Auto-regulation of Secretory Flux by Sensing and Responding to the Folded Cargo Protein Load in the Endoplasmic Reticulum.

Maintaining the optimal performance of cell processes and organelles is the task of auto-regulatory systems. Here we describe an auto-regulatory device that helps to maintain homeostasis of the endoplasmic reticulum (ER) by adjusting the secretory flux to the cargo load. The cargo-recruiting subunit of the coatomer protein II (COPII) coat, Sec24, doubles as a sensor of folded cargo and, upon cargo binding, acts as a guanine nucleotide exchange factor to activate the signaling protein Gα12 at the ER exit sites (ERESs). This step, in turn, activates a complex signaling network that activates and coordinates the ER export machinery and attenuates proteins synthesis, thus preventing large fluctuations of folded and potentially active cargo that could be harmful to the cell or the organism. We call this mechanism AREX (autoregulation of ER export) and expect that its identification will aid our understanding of human physiology and diseases that develop from secretory dysfunction.

Authenticity of probiotic foods and dietary supplements: A pivotal issue to address.

The market of probiotic foods and supplements is growing rapidly but frequently the commercialized products are not compliant with their labels in terms of claimed probiotic strain(s) and labeled number of viable probiotic cells, thus mining the authenticity of these probiotic products.In this review, we provide an up-to-date overview of: (i) the current regulatory aspects, (ii) the consistency of probiotic foods and supplements with their labels, (iii) the implications of mislabeling on the quality, safety and functionality of these products and (iv) the available and most promising methods to assess the authenticity of these products, taking into account the need to discriminate among the different physiological states probiotics might be in the carrier matrices. It arises that authenticity of probiotic foods and supplements is an urgent issue, of industrial and legislation relevance, that need to be addressed. A plethora of methods are available to reach this goal, each with its own advantages and disadvantages. Protocols that combine the use of propidium monoazide (PMA) with metagenomics or polyphasic approaches including the PMA real time PCR or flow cytometry (for the viability assessment) and the whole genome sequence analysis (for the identification and typing of the probiotic strain) are the most promising that should be standardized and used by producers and regulators.

webPSN v2.0: a webserver to infer fingerprints of structural communication in biomacromolecules.

A mixed Protein Structure Network (PSN) and Elastic Network Model-Normal Mode Analysis (ENM-NMA)-based strategy (i.e. PSN-ENM) was developed to investigate structural communication in bio-macromolecules. Protein Structure Graphs (PSGs) are computed on a single structure, whereas information on system dynamics is supplied by ENM-NMA. The approach was implemented in a webserver (webPSN), which was significantly updated herein. The webserver now handles both proteins and nucleic acids and relies on an internal upgradable database of network parameters for ions and small molecules in all PDB structures. Apart from the radical restyle of the server and some changes in the calculation setup, other major novelties concern the possibility to: a) compute the differences in nodes, links, and communication pathways between two structures (i.e. network difference) and b) infer links, hubs, communities, and metapaths from consensus networks computed on a number of structures. These new features are useful to identify commonalties and differences between two different functional states of the same system or structural-communication signatures in homologous or analogous systems. The output analysis relies on 3D-representations, interactive tables and graphs, also available for download. Speed and accuracy make this server suitable to comparatively investigate structural communication in large sets of bio-macromolecular systems. URL: http://webpsn.hpc.unimore.it.

Induced Pluripotent Stem Cell (iPSC) Lines from a Family with Resistant Epileptic Encephalopathy Caused by Compound Heterozygous Mutations in SZT2 Gene.

Mutations in the SZT2 gene have been associated with developmental and epileptic encephalopathy-18, a rare severe autosomal recessive neurologic disorder, characterized by psychomotor impairment/intellectual disability, dysmorphic facial features and early onset of refractory seizures. Here we report the generation of the first induced pluripotent stem cell (iPSC) lines from a patient with treatment-resistant epilepsy, carrying compound heterozygous mutations in SZT2 (Mut1: c.498G>T and Mut2: c.6553C>T), and his healthy heterozygous parents. Peripheral blood mononuclear cells were reprogrammed by a non-integrating Sendai virus-based reprogramming system. The generated human iPSC lines exhibited expression of the main pluripotency markers, the potential to differentiate into all three germ layers and presented a normal karyotype. These lines represent a valuable resource to study neurodevelopmental alterations, and to obtain mature, pathology-relevant neuronal populations as an in vitro model to perform functional assays and test the patient's responsiveness to novel antiepileptic treatments.

Structural aspects of rod opsin and their implication in genetic diseases.

Vision in dim-light conditions is triggered by photoactivation of rhodopsin, the visual pigment of rod photoreceptor cells. Rhodopsin is made of a protein, the G protein coupled receptor (GPCR) opsin, and the chromophore 11-cis-retinal. Vertebrate rod opsin is the GPCR best characterized at the atomic level of detail. Since the release of the first crystal structure 20 years ago, a huge number of structures have been released that, in combination with valuable spectroscopic determinations, unveiled most aspects of the photobleaching process. A number of spontaneous mutations of rod opsin have been found linked to vision-impairing diseases like autosomal dominant or autosomal recessive retinitis pigmentosa (adRP or arRP, respectively) and autosomal congenital stationary night blindness (adCSNB). While adCSNB is mainly caused by constitutive activation of rod opsin, RP shows more variegate determinants affecting different aspects of rod opsin function. The vast majority of missense rod opsin mutations affects folding and trafficking and is linked to adRP, an incurable disease that awaits light on its molecular structure determinants. This review article summarizes all major structural information available on vertebrate rod opsin conformational states and the insights gained so far into the structural determinants of adCSNB and adRP linked to rod opsin mutations. Strategies to design small chaperones with therapeutic potential for selected adRP rod opsin mutants will be discussed as well.

