The last two transmembrane helices in the APC-type FurE transporter act as an intramolecular chaperone essential for concentrative ER-exit.

FurE is a H+ symporter specific for the cellular uptake of uric acid, allantoin, uracil, and toxic nucleobase analogues in the fungus Aspergillus nidulans. Being member of the NCS1 protein family, FurE is structurally related to the APC-superfamily of transporters. APC-type transporters are characterised by a 5+5 inverted repeat fold made of ten transmembrane segments (TMS1-10) and function through the rocking-bundle mechanism. Most APC-type transporters possess two extra C-terminal TMS segments (TMS11-12), the function of which remains elusive. Here we present a systematic mutational analysis of TMS11-12 of FurE and show that two specific aromatic residues in TMS12, Trp473 and Tyr484, are essential for ER-exit and trafficking to the plasma membrane (PM). Molecular modeling shows that Trp473 and Tyr484 might be essential through dynamic interactions with residues in TMS2 (Leu91), TMS3 (Phe111), TMS10 (Val404, Asp406) and other aromatic residues in TMS12. Genetic analysis confirms the essential role of Phe111, Asp406 and TMS12 aromatic residues in FurE ER-exit. We further show that co-expression of FurE-Y484F or FurE-W473A with wild-type FurE leads to a dominant negative phenotype, compatible with the concept that FurE molecules oligomerize or partition in specific microdomains to achieve concentrative ER-exit and traffic to the PM. Importantly, truncated FurE versions lacking TMS11-12 are unable to reproduce a negative effect on the trafficking of co-expressed wild-type FurE. Overall, we show that TMS11-12 acts as an intramolecular chaperone for proper FurE folding, which seems to provide a structural code for FurE partitioning in ER-exit sites.

Structural principles of peptide-centric chimeric antigen receptor recognition guide therapeutic expansion.

Peptide-centric chimeric antigen receptors (PC-CARs) recognize oncoprotein epitopes displayed by cell-surface human leukocyte antigens (HLAs) and offer a promising strategy for targeted cancer therapy. We have previously developed a PC-CAR targeting a neuroblastoma-associated PHOX2B peptide, leading to robust tumor cell lysis restricted by two common HLA allotypes. Here, we determine the 2.1-angstrom crystal structure of the PC-CAR-PHOX2B-HLA-A*24:02-ß2m complex, which reveals the basis for antigen-specific recognition through interactions with CAR complementarity-determining regions (CDRs). This PC-CAR adopts a diagonal docking mode, where interactions with both conserved and polymorphic HLA framework residues permit recognition of multiple HLA allotypes from the A9 serological cross-reactive group, covering a combined global population frequency of up to 46.7%. Biochemical binding assays, molecular dynamics simulations, and structural and functional analyses demonstrate that high-affinity PC-CAR recognition of cross-reactive pHLAs necessitates the presentation of a specific peptide backbone, where subtle structural adaptations of the peptide are critical for high-affinity complex formation, and CAR T cell killing. Our results provide a molecular blueprint for engineering CARs with optimal recognition of tumor-associated antigens in the context of different HLAs, while minimizing cross-reactivity with self-epitopes.

Uracil/H+ Symport by FurE Refines Aspects of the Rocking-bundle Mechanism of APC-type Transporters.

Transporters mediate the uptake of solutes, metabolites and drugs across the cell membrane. The eukaryotic FurE nucleobase/H+ symporter of Aspergillus nidulans has been used as a model protein to address structure-function relationships in the APC transporter superfamily, members of which are characterized by the LeuT-fold and seem to operate by the so-called 'rocking-bundle' mechanism. In this study, we reveal the binding mode, translocation and release pathway of uracil/H+ by FurE using path collective variable, funnel metadynamics and rational mutational analysis. Our study reveals a stepwise, induced-fit, mechanism of ordered sequential transport of proton and uracil, which in turn suggests that FurE, functions as a multi-step gated pore, rather than employing 'rocking' of compact domains, as often proposed for APC transporters. Finally, our work supports that specific residues of the cytoplasmic N-tail are involved in substrate translocation, in line with their essentiality for FurE function.

A Chicken Tapasin ortholog can chaperone empty HLA-B∗37:01 molecules independent of other peptide-loading components.

