Rescue of secretion of rare-disease-associated misfolded mutant glycoproteins in UGGT1 knock-out mammalian cells.

Endoplasmic reticulum (ER) retention of misfolded glycoproteins is mediated by the ER-localized eukaryotic glycoprotein secretion checkpoint, UDP-glucose glycoprotein glucosyl-transferase (UGGT). The enzyme recognizes a misfolded glycoprotein and flags it for ER retention by re-glucosylating one of its N-linked glycans. In the background of a congenital mutation in a secreted glycoprotein gene, UGGT-mediated ER retention can cause rare disease, even if the mutant glycoprotein retains activity ("responsive mutant"). Using confocal laser scanning microscopy, we investigated here the subcellular localization of the human Trop-2-Q118E, E227K and L186P mutants, which cause gelatinous drop-like corneal dystrophy (GDLD). Compared with the wild-type Trop-2, which is correctly localized at the plasma membrane, these Trop-2 mutants are retained in the ER. We studied fluorescent chimeras of the Trop-2 Q118E, E227K and L186P mutants in mammalian cells harboring CRISPR/Cas9-mediated inhibition of the UGGT1 and/or UGGT2 genes. The membrane localization of the Trop-2 Q118E, E227K and L186P mutants was successfully rescued in UGGT1-/- cells. UGGT1 also efficiently reglucosylated Trop-2-Q118E-EYFP in cellula. The study supports the hypothesis that UGGT1 modulation would constitute a novel therapeutic strategy for the treatment of pathological conditions associated to misfolded membrane glycoproteins (whenever the mutation impairs but does not abrogate function), and it encourages the testing of modulators of ER glycoprotein folding quality control as broad-spectrum rescue-of-secretion drugs in rare diseases caused by responsive secreted glycoprotein mutants.

Characterizing the protein-protein interaction between MDM2 and 14-3-3σ; proof of concept for small molecule stabilization.

Mouse Double Minute 2 (MDM2) is a key negative regulator of the tumor suppressor protein p53. MDM2 overexpression occurs in many types of cancer and results in the suppression of WT p53. The 14-3-3 family of adaptor proteins are known to bind MDM2 and the 14-3-3σ isoform controls MDM2 cellular localization and stability to inhibit its activity. Therefore, small molecule stabilization of the 14-3-3σ/MDM2 protein-protein interaction (PPI) is a potential therapeutic strategy for the treatment of cancer. Here, we provide a detailed biophysical and structural characterization of the phosphorylation-dependent interaction between 14-3-3σ and peptides that mimic the 14-3-3 binding motifs within MDM2. The data show that di-phosphorylation of MDM2 at S166 and S186 is essential for high affinity 14-3-3 binding and that the binary complex formed involves one MDM2 di-phosphorylated peptide bound to a dimer of 14-3-3σ. However, the two phosphorylation sites do not simultaneously interact so as to bridge the 14-3-3 dimer in a 'multivalent' fashion. Instead, the two phosphorylated MDM2 motifs 'rock' between the two binding grooves of the dimer, which is unusual in the context of 14-3-3 proteins. In addition, we show that the 14-3-3σ-MDM2 interaction is amenable to small molecule stabilization. The natural product fusicoccin A forms a ternary complex with a 14-3-3σ dimer and an MDM2 di-phosphorylated peptide resulting in the stabilization of the 14-3-3σ/MDM2 PPI. This work serves as a proof-of-concept of the drugability of the 14-3-3/MDM2 PPI and paves the way toward the development of more selective and efficacious small molecule stabilizers.

A quinolin-8-ol sub-millimolar inhibitor of UGGT, the ER glycoprotein folding quality control checkpoint.

Misfolded glycoprotein recognition and endoplasmic reticulum (ER) retention are mediated by the ER glycoprotein folding quality control (ERQC) checkpoint enzyme, UDP-glucose glycoprotein glucosyltransferase (UGGT). UGGT modulation is a promising strategy for broad-spectrum antivirals, rescue-of-secretion therapy in rare disease caused by responsive mutations in glycoprotein genes, and many cancers, but to date no selective UGGT inhibitors are known. The small molecule 5-[(morpholin-4-yl)methyl]quinolin-8-ol (5M-8OH-Q) binds a CtUGGTGT24 "WY" conserved surface motif conserved across UGGTs but not present in other GT24 family glycosyltransferases. 5M-8OH-Q has a 47 µM binding affinity for CtUGGTGT24in vitro as measured by ligand-enhanced fluorescence. In cellula, 5M-8OH-Q inhibits both human UGGT isoforms at concentrations higher than 750 µM. 5M-8OH-Q binding to CtUGGTGT24 appears to be mutually exclusive to M5-9 glycan binding in an in vitro competition experiment. A medicinal program based on 5M-8OH-Q will yield the next generation of UGGT inhibitors.

