps also suggest new treatment strategies at an earlier stage.

Membrane insertion and topology of the amino-terminal domain TMD0 of multidrug-resistance associated protein 6 (MRP6).

The function of the ATP-binding cassette transporter MRP6 is unknown but mutations in its gene cause pseudoxanthoma elasticum. We have investigated the membrane topology of the N-terminal transmembrane domain TMD0 of MRP6 and the membrane integration and orientation propensities of its transmembrane segments (TMs) by glycosylation mapping. Results demonstrate that TMD0 has five TMs, an Nout-Cin topology and that the less hydrophobic TMs have strong preference for their orientation in the membrane that affects the neighboring TMs. Two disease-causing mutations changing the number of positive charges in the loops of TMD0 did not affect the membrane insertion efficiencies of the adjacent TMs.

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.