Human Tapasin (hTapasin) is the main chaperone of MHC-I molecules, enabling peptide loading and antigen repertoire optimization across HLA allotypes. However, it is restricted to the endoplasmic reticulum (ER) lumen as part of the protein loading complex (PLC), and therefore is highly unstable when expressed in recombinant form. Additional stabilizing co-factors such as ERp57 are required to catalyze peptide exchange in vitro, limiting uses for the generation of pMHC-I molecules of desired antigen specificities. Here, we show that the chicken Tapasin (chTapasin) ortholog can be expressed recombinantly at high yields in a stable form, independent of co-chaperones. chTapasin can bind the human HLA-B∗37:01 with low micromolar-range affinity to form a stable tertiary complex. Biophysical characterization by methyl-based NMR methods reveals that chTapasin recognizes a conserved ß2m epitope on HLA-B∗37:01, consistent with previously solved X-ray structures of hTapasin. Finally, we provide evidence that the B∗37:01/chTapasin complex is peptide-receptive and can be dissociated upon binding of high-affinity peptides. Our results highlight the use of chTapasin as a stable scaffold for protein engineering applications aiming to expand the ligand exchange function on human MHC-I and MHC-like molecules.

A Chicken Tapasin ortholog can chaperone empty HLA molecules independently of other peptide-loading components.

Human Tapasin (hTapasin) is the main chaperone of MHC-I molecules, enabling peptide loading and antigen repertoire optimization across HLA allotypes. However, it is restricted to the endoplasmic reticulum (ER) lumen as part of the protein loading complex (PLC) and therefore is highly unstable when expressed in recombinant form. Additional stabilizing co-factors such as ERp57 are required to catalyze peptide exchange in vitro , limiting uses for the generation of pMHC-I molecules of desired antigen specificities. Here, we show that the chicken Tapasin (chTapasin) ortholog can be expressed recombinantly at high yields in stable form, independently of co-chaperones. chTapasin can bind the human HLA-B * 37:01 with low micromolar-range affinity to form a stable tertiary complex. Biophysical characterization by methyl-based NMR methods reveals that chTapasin recognizes a conserved ß 2 m epitope on HLA-B * 37:01, consistent with previously solved X-ray structures of hTapasin. Finally, we provide evidence that the B * 37:01/chTapasin complex is peptide-receptive and can be dissociated upon binding of high-affinity peptides. Our results highlight the use of chTapasin as a stable scaffold for future protein engineering applications aiming to expand the ligand exchange function on human MHC-I and MHC-like molecules.

Structural principles of peptide-centric Chimeric Antigen Receptor recognition guide therapeutic expansion.

Peptide-Centric Chimeric Antigen Receptors (PC-CARs), which recognize oncoprotein epitopes displayed by human leukocyte antigens (HLAs) on the cell surface, offer a promising strategy for targeted cancer therapy 1 . We have previously developed a PC-CAR targeting a neuroblastoma- associated PHOX2B peptide, leading to robust tumor cell lysis restricted by two common HLA allotypes 2 . Here, we determine the 2.1 Å structure of the PC-CAR:PHOX2B/HLA-A*24:02/ß2m complex, which reveals the basis for antigen-specific recognition through interactions with CAR complementarity-determining regions (CDRs). The PC-CAR adopts a diagonal docking mode, where interactions with both conserved and polymorphic HLA framework residues permit recognition of multiple HLA allotypes from the A9 serological cross-reactivity group, covering a combined American population frequency of up to 25.2%. Comprehensive characterization using biochemical binding assays, molecular dynamics simulations, and structural and functional analyses demonstrate that high-affinity PC-CAR recognition of cross-reactive pHLAs necessitates the presentation of a specific peptide backbone, where subtle structural adaptations of the peptide are critical for high-affinity complex formation and CAR-T cell killing. Our results provide a molecular blueprint for engineering CARs with optimal recognition of tumor-associated antigens in the context of different HLAs, while minimizing cross-reactivity with self-epitopes.

Xeno interactions between MHC-I proteins and molecular chaperones enable ligand exchange on a broad repertoire of HLA allotypes.