Rescue of secretion of a rare-disease associated mis-folded mutant glycoprotein in UGGT1 knock-out mammalian cells.

Endoplasmic reticulum (ER) retention of mis-folded glycoproteins is mediated by the ERlocalised eukaryotic glycoprotein secretion checkpoint, UDP-glucose glycoprotein glucosyl-transferase (UGGT). The enzyme recognises a mis-folded glycoprotein and flags it for ER retention by reglucosylating one of its N-linked glycans. In the background of a congenital mutation in a secreted glycoprotein gene, UGGT-mediated ER retention can cause rare disease even if the mutant glycoprotein retains activity ("responsive mutant"). Here, we investigated the subcellular localisation of the human Trop-2 Q118E variant, which causes gelatinous droplike corneal dystrophy (GDLD). Compared with the wild type Trop-2, which is correctly localised at the plasma membrane, the Trop-2-Q118E variant is found to be heavily retained in the ER. Using Trop-2-Q118E, we tested UGGT modulation as a rescue-of-secretion therapeutic strategy for congenital rare disease caused by responsive mutations in genes encoding secreted glycoproteins. We investigated secretion of a EYFP-fusion of Trop-2-Q118E by confocal laser scanning microscopy. As a limiting case of UGGT inhibition, mammalian cells harbouring CRISPR/Cas9-mediated inhibition of the UGGT1 and/or UGGT2 gene expressions were used. The membrane localisation of the Trop-2-Q118E-EYFP mutant was successfully rescued in UGGT1-/- and UGGT1/2-/- cells. UGGT1 also efficiently reglucosylated Trop-2-Q118E-EYFP in cellula. The study supports the hypothesis that UGGT1 modulation constitutes a novel therapeutic strategy for the treatment of Trop-2-Q118E associated GDLD, and it encourages the testing of modulators of ER glycoprotein folding Quality Control (ERQC) as broad-spectrum rescueof-secretion drugs in rare diseases caused by responsive secreted glycoprotein mutants.

Protein target highlights in CASP15: Analysis of models by structure providers.

We present an in-depth analysis of selected CASP15 targets, focusing on their biological and functional significance. The authors of the structures identify and discuss key protein features and evaluate how effectively these aspects were captured in the submitted predictions. While the overall ability to predict three-dimensional protein structures continues to impress, reproducing uncommon features not previously observed in experimental structures is still a challenge. Furthermore, instances with conformational flexibility and large multimeric complexes highlight the need for novel scoring strategies to better emphasize biologically relevant structural regions. Looking ahead, closer integration of computational and experimental techniques will play a key role in determining the next challenges to be unraveled in the field of structural molecular biology.

3D-Informed Targeting of the Trop-2 Signal-Activation Site Drives Selective Cancer Vulnerability.

Next-generation Trop-2-targeted therapy against advanced cancers is hampered by expression of Trop-2 in normal tissues. We discovered that Trop-2 undergoes proteolytic activation by ADAM10 in cancer cells, leading to the exposure of a previously inaccessible protein groove flanked by two N-glycosylation sites. We designed a recognition strategy for this region, to drive selective cancer vulnerability in patients. Most undiscriminating anti-Trop-2 mAbs recognize a single immunodominant epitope. Hence, we removed it by deletion mutagenesis. Cancer-specific, glycosylation-prone mAbs were selected by ELISA, bio-layer interferometry, flow cytometry, confocal microscopy for differential binding to cleaved/activated, wild-type and glycosylation site-mutagenized Trop-2. The resulting 2G10 mAb family binds Trop-2-expressing cancer cells, but not Trop-2 on normal cells. We humanized 2G10 by state-of-the-art complementarity determining region grafting/re-modeling, yielding Hu2G10. This antibody binds cancer-specific, cleaved/activated Trop-2 with Kd < 10-12 mol/L, and uncleaved/wtTrop-2 in normal cells with Kd 3.16×10-8 mol/L, thus promising an unprecedented therapeutic index in patients. In vivo, Hu2G10 ablates growth of Trop-2-expressing breast, colon, prostate cancers, but shows no evidence of systemic toxicity, paving the way for a paradigm shift in Trop-2-targeted therapy.

