Structure of a Complete Mediator-RNA Polymerase II Pre-Initiation Complex.

A complete, 52-protein, 2.5 million dalton, Mediator-RNA polymerase II pre-initiation complex (Med-PIC) was assembled and analyzed by cryo-electron microscopy and by chemical cross-linking and mass spectrometry. The resulting complete Med-PIC structure reveals two components of functional significance, absent from previous structures, a protein kinase complex and the Mediator-activator interaction region. It thereby shows how the kinase and its target, the C-terminal domain of the polymerase, control Med-PIC interaction and transcription.

The mammalian cytosolic thioredoxin reductase pathway acts via a membrane protein to reduce ER-localised proteins.

Folding of proteins entering the mammalian secretory pathway requires the insertion of the correct disulfides. Disulfide formation involves both an oxidative pathway for their insertion and a reductive pathway to remove incorrectly formed disulfides. Reduction of these disulfides is crucial for correct folding and degradation of misfolded proteins. Previously, we showed that the reductive pathway is driven by NADPH generated in the cytosol. Here, by reconstituting the pathway using purified proteins and ER microsomal membranes, we demonstrate that the thioredoxin reductase system provides the minimal cytosolic components required for reducing proteins within the ER lumen. In particular, saturation of the pathway and its protease sensitivity demonstrates the requirement for a membrane protein to shuttle electrons from the cytosol to the ER. These results provide compelling evidence for the crucial role of the cytosol in regulating ER redox homeostasis, ensuring correct protein folding and facilitating the degradation of misfolded ER proteins.

Protein secondary structure determines the temporal relationship between folding and disulfide formation.

How and when disulfide bonds form in proteins relative to the stage of their folding is a fundamental question in cell biology. Two models describe this relationship: the folded precursor model, in which a nascent structure forms before disulfides do, and the quasi-stochastic model, where disulfides form prior to folding. Here we investigated oxidative folding of three structurally diverse substrates, ß2-microglobulin, prolactin, and the disintegrin domain of ADAM metallopeptidase domain 10 (ADAM10), to understand how these mechanisms apply in a cellular context. We used a eukaryotic cell-free translation system in which we could identify disulfide isomers in stalled translation intermediates to characterize the timing of disulfide formation relative to translocation into the endoplasmic reticulum and the presence of non-native disulfides. Our results indicate that in a domain lacking secondary structure, disulfides form before conformational folding through a process prone to nonnative disulfide formation, whereas in proteins with defined secondary structure, native disulfide formation occurs after partial folding. These findings reveal that the nascent protein structure promotes correct disulfide formation during cotranslational folding.

Cytosolic thioredoxin reductase 1 is required for correct disulfide formation in the ER.

Folding of proteins entering the secretory pathway in mammalian cells frequently requires the insertion of disulfide bonds. Disulfide insertion can result in covalent linkages found in the native structure as well as those that are not, so-called non-native disulfides. The pathways for disulfide formation are well characterized, but our understanding of how non-native disulfides are reduced so that the correct or native disulfides can form is poor. Here, we use a novel assay to demonstrate that the reduction in non-native disulfides requires NADPH as the ultimate electron donor, and a robust cytosolic thioredoxin system, driven by thioredoxin reductase 1 (TrxR1 or TXNRD1). Inhibition of this reductive pathway prevents the correct folding and secretion of proteins that are known to form non-native disulfides during their folding. Hence, we have shown for the first time that mammalian cells have a pathway for transferring reducing equivalents from the cytosol to the ER, which is required to ensure correct disulfide formation in proteins entering the secretory pathway.

Role of integrative structural biology in understanding transcriptional initiation.