Immunological chaperones tapasin and TAP binding protein, related (TAPBPR) play key roles in antigenic peptide optimization and quality control of nascent class I major histocompatibility complex (MHC-I) molecules. The polymorphic nature of MHC-I proteins leads to a range of allelic dependencies on chaperones for assembly and cell-surface expression, limiting chaperone-mediated peptide exchange to a restricted set of human leukocyte antigen (HLA) allotypes. Here, we demonstrate and characterize xeno interactions between a chicken TAPBPR ortholog and a complementary repertoire of HLA allotypes, relative to its human counterpart. We find that TAPBPR orthologs recognize empty MHC-I with broader allele specificity and facilitate peptide exchange by maintaining a reservoir of receptive molecules. Deep mutational scanning of human TAPBPR further identifies gain-of-function mutants, resembling the chicken sequence, which can enhance HLA-A*01:01 expression in situ and promote peptide exchange in vitro. These results highlight that polymorphic sites on MHC-I and chaperone surfaces can be engineered to manipulate their interactions, enabling chaperone-mediated peptide exchange on disease-relevant HLA alleles.

Decoupling peptide binding from T cell receptor recognition with engineered chimeric MHC-I molecules.

Major Histocompatibility Complex class I (MHC-I) molecules display self, viral or aberrant epitopic peptides to T cell receptors (TCRs), which employ interactions between complementarity-determining regions with both peptide and MHC-I heavy chain 'framework' residues to recognize specific Human Leucocyte Antigens (HLAs). The highly polymorphic nature of the HLA peptide-binding groove suggests a malleability of interactions within a common structural scaffold. Here, using structural data from peptide:MHC-I and pMHC:TCR structures, we first identify residues important for peptide and/or TCR binding. We then outline a fixed-backbone computational design approach for engineering synthetic molecules that combine peptide binding and TCR recognition surfaces from existing HLA allotypes. X-ray crystallography demonstrates that chimeric molecules bridging divergent HLA alleles can bind selected peptide antigens in a specified backbone conformation. Finally, in vitro tetramer staining and biophysical binding experiments using chimeric pMHC-I molecules presenting established antigens further demonstrate the requirement of TCR recognition on interactions with HLA framework residues, as opposed to interactions with peptide-centric Chimeric Antigen Receptors (CARs). Our results underscore a novel, structure-guided platform for developing synthetic HLA molecules with desired properties as screening probes for peptide-centric interactions with TCRs and other therapeutic modalities.

TAPBPR employs a ligand-independent docking mechanism to chaperone MR1 molecules.

Chaperones tapasin and transporter associated with antigen processing (TAP)-binding protein related (TAPBPR) associate with the major histocompatibility complex (MHC)-related protein 1 (MR1) to promote trafficking and cell surface expression. However, the binding mechanism and ligand dependency of MR1/chaperone interactions remain incompletely characterized. Here in vitro, biochemical and computational studies reveal that, unlike MHC-I, TAPBPR recognizes MR1 in a ligand-independent manner owing to the absence of major structural changes in the MR1 α2-1 helix between empty and ligand-loaded molecules. Structural characterization using paramagnetic nuclear magnetic resonance experiments combined with restrained molecular dynamics simulations reveals that TAPBPR engages conserved surfaces on MR1 to induce similar adaptations to those seen in MHC-I/TAPBPR co-crystal structures. Finally, nuclear magnetic resonance relaxation dispersion experiments using 19F-labeled diclofenac show that TAPBPR can affect the exchange kinetics of noncovalent metabolites with the MR1 groove, serving as a catalyst. Our results support a role of chaperones in stabilizing nascent MR1 molecules to enable loading of endogenous or exogenous cargo.

Device use errors among patients with asthma and COPD and the role of training: a real-life study.