Cadmium treatment induces endoplasmic reticulum stress and unfolded protein response in Arabidopsisthaliana.

We report about the response of Arabidopsis thaliana to chronic and temporary Cd2+ stress, and the Cd2+ induced activation of ER stress and unfolded protein response (UPR). Cd2+-induced UPR proceeds mainly through the bZIP60 arm, which in turn activates relevant ER stress marker genes such as BiP3, CNX, PDI5 and ERdj3B in a concentration- (chronic stress) or time- (temporary stress) dependent manner. A more severe Cd-stress triggers programmed cell death (PCD) through the activation of the NAC089 transcription factor. Toxic effects of Cd2+ exposure are reduced in the Atbzip28/bzip60 double mutant in terms of primary root length and fresh shoot weight, likely due to reduced UPR and PCD activation. We also hypothesised that the enhanced Cd2+ tolerance of the Atbzip28/bzip60 double mutant is due to an increase in brassinosteroids signaling, since the amount of the brassinosteroid insensitive1 receptor (BRI1) protein decreases under Cd2+ stress only in Wt plants. These data highlight the complexity of the UPR pathway, since the ER stress response is strictly related to the type of the treatment applied and the multifaceted connections of ER signaling. The reduced sensing of Cd2+ stress in plants with UPR defects can be used as a novel strategy for phytoremediation.

Fusarium verticillioides NAT1 (FDB2) N-malonyltransferase is structurally, functionally and phylogenetically distinct from its N-acetyltransferase (NAT) homologues.

Fusarium endophytes damage cereal crops and contaminate produce with mycotoxins. Those fungi overcome the main chemical defence of host via detoxification by a malonyl-CoA-dependent enzyme homologous to xenobiotic metabolizing arylamine N-acetyltransferase (NAT). In Fusarium verticillioides (teleomorph Gibberella moniliformis, GIBMO), this N-malonyltransferase activity is attributed to (GIBMO)NAT1, and the fungus has two additional isoenzymes, (GIBMO)NAT3 (N-acetyltransferase) and (GIBMO)NAT2 (unknown function). We present the crystallographic structure of (GIBMO)NAT1, also modelling other fungal NAT homologues. Monomeric (GIBMO)NAT1 is distinctive, with access to the catalytic core through two "tunnel-like" entries separated by a "bridge-like" helix. In the quaternary arrangement, (GIBMO)NAT1 monomers interact in pairs along an extensive interface whereby one entry of each monomer is covered by the N-terminus of the other monomer. Although monomeric (GIBMO)NAT1 apparently accommodates acetyl-CoA better than malonyl-CoA, dimerization changes the active site to allow malonyl-CoA to reach the catalytic triad (Cys110, His158 and Asp173) via the single uncovered entry, and anchor its terminal carboxyl-group via hydrogen bonds to Arg109, Asn157 and Thr261. Lacking a terminal carboxyl-group, acetyl-CoA cannot form such stabilizing interactions, while longer acyl-CoAs enter the active site but cannot reach catalytic Cys. Other NAT isoenzymes lack such structural features, with (GIBMO)NAT3 resembling bacterial NATs and (GIBMO)NAT2 adopting a structure intermediate between (GIBMO)NAT1 and (GIBMO)NAT3. Biochemical assays confirmed differential donor substrate preference of (GIBMO)NAT isoenzymes, with phylogenetic analysis demonstrating evolutionary separation. Given the role of (GIBMO)NAT1 in enhancing Fusarium pathogenicity, unravelling the structure and function of this enzyme may benefit research into more targeted strategies for pathogen control.

Quantifying volume and high-speed technical actions of professional soccer players using foot-mounted inertial measurement units.