Integrative structural biology combines data from multiple experimental techniques to generate complete structural models for the biological system of interest. Most commonly cross-linking data sets are employed alongside electron microscopy maps, crystallographic structures, and other data by computational methods that integrate all known information and produce structural models at a level of resolution that is appropriate to the input data. The precision of these modelled solutions is limited by the sparseness of cross-links observed, the length of the cross-linking reagent, the ambiguity arisen from the presence of multiple copies of the same protein, and structural and compositional heterogeneity. In recent years integrative structural biology approaches have been successfully applied to a range of RNA polymerase II complexes. Here we will provide a general background to integrative structural biology, a description of how it should be practically implemented and how it has furthered our understanding of the biology of large transcriptional assemblies. Finally, in the context of recent breakthroughs in microscope and direct electron detector technology, where increasingly EM is capable of resolving structural features directly without the aid of other structural techniques, we will discuss the future role of integrative structural techniques.

Changing Prevalence of Lower Airway Infections in Young Children with Cystic Fibrosis.

Rationale: Historical studies suggest that airway infection in cystic fibrosis initiates with Staphylococcus aureus and Haemophilus influenzae, with later emergence of Pseudomonas aeruginosa. Aspergillus species are regarded as relatively infrequent, late-occurring infections.Objectives: To assess the prevalence and change in prevalence of early lower airway infections in a modern cohort of children with cystic fibrosis.Methods: All infants diagnosed with cystic fibrosis after newborn screening participating in the Australian Respiratory Early Surveillance Team for Cystic Fibrosis (AREST CF) cohort study between 2000 and 2018 were included. Participants prospectively underwent BAL at 3-6 months, 1 year, and annually up to 6 years of age. Lower airway infection prevalence was described. Changes in prevalence patterns were assessed longitudinally using generalized estimating equations controlling for age and repeated visits.Measurements and Main Results: A total of 380 infants underwent 1,759 BALs. The overall prevalence and median age of first acquisition of the most common infections were as follows: S. aureus, 11%, 2.5 years; P. aeruginosa, 8%, 2.4 years; Aspergillus species, 11%, 3.2 years; and H. influenzae, 9%, 3.1 years. During the study, a significant decrease in prevalence of P. aeruginosa (P < 0.001) and S. aureus (P < 0.001) was observed with a significant change toward more aggressive treatment. Prevalence of Aspergillus infections did not significantly change (P = 0.669).Conclusions:Aspergillus species and P. aeruginosa are commonly present in the lower airways from infancy. The decrease in prevalence of P. aeruginosa and S. aureus since 2000, coinciding with a more aggressive therapeutic approach, has resulted in Aspergillus becoming the most commonly isolated pathogen in young children. Further research is warranted to understand the implication of these findings.

Identification of an Immortalized Human Airway Epithelial Cell Line with Dyskinetic Cilia.

Primary ciliary dyskinesia is an inherited, currently incurable condition. In the respiratory system, primary ciliary dyskinesia causes impaired functioning of the mucociliary escalator, leading to nasal congestion, cough, and recurrent otitis media, and commonly progresses to cause more serious and permanent damage, including hearing deficits, chronic sinusitis, and bronchiectasis. New treatment options for the condition are thus necessary. In characterizing an immortalized human bronchial epithelial cell line (BCi-NS1.1) grown at an air-liquid interface to permit differentiation, we have identified that these cells have dyskinetic motile cilia. The cells had a normal male karyotype, and phenotypic markers of epithelial cell differentiation emerged, as previously shown. Ciliary beat frequency (CBF) as assessed by high-speed videomicroscopy was lower than normal (4.4 Hz). Although changes in CBF induced by known modulators were as expected, the cilia displayed a dyskinetic, circular beat pattern characteristic of central microtubular agenesis with outer doublet transposition. This ultrastructural defect was confirmed by electron microscopy. We propose that the BCi-NS1.1 cell line is a useful model system for examination of modulators of CBF and more specifically could be used to screen for novel drugs with the ability to enhance CBF and perhaps repair a dyskinetic ciliary beat pattern.

Folding of a single domain protein entering the endoplasmic reticulum precedes disulfide formation.