Background and objectives: Administration of inhaled medication for asthma and COPD is often difficult and incorrect device use is associated with unfavorable outcomes. We aimed to evaluate device use errors in asthma and COPD patients and to associate incorrect use with the patient's characteristics and medical history.Methods: Demographics and medical history were recorded. The use of each prescribed device was evaluated according to predefined steps.Results: 607 patients (49.9% male, median age (IQR) 63 (51, 70) years performed 663 demonstrations (56 patients were using 2 different types of devices). 51.4% were treated for asthma and 48.6% for COPD. 79.6% of demonstrations were performed using DPIs. Errors were documented on 41.2% of demonstrations and were associated with the type of device, p < 0.001. Elderly patients were less frequently using their devices correctly compared to younger patients, 50.8% vs 62.2%, respectively, p = 0.007. Correct demonstrations were more among asthmatics compared to COPD patients 63.1% vs 54.5%, p = 0.024. Incorrect use was associated with more acute exacerbations in the preceding year [median(IQR), 1(0, 2) vs 1(0, 1)], for incorrect and correct use, respectively, p < 0.001. Upon demonstration, 15.5% of patients have never been trained (i.e., undergone actual demonstrations and observation while using their device) by anyone. Errors occurred more frequently among patients who reported not to be trained compared to those who were trained, 67.0% vs 14.6%, respectively, p < 0.001. The commonest error was associated with the inspiration maneuver and accounted for the 48.3% of errors in the DPIs and 53.0% of errors in the MDIs.Conclusion: Device use errors are common and associated with unfavorable outcomes. Trained patients were more likely to use the device correctly.

Prevalence, distribution and clinical significance of joints, muscles and bones in sarcoidosis: an 18F-FDG-PET/CT study.

OBJECTIVES: In Sarcoidosis joints-muscles-bones (JMBs) localizations are of the least common. 18F-FDG-PET/CT imaging revolutionized detection of JMBs involvement by adding metabolic activity information and allowing for a comprehensive, whole-body mapping of the disease. AIM AND METHODS: This study investigated prevalence, distribution, and clinical significance of JMBs sarcoidosis in 195 consecutive patients that underwent 18F-FDG PET/CT examination. RESULTS: Joint and bone involvement were encountered in 15% of patients with a mean of the maximum-standardized-uptake-value (SUVmax) of 6.1. Most common location was the axial skeleton. Hypercalciuria was significantly more frequent in patients with osseous involvement (p = 0.003). Muscle activity (SUVmax = 2.4) was encountered in 20% of the patients, most frequently in treatment-naïve (p = 0.02). The muscles of the lower extremities were affected the most. Muscle and bone localization coexist in 50% of the cases. JMBs disease was almost asymptomatic, not related to chronicity but to pulmonary, nodal, and systemic disease. Long-term follow-up and treatment response of affected patients confirmed sarcoidosis. CONCLUSION: 18F-FDG-PET/CT revealed JMBs localizations and coexistence with other organ sites supporting the concept that sarcoidosis is a systemic disease. By allowing an integrative interpretation of multi-organ involvement in the context of a pattern highly suggestive of sarcoidosis, it strongly keeps-off the diagnosis of malignancy.

Mycophenolate mofetil as an alternative treatment in sarcoidosis.

INTRODUCTION: In sarcoidosis although no better drug therapy than corticosteroids (CS) has emerged, alternative immunosuppressive agents are used when indicated. Mycophenolate mofetil (MMF) presents rapid action, a considerable safety profile and absence of lung toxicity. Few data exist so far on its use in patients with sarcoidosis. This is a retrospective study on the effectiveness and safety of MMF in patients with sarcoidosis. MATERIALS AND METHODS: All patients with biopsy proven sarcoidosis treated for at least 1 year with MMF from 2008 to 2017 in our department are evaluated. RESULTS: Eight patients with both pulmonary and extrapulmonary disease are included in the analysis. During follow-up, symptoms and chest radiological findings improved in all. A statistically significant improvement of FEV1 and FVC is reported (p = 0.010 and p = 0.021 respectively). Cardiac and renal disease resolved during treatment while dermal disease significantly improved. MMF permitted CS dose reduction from 15.0 (10.0, 35.0) to 2.5 (0.0, 5.0) mg prednisolone (or equivalent), p = 0.016. All patients but one, tolerated well MMF. CONCLUSION: MMF as an alternative drug in systemic sarcoidosis, proved safe and effective, permitting the reduction of the dose of oral CS and leading to clinical, functional and radiological improvement.

Myelodysplastic syndromes and idiopathic pulmonary fibrosis: a dangerous liaison.