AIMS: The aims of the study were two-fold: i) examine the validity and reliability of high-speed kicking actions using foot-mounted inertial measurement unit's (IMU), ii) quantify soccer players within-microcycle and inter-positional differences in both the frequency and speed of technical actions. METHODS: During the in-season phase (25 weeks) of the UK domestic season, 21 professional soccer player ball releases, high-speed ball releases and ball release index were analysed. Pearson product-moment correlation coefficient and confidence intervals were used to determine the validity between the systems, whilst a general linear mixed model analysis approach was used to establish estimated marginal mean values for total ball releases, high-speed ball releases and ball release index. RESULTS: Good concurrent validity was observed for ball release velocity and high-speed kicks against a high-speed camera (r2- 0.96, CI 0.93-0.98). Ball releases, high-speed ball releases and ball release index all showed main effects for fixture proximity (p>0.001), playing positions (p>0.001) and across different training categories (p>0.001). The greatest high-speed ball releases were observed on a match-day (MD)+1 (17.6 ± 11.9; CI- 16.2 to 19) and MD-2 (16.8 ± 15; CI- 14.9 to 18.7), with MD+1 exhibiting the highest number of ball releases (161.1 ± 51.2; CI- 155.0 to 167.2) and ball release index (145.5 ± 45.2; CI- 140.1 to 150.9) across all fixture proximities. Possessions (0.3 ± 0.9; CI- 0.3 to 0.4) and small-sided games (1.4 ± 1.6; CI- 1.4 to 1.5), had the lowest values for high-speed ball releases with technical (6.1 ± 7.2; CI- 5.7 to 6.6) and tactical (10.0 ± 10.5; CI- 6.9 to 13.1) drills showing the largest high-speed ball releases. CONCLUSIONS: The present study provides novel information regarding the quantification of technical actions of professional soccer players. Insights into absolute and relative frequency and intensity of releases in different drill types, provide practitioners with valuable information on technical outputs that can be manipulated during the process of planning training programmes to produce desired outcomes. Both volume and speed of ball release actions should be measured, when monitoring the technical actions in training according to fixture proximity, drill type and player position to permit enhanced training prescription.

Crystal polymorphism in fragment-based lead discovery of ligands of the catalytic domain of UGGT, the glycoprotein folding quality control checkpoint.

None of the current data processing pipelines for X-ray crystallography fragment-based lead discovery (FBLD) consults all the information available when deciding on the lattice and symmetry (i.e., the polymorph) of each soaked crystal. Often, X-ray crystallography FBLD pipelines either choose the polymorph based on cell volume and point-group symmetry of the X-ray diffraction data or leave polymorph attribution to manual intervention on the part of the user. Thus, when the FBLD crystals belong to more than one crystal polymorph, the discovery pipeline can be plagued by space group ambiguity, especially if the polymorphs at hand are variations of the same lattice and, therefore, difficult to tell apart from their morphology and/or their apparent crystal lattices and point groups. In the course of a fragment-based lead discovery effort aimed at finding ligands of the catalytic domain of UDP-glucose glycoprotein glucosyltransferase (UGGT), we encountered a mixture of trigonal crystals and pseudotrigonal triclinic crystals-with the two lattices closely related. In order to resolve that polymorphism ambiguity, we have written and described here a series of Unix shell scripts called CoALLA (crystal polymorph and ligand likelihood-based assignment). The CoALLA scripts are written in Unix shell and use autoPROC for data processing, CCP4-Dimple/REFMAC5 and BUSTER for refinement, and RHOFIT for ligand docking. The choice of the polymorph is effected by carrying out (in each of the known polymorphs) the tasks of diffraction data indexing, integration, scaling, and structural refinement. The most likely polymorph is then chosen as the one with the best structure refinement Rfree statistic. The CoALLA scripts further implement a likelihood-based ligand assignment strategy, starting with macromolecular refinement and automated water addition, followed by removal of the water molecules that appear to be fitting ligand density, and a final round of refinement after random perturbation of the refined macromolecular model, in order to obtain unbiased difference density maps for automated ligand placement. We illustrate the use of CoALLA to discriminate between H3 and P1 crystals used for an FBLD effort to find fragments binding to the catalytic domain of Chaetomium thermophilum UGGT.

Ten things I `hate' about refinement.

Macromolecular refinement is an optimization process that aims to produce the most likely macromolecular structural model in the light of experimental data. As such, macromolecular refinement is one of the most complex optimization problems in wide use. Macromolecular refinement programs have to deal with the complex relationship between the parameters of the atomic model and the experimental data, as well as a large number of types of prior knowledge about chemical structure. This paper draws attention to areas of unfinished business in the field of macromolecular refinement. In it, we describe ten refinement topics that we think deserve attention and discuss directions leading to macromolecular refinement software that would make the best use of modern computer resources to meet the needs of structural biologists of the twenty-first century.