The relationship between protein synthesis, folding, and disulfide formation within the endoplasmic reticulum (ER) is poorly understood. Previous studies have suggested that pre-existing disulfide links are absolutely required to allow protein folding and, conversely, that protein folding occurs prior to disulfide formation. To address the question of what happens first within the ER, that is, protein folding or disulfide formation, we studied folding events at the early stages of polypeptide chain translocation into the mammalian ER using stalled translation intermediates. Our results demonstrate that polypeptide folding can occur without complete domain translocation. Protein disulfide isomerase (PDI) interacts with these early intermediates, but disulfide formation does not occur unless the entire sequence of the protein domain is translocated. This is the first evidence that folding of the polypeptide chain precedes disulfide formation within a cellular context and highlights key differences between protein folding in the ER and refolding of purified proteins.

Matching cross-linked peptide spectra: only as good as the worse identification.

Chemical cross-linking mass spectrometry identifies interacting surfaces within a protein assembly through labeling with bifunctional reagents and identifying the covalently modified peptides. These yield distance constraints that provide a powerful means to model the three-dimensional structure of the assembly. Bioinformatic analysis of cross-linked data resulting from large protein assemblies is challenging because each cross-linked product contains two covalently linked peptides, each of which must be correctly identified from a complex matrix of potential confounders. Protein Prospector addresses these issues through a complementary mass modification strategy in which each peptide is searched and identified separately. We demonstrate this strategy with an analysis of RNA polymerase II. False discovery rates (FDRs) are assessed via comparison of cross-linking data to crystal structure, as well as by using a decoy database strategy. Parameters that are most useful for positive identification of cross-linked spectra are explored. We find that fragmentation spectra generally contain more product ions from one of the two peptides constituting the cross-link. Hence, metrics reflecting the quality of the spectral match to the less confident peptide provide the most discriminatory power between correct and incorrect matches. A support vector machine model was built to further improve classification of cross-linked peptide hits. Furthermore, the frequency with which peptides cross-linked via common acylating reagents fragment to produce diagnostic, cross-linker-specific ions is assessed. The threshold for successful identification of the cross-linked peptide product depends upon the complexity of the sample under investigation. Protein Prospector, by focusing the reliability assessment on the least confident peptide, is better able to control the FDR for results as larger complexes and databases are analyzed. In addition, when FDR thresholds are calculated separately for intraprotein and interprotein results, a further improvement in the number of unique cross-links confidently identified is achieved. These improvements are demonstrated on two previously published cross-linking datasets.

Deconvolution method for specific and nonspecific binding of ligand to multiprotein complex by native mass spectrometry.

In native mass spectrometry, it has been difficult to discriminate between specific bindings of a ligand to a multiprotein complex target from the nonspecific interactions. Here, we present a deconvolution model that consists of two levels of data reduction. At the first level, the apparent association binding constants are extracted from the measured intensities of the target/ligand complexes by varying ligand concentration. At the second level, two functional forms representing the specific and nonspecific binding events are fit to the apparent binding constants obtained from the first level of modeling. Using this approach, we found that a power-law distribution described nonspecific binding of α-amanitin to yeast RNA polymerase II. Moreover, treating the concentration of the multiprotein complex as a fitting parameter reduced the impact of inaccuracies in this experimental measurement on the apparent association constants. This model improves upon current methods for separating specific and nonspecific binding to large, multiprotein complexes in native mass spectrometry, by modeling nonspecific binding with a power-law function.

Clinical audit results in earlier nutritional intervention in malnourished children with cystic fibrosis with improved outcome.