Previous studies have shown that the co-existence of bone marrow failure and pulmonary fibrosis in a single patient or in a family is suggestive of telomere related genes (TRG) germline mutations. This study presents the genetic background, clinical characteristics, and outcome of a group of five Greek patients co-affected with IPF and MDS. Four out of five patients developed an IPF acute exacerbation that was not reversible. We failed to detect any mutation in the TERT, TERC, DKC1, TINF2, RTEL1, PARN, NAF1, ACD, NHP2 and NOP10 genes in any patient. Moreover, telomere length was normal in the two patients tested. This could suggest that although the co-occurence of IPF and MDS are suggestive of TRG mutation in patients < 65 years old, in the elderly it may occur without germline mutations and could negatively affect prognosis. Physicians should be aware for possible IPF deterioration and therapeutic options for MDS should be wisely considered.

Cytosolic N- and C-Termini of the Aspergillus nidulans FurE Transporter Contain Distinct Elements that Regulate by Long-Range Effects Function and Specificity.

FurE, a member of the NCS1 family, is an Aspergillus nidulans transporter specific for uracil, allantoin and uric acid. Recently, we showed that C- or N-terminally truncated FurE versions are blocked for endocytosis and surprisingly show modified substrate specificities. Bifluorescence complementation assays and genetic analyses supported the idea that C- and N-termini interact dynamically and through this interaction regulate selective substrate translocation. Here we functionally dissect and define distinct motifs crucial for endocytosis, transport activity, substrate specificity and folding, in both cytosolic termini of FurE. Subsequently, we obtain novel genetic and in silico evidence indicating that the molecular dynamics of specific N- and C-terminal regions exert long-range effects on the gating mechanism responsible for substrate selection, via pH-dependent interactions with other internal cytosolic loops and membrane lipids. Our work shows that expanded cytoplasmic termini, acquired through evolution mostly in eukaryotic transporters, provide novel specific functional roles.

Structure-activity relationships in fungal nucleobases transporters as dissected by the inhibitory effects of novel purine analogues.

We have previously rationally designed, synthesized and tested a number of 3-deazapurine analogues, which inhibit the ubiquitous fungal nucleobase transporter FcyB, through binding in its major substrate binding site, by specifically interacting with Asn163. Here, in an effort to further understand the molecular details of structure-activity relationships in all three major nucleobase transporters of fungi, we extend this study by designing, based on our previous experience, synthesizing and testing further 3-deazapurine analogues. We thus identify seven new compounds with relatively high affinity (19-106 µΜ) for the FcyB binding site. Importantly, four of these compounds can also efficiently inhibit AzgA, a structurally and evolutionary distinct, but functionally similar, purine transporter. Contrastingly, none of the new compounds tested had any effect on the transport activity of the uric acid-xanthine transporter UapA, albeit this being a structural homologue of AzgA. Besides the apparent importance for understanding how nucleobase transporter specificity is determined at the molecular level, our work might constitute a critical step in the design of novel purine-related antifungals.

Substrate Specificity of the FurE Transporter Is Determined by Cytoplasmic Terminal Domain Interactions.

FurE, a member of the Nucleobase Cation Symporter 1 transporter family in Aspergillus nidulans, is specific for allantoin, uric acid (UA), uracil, and related analogs. Herein, we show that C- or N-terminally-truncated FurE transporters (FurE-ΔC or FurE-ΔΝ) present increased protein stability, but also an inability for UA transport. To better understand the role of cytoplasmic terminal regions, we characterized genetic suppressors that restore FurE-ΔC-mediated UA transport. Suppressors map in the periphery of the substrate-binding site [Thr133 in transmembrane segment (TMS)3 and Val343 in TMS8], an outward-facing gate (Ser296 in TMS7, Ile371 in TMS9, and Tyr392 and Leu394 in TMS10), or in flexible loops (Asp26 in LN, Gly222 in L5, and Asn308 in L7). Selected suppressors were also shown to restore the wild-type specificity of FurE-ΔΝ, suggesting that both C- and/or N-terminal domains are involved in intramolecular dynamics critical for substrate selection. A direct, substrate-sensitive interaction of C- and/or N-terminal domains was supported by bimolecular fluorescence complementation assays. To our knowledge, this is the first case where not only the function, but also the specificity, of a eukaryotic transporter is regulated by its terminal cytoplasmic regions.