Cooperative stabilisation of 14-3-3σ protein-protein interactions via covalent protein modification.

14-3-3 proteins are an important family of hub proteins that play important roles in many cellular processes via a large network of interactions with partner proteins. Many of these protein-protein interactions (PPI) are implicated in human diseases such as cancer and neurodegeneration. The stabilisation of selected 14-3-3 PPIs using drug-like 'molecular glues' is a novel therapeutic strategy with high potential. However, the examples reported to date have a number of drawbacks in terms of selectivity and potency. Here, we report that WR-1065, the active species of the approved drug amifostine, covalently modifies 14-3-3σ at an isoform-unique cysteine residue, Cys38. This modification leads to isoform-specific stabilisation of two 14-3-3σ PPIs in a manner that is cooperative with a well characterised molecular glue, fusicoccin A. Our findings reveal a novel stabilisation mechanism for 14-3-3σ, an isoform with particular involvement in cancer pathways. This mechanism can be exploited to harness the enhanced potency conveyed by covalent drug molecules and dual ligand cooperativity. This is demonstrated in two cancer cell lines whereby the cooperative behaviour of fusicoccin A and WR-1065 leads to enhanced efficacy for inducing cell death and attenuating cell growth.

Anharmonic Thermal Motion Modelling in the Experimental XRD Charge Density Determination of 1-Methyluracil at T = 23 K.

The experimental electron density distribution (EDD) of 1-methyluracil (1-MUR) was obtained by single crystal X-ray diffraction (XRD) experiments at 23 K. Four different structural models fitting an extensive set of XRD data to a resolution of (sinθ/λ)max = 1.143 Å-1 are compared. Two of the models include anharmonic temperature factors, whose inclusion is supported by the Hamilton test at a 99.95% level of confidence. Positive Fourier residuals up to 0.5 eÅ-3 in magnitude were found close to the methyl group and in the region of hydrogen bonds. Residual density analysis (RDA) and molecular dynamics simulations in the solid-state demonstrate that these residuals can be likely attributed to unresolved disorder, possibly dynamical and long-range in nature. Atomic volumes and charges, molecular moments up to hexadecapoles, as well as maps of the molecular electrostatic potential were obtained from distributed multipole analysis of the EDD. The derived electrostatic properties neither depend on the details of the multipole model, nor are significantly affected by the explicit inclusion of anharmonicity in the least-squares model. The distribution of atomic charges in 1-MUR is not affected by the crystal environment in a significant way. The quality of experimental findings is discussed in light of in-crystal and gas-phase quantum simulations.

Clamping, bending, and twisting inter-domain motions in the misfold-recognizing portion of UDP-glucose: Glycoprotein glucosyltransferase.

UDP-glucose:glycoprotein glucosyltransferase (UGGT) flags misfolded glycoproteins for ER retention. We report crystal structures of full-length Chaetomium thermophilum UGGT (CtUGGT), two CtUGGT double-cysteine mutants, and its TRXL2 domain truncation (CtUGGT-ΔTRXL2). CtUGGT molecular dynamics (MD) simulations capture extended conformations and reveal clamping, bending, and twisting inter-domain movements. We name "Parodi limit" the maximum distance on the same glycoprotein between a site of misfolding and an N-linked glycan that can be reglucosylated by monomeric UGGT in vitro, in response to recognition of misfold at that site. Based on the MD simulations, we estimate the Parodi limit as around 70-80 Å. Frequency distributions of distances between glycoprotein residues and their closest N-linked glycosylation sites in glycoprotein crystal structures suggests relevance of the Parodi limit to UGGT activity in vivo. Our data support a "one-size-fits-all adjustable spanner" UGGT substrate recognition model, with an essential role for the UGGT TRXL2 domain.

Partial catalytic Cys oxidation of human GAPDH to Cys-sulfonic acid.