AIM: The association between nutritional status, pulmonary function and survival in cystic fibrosis (CF) is well established. A previous case series from the Royal Children's Hospital, Melbourne (RCH), demonstrated suboptimal referral practices and highlighted the importance of early nutritional interventions in children with CF. Various qualitative changes were made to our CF service, and this study assesses the effects of these practice changes timing of gastrostomy and clinical outcome in patients who underwent gastrostomy insertion. METHOD: Clinical audit of all CF patients who had undergone gastrostomy insertion from 2002 to 2010 at Royal Children's Hospital. Clinical data, including nutritional parameters, respiratory function and survival, were collected at 2 years prior and 2 years post gastrostomy insertion. Data were compared with the previous study from 1989 to 1997. RESULTS: Patients with CF who underwent gastrostomy insertion between 2002 and 2010 (n = 22) had higher weight-for-age scores (-1.5 ± 0.68 vs. -2.67 ± 1.06; P = 0.0001) and higher forced expiratory volume in 1 s (68% ± 22 vs. 52% ± 18.5; P = 0.006), compared with the cohort from 1989 to 1997 (n = 37). These differences were maintained at 2-year follow-up. Pseudomonas aeruginosa colonisation rate was 100% in 1989-1997 vs. 41% in 2002-2010; P = 0.0001. The 2-year survival post-gastrostomy insertion improved from 70% to 100%; P = 0.004. CONCLUSION: Earlier referral of patients in the recent cohort resulted in sustained improvements in weight-for-age and lung function. Survival at 2 years post-procedure was significantly improved. This study confirms the value of clinical audits and subsequent re-evaluation of clinical services.

Structure of the mediator head module bound to the carboxy-terminal domain of RNA polymerase II.

The X-ray crystal structure of the Head module, one-third of the Mediator of transcriptional regulation, has been determined as a complex with the C-terminal domain (CTD) of RNA polymerase II. The structure reveals multiple points of interaction with an extended conformation of the CTD; it suggests a basis for regulation by phosphorylation of the CTD. Biochemical studies show a requirement for Mediator-CTD interaction for transcription.

Tfb6, a previously unidentified subunit of the general transcription factor TFIIH, facilitates dissociation of Ssl2 helicase after transcription initiation.

General transcription factor TFIIH, previously described as a 10-subunit complex, is essential for transcription and DNA repair. An eleventh subunit now identified, termed Tfb6, exhibits 45% sequence similarity to human nuclear mRNA export factor 5. Tfb6 dissociates from TFIIH as a heterodimer with the Ssl2 subunit, a DNA helicase that drives promoter melting for the initiation of transcription. Tfb6 does not, however, dissociate Ssl2 from TFIIH in the context of a fully assembled transcription preinitiation complex. Our findings suggest a dynamic state of Ssl2, allowing its engagement in multiple cellular processes.

Post-translational membrane insertion of an endogenous YidC substrate.

Membrane protein insertion is controlled by proteinaceous factors embedded in the lipid bilayer. Bacterial inner membrane proteins utilise the Sec translocon as the major facilitator of insertion; however some proteins are Sec independent and instead require only YidC. A common feature of YidC substrates is the exposure of a signal anchor sequence when translation is close to completion; this allows minimal time for targeting and favours a post-translational insertion mechanism. Despite this there is little evidence of YidC's post-translational activity. Here we develop an experimental system that uncouples translation and insertion of the endogenous YidC substrate F0c (subunit c of the F0F1 ATP synthase). In this process we (i) develop a novel one step purification method for YidC, including an on column membrane reconstitution, (ii) isolate a soluble form of F0c and (iii) show that incubation of F0c with YidC proteoliposomes results in a high level of membrane integration. Conformational analyses of inserted F0c through Blue Native PAGE and fluorescence quenching reveal a native, oligomerised structure. These data show that YidC can act as a post-translational insertase, a finding which could explain the absence of a ribosome binding domain on YidC. This correlates with the post-translational activity of other YidC family members lacking the ribosome binding domain.

Shared Pseudomonas aeruginosa genotypes are common in Australian cystic fibrosis centres.