Combined pulmonary fibrosis and emphysema: The many aspects of a cohabitation contract.

Combined pulmonary fibrosis and emphysema (CPFE) is a clinical entity characterized by the coexistence of upper lobe emphysema and lower lobe fibrosis. Patients with this condition experience severe dyspnea and impaired gas exchange with preserved lung volumes. The diagnosis of the CPFE syndrome is based on HRCT imaging, showing the coexistence of emphysema and pulmonary fibrosis both in varying extent and locations within the lung parenchyma. Individual genetic background seem to predispose to the development of the disease. The risk of the development of pulmonary hypertension in patients with CPFE is high and related to poor prognosis. CPFE patients also present a high risk of lung cancer. Mortality is significant in patients with CPFE and median survival is reported between 2.1 and 8.5 years. Currently, no specific recommendations are available regarding the management of patients with CPFE. In this review we provide information on the existing knowledge on CPFE regarding the pathophysiology, clinical manifestations, imaging, complications, possible therapeutic interventions and prognosis of the disease.

Serum Levels of Surfactant Proteins in Patients with Combined Pulmonary Fibrosis and Emphysema (CPFE).

INTRODUCTION: Emphysema and idiopathic pulmonary fibrosis (IPF) present either per se or coexist in combined pulmonary fibrosis and emphysema (CPFE). Serum surfactant proteins (SPs) A, B, C and D levels may reflect lung damage. We evaluated serum SP levels in healthy controls, emphysema, IPF, and CPFE patients and their associations to disease severity and survival. METHODS: 122 consecutive patients (31 emphysema, 62 IPF, and 29 CPFE) and 25 healthy controls underwent PFTs, ABG-measurements, 6MWT and chest HRCT. Serum levels of SPs were measured. Patients were followed-up for 1-year. RESULTS: SP-A and SP-D levels differed between groups (p = 0.006 and p<0.001 respectively). In post-hoc analysis, SP-A levels differed only between controls and CPFE (p<0.05) and CPFE and emphysema (p<0.05). SP-D differed between controls and IPF or CPFE (p<0.001 for both comparisons). In IPF SP-B correlated to pulmonary function while SP-A, correlated to the Composite Physiological Index (CPI). Controls current smokers had higher SP-A and SP-D levels compared to non-smokers (p = 0.026 and p = 0.023 respectively). SP-D levels were higher in CPFE patients with extended emphysema (p = 0.042). In patients with IPF, SP-B levels at the upper quartile of its range (≥26 ng/mL) presented a weak association with reduced survival (p = 0.05). CONCLUSION: In conclusion, serum SP-A and SP-D levels were higher where fibrosis exists or coexists and related to disease severity, suggesting that serum SPs relate to alveolar damage in fibrotic lungs and may reflect either local overproduction or overleakage. The weak association between high levels of SP-B and survival needs further validation in clinical trials.

Surfactant Proteins in Smoking-Related Lung Disease.

Pulmonary surfactant is a highly surface-active mixture of proteins and lipids that is synthesized and secreted in the alveoli by type II epithelial cells and is found in the fluid lining the alveolar surface. The protein part of surfactant constitutes two hydrophilic proteins (SP-A and SP-D) that regulate surfactant metabolism and have immunologic functions, and two hydrophobic proteins (SP-B and SP-C), which play a direct role in the organization of the surfactant structure in the interphase and in the stabilization of the lipid layers during the respiratory cycle. Several studies have shown that cigarette smoke seems to affect, in several ways, both surfactant homeostasis and function. The alterations in surfactants' biophysical properties caused by cigarette smoking, contribute to the development of several smoking related lung diseases. In this review we provide information on biochemical and physiological aspects of the pulmonary surfactant and on its possible association with the development of two major chronic diseases of the lung known to be related to smoking, i.e. chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF). Additional information on the possible role of surfactant protein alterations and/or dysfunction in the combination of these two conditions, recently described as combined pulmonary fibrosis and emphysema (CPFE) are also provided.