Background: n-Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) catalyses the NAD +-dependent oxidative phosphorylation of n-glyceraldehyde-3-phosphate to 1,3-diphospho-n-glycerate and its reverse reaction in glycolysis and gluconeogenesis. Methods: Four distinct crystal structures of human n-Glyceraldehyde-3-phosphate dehydrogenase ( HsGAPDH) have been determined from protein purified from the supernatant of HEK293F human epithelial kidney cells. Results: X-ray crystallography and mass-spectrometry indicate that the catalytic cysteine of the protein ( HsGAPDH Cys152) is partially oxidised to cysteine S-sulfonic acid. The average occupancy for the Cys152-S-sulfonic acid modification over the 20 crystallographically independent copies of HsGAPDH across three of the crystal forms obtained is 0.31±0.17. Conclusions: The modification induces no significant structural changes on the tetrameric enzyme, and only makes aspecific contacts to surface residues in the active site, in keeping with the hypothesis that the oxidising conditions of the secreted mammalian cell expression system result in HsGAPDH catalytic cysteine S-sulfonic acid modification and irreversible inactivation of the enzyme.

Erratum to "Modulation of ERQC and ERAD: A Broad-Spectrum Spanner in the Works of Cancer Cells?"

[This corrects the article DOI: 10.1155/2019/8384913.].

Targeting Endoplasmic Reticulum α-Glucosidase I with a Single-Dose Iminosugar Treatment Protects against Lethal Influenza and Dengue Virus Infections.

Influenza and dengue viruses present a growing global threat to public health. Both viruses depend on the host endoplasmic reticulum (ER) glycoprotein folding pathway. In 2014, Sadat et al. reported two siblings with a rare genetic defect in ER α-glucosidase I (ER Glu I) who showed resistance to viral infections, identifying ER Glu I as a key antiviral target. Here, we show that a single dose of UV-4B (the hydrochloride salt form of N-(9'-methoxynonyl)-1-deoxynojirimycin; MON-DNJ) capable of inhibiting Glu I in vivo is sufficient to prevent death in mice infected with lethal viral doses, even when treatment is started as late as 48 h post infection. The first crystal structure of mammalian ER Glu I will constitute the basis for the development of potent and selective inhibitors. Targeting ER Glu I with UV-4B-derived compounds may alter treatment paradigms for acute viral disease through development of a single-dose therapeutic regime.

Modulation of ERQC and ERAD: A Broad-Spectrum Spanner in the Works of Cancer Cells?

Endoplasmic reticulum glycoprotein folding quality control (ERQC) and ER-associated degradation (ERAD) preside over cellular glycoprotein secretion and maintain steady glycoproteostasis. When cells turn malignant, cancer cell plasticity is affected and supported either by point mutations, preferential isoform selection, altered expression levels, or shifts to conformational equilibria of a secreted glycoprotein. Such changes are crucial in mediating altered extracellular signalling, metabolic behavior, and adhesion properties of cancer cells. It is therefore conceivable that interference with ERQC and/or ERAD can be used to selectively damage cancers. Indeed, inhibitors of the late stages of ERAD are already in the clinic against cancers such as multiple myeloma. Here, we review recent advances in our understanding of the complex relationship between glycoproteostasis and cancer biology and discuss the potential of ERQC and ERAD modulators for the selective targeting of cancer cell plasticity.

Early management of COPD: where are we now and where do we go from here? A Delphi consensus project.

PURPOSE: There is a lack of consensus on the most appropriate early diagnostic strategy, criteria for early access to treatment and follow-up approach for patients with COPD. MATERIALS AND METHODS: A Delphi consensus project investigated the early management of COPD. We formulated two questionnaires for completion by pneumologists in Italy. RESULTS: A total of 207 specialists completed questionnaire 1 and 184 of them questionnaire 2, between November 2016 and October 2017. Early diagnosis of COPD was considered uncommon for 93.2% of the expert panel. Regardless of the definition of "early diagnosis" - a diagnosis made before the clinical manifestation of the disease for most responders (60.4%) - experts were confident of the positive effects of early disease management, which they consider is effective in modifying the natural history of the disease. Lack of awareness of the disease was considered the first limiting factor to early COPD management for 78% of respondents. The most effective steps to reduce functional decline were considered to be smoking cessation, followed by long-acting ß2-agonist (LABA)/long-acting muscarinic antagonist (LAMA), LAMA, LABA, and finally inhaled corticosteroid/LABA (P<0.01 for each paired comparison). Specialists considered it "inappropriate" for general practitioners to perform both the early diagnosis and therapy of COPD without the involvement of a specialist. CONCLUSION: Early management of COPD is uncommon, and although data on the effects of early disease management on long-term outcomes are limited, Italian experts are confident of the clinical efficacy of this approach.