Recent molecular-typing studies suggest cross-infection as one of the potential acquisition pathways for Pseudomonas aeruginosa in patients with cystic fibrosis (CF). In Australia, there is only limited evidence of unrelated patients sharing indistinguishable P. aeruginosa strains. We therefore examined the point-prevalence, distribution, diversity and clinical impact of P. aeruginosa strains in Australian CF patients nationally. 983 patients attending 18 Australian CF centres provided 2887 sputum P. aeruginosa isolates for genotyping by enterobacterial repetitive intergenic consensus-PCR assays with confirmation by multilocus sequence typing. Demographic and clinical details were recorded for each participant. Overall, 610 (62%) patients harboured at least one of 38 shared genotypes. Most shared strains were in small patient clusters from a limited number of centres. However, the two predominant genotypes, AUST-01 and AUST-02, were widely dispersed, being detected in 220 (22%) and 173 (18%) patients attending 17 and 16 centres, respectively. AUST-01 was associated with significantly greater treatment requirements than unique P. aeruginosa strains. Multiple clusters of shared P. aeruginosa strains are common in Australian CF centres. At least one of the predominant and widespread genotypes is associated with increased healthcare utilisation. Longitudinal studies are now needed to determine the infection control implications of these findings.

Deletion of the Parkin co-regulated gene causes defects in ependymal ciliary motility and hydrocephalus in the quakingviable mutant mouse.

The quakingviable mouse (qkv) is a spontaneous recessive mouse mutant with a deletion of approximately 1.1 Mb in the proximal region of chromosome 17. The deletion affects the expression of three genes; quaking (Qk), Parkin-coregulated gene (Pacrg) and parkin (Park2). The resulting phenotype, which includes dysmyelination of the central nervous system and male sterility, is due to reduced expression of Qk and a complete lack of Pacrg expression, respectively. Pacrg is required for correct development of the spermatozoan flagella, a specialized type of motile cilia. In vertebrates, motile cilia are required for multiple functions related to cellular movement or movement of media over a stationary cell surface. To investigate the potential role of PACRG in motile cilia we analysed qkv mutant mice for evidence of cilial dysfunction. Histological and magnetic resonance imaging analyses demonstrated that qkv mutant mice were affected by acquired, communicating hydrocephalus (HC). Structural analysis of ependymal cilia demonstrated that the 9 + 2 arrangement of axonemal microtubules was intact and that both the density of ciliated cells and cilia length was similar to wild-type littermates. Cilia function studies showed a reduction in ependymal cilial beat frequency and cilial mediated flow in qkv mutant mice compared with wild-type littermate controls. Moreover, transgenic expression of Pacrg was necessary and sufficient to correct this deficit and rescue the HC phenotype in the qkv mutant. This study provides novel in vivo evidence that Pacrg is required for motile cilia function and may be involved in the pathogenesis of human ciliopathies, such as HC, asthenospermia and primary ciliary dyskinesia.

Progression of early structural lung disease in young children with cystic fibrosis assessed using CT.

BACKGROUND: Cross-sectional studies implicate neutrophilic inflammation and pulmonary infection as risk factors for early structural lung disease in infants and young children with cystic fibrosis (CF). However, the longitudinal progression in a newborn screened population has not been investigated. AIM: To determine whether early CF structural lung disease persists and progresses over 1 year and to identify factors associated with radiological persistence and progression. METHODS: 143 children aged 0.2-6.5 years with CF from a newborn screened population contributed 444 limited slice annual chest CT scans for analysis that were scored for bronchiectasis and air trapping and analysed as paired scans 1 year apart. Logistic and linear regression models, using generalised estimating equations to account for multiple measures, determined associations between persistence and progression over 1 year and age, sex, severe cystic fibrosis transmembrane regulator (CFTR) genotype, pancreatic sufficiency, current respiratory symptoms, and neutrophilic inflammation and infection measured by bronchoalveolar lavage. RESULTS: Once detected, bronchiectasis persisted in 98/133 paired scans (74%) and air trapping in 178/220 (81%). The extent of bronchiectasis increased in 139/227 (63%) of paired scans and air trapping in 121/264 (47%). Radiological progression of bronchiectasis and air trapping was associated with severe CFTR genotype, worsening neutrophilic inflammation and pulmonary infection. DISCUSSION: CT-detected structural lung disease identified in infants and young children with CF persists and progresses over 1 year in most cases, with deteriorating structural lung disease associated with worsening inflammation and pulmonary infection. Early intervention is required to prevent or arrest the progression of structural lung disease in young children with CF.