Defining type 2 asthma and patients eligible for dupilumab in Italy: a biomarker-based analysis.

BACKGROUND: Asthma is a chronic disease characterized by airway hyperresponsiveness, inflammation and mucus production. In Type 2 asthma, two phenotypic components are often co-expressed (eosinophilic and allergic). Elevated biomarker levels, such as eosinophils (EOS), fraction of exhaled nitric oxide (FeNO) and immunoglobulin E (IgE), are key clinical indicators of Type 2 inflammation. Dupilumab has been recently approved for the treatment of uncontrolled severe Type 2 asthma. Type 2 asthma includes allergic and/or eosinophilic phenotypes. The aim of this analysis was to estimate the dupilumab-eligible population in Italy and characterize it by expected biomarker status. METHODS: A 4-step approach was carried out to calculate dupilumab-eligible population. The approach consisted in: (1) estimating the total number of asthma patients in Italy (using 2016-2017 Italian-adapted Global Initiative for Asthma -GINA- guidelines); (2) estimating the number of severe asthma patients with poorly controlled or uncontrolled disease (using the findings of two recent administrative claim analyses conducted in Italy); (3) stratifying the severe uncontrolled population by biomarker levels (EOS, FeNO and IgE) according to the outcomes of the QUEST trial (a clinical study assessing the efficacy of dupilumab in patients with uncontrolled moderate-to-severe asthma; NCT02414854); (4) identifying the sub-populations of severe uncontrolled asthma patients characterised by raised blood EOS and/or FeNO level (thus indicated to receive dupilumab). RESULTS: According to these estimates, about 3.3 million asthmatic patients live in Italy (6.10% of the population). Of them, almost 20 thousand (N = 19,960) have uncontrolled severe asthma. Dupilumab-eligible patients would be N = 15,988, corresponding to 80.1% of the total uncontrolled severe population. Most of these patients (89.3%; N = 14,271) have at least an increase of EOS level, while slightly more than half (51.9%; N = 8,303) have raised levels of both biomarkers. Increased FeNO levels without increased EOS are observed less frequently (N = 1,717; 10.7% of the eligible population). CONCLUSIONS: There is a strong rationale for testing all asthma biomarkers during diagnosis and disease follow-up. Given the large availability and the limited costs, these tests are cost-effective tools to detect severe Type 2 asthma, stratify patients by phenotype, and drive appropriate treatment decisions.

Using 2nd generation basal insulins in type 2 diabetes: Costs and savings in a comparative economic analysis in Italy, based on the BRIGHT study.

BACKGROUND AND AIMS: To evaluate the economic impact of using 2nd generation basal insulin analogs, Glargine 300 Units/ml (Gla-300) vs Degludec 100 Units/ml (IDeg-100), in patients with type 2 diabetes (T2D). METHODS AND RESULTS: An economic analysis was conducted using findings from the BRIGHT study (the first controlled, head-to-head study comparing Gla-300 vs IDeg-100), and costs for the Italian National Healthcare Service (NHS). A cost-minimization analysis (CMA) and a budget impact analysis (BIA) were conducted. Only pharmacological costs were included in the analysis. The CMA estimated patient treatment costs at 24 weeks and 1 year; the BIA assessed the economic impact of treating the overall Italian population of T2D insulin-naïve patients, who initiated insulin treatment during the period September 2017-August 2018 (N = 55 318). In the BIA, four different scenarios were compared: i) all patients receive IDeg-100 (Scenario A); ii) 61% of patients receive Gla-300, 39% IDeg-100 (Scenario B); iii) 80% of patients receive Gla-300, 20% IDeg-100 (Scenario C); iv) all patients treated with Gla-300 (Scenario D). The average treatment costs per patient were lower with Gla-300 vs IDeg-100 (at 24 weeks: €129 vs €161; at 1 year: €324 vs €409, respectively). Results of the BIA showed that comparing Scenario D vs Scenario A, total savings would amount to €1.76 million at 24 weeks, €4.73 million at 1 year, €5.53 million at 2 years. CONCLUSION: A larger use of Gla-300 vs IDeg-100 for the treatment of T2D patients would lead to a relevant reduction of therapy costs in Italy.

Phenotype and genomic background of Arcobacter butzleri strains and taxogenomic assessment of the species.

In this study the phenotypic and genomic characterization of two Arcobacter butzleri (Ab) strains (Ab 34_O and Ab 39_O) isolated from pre-cut ready-to-eat vegetables were performed. Results provided useful data about their taxonomy and their overall virulence potential with particular reference to the antibiotic and heavy metal susceptibility. These features were moreover compared with those of two Ab strains isolated from shellfish and a genotaxonomic assessment of the Ab species was performed. The two Ab isolated from vegetables were confirmed to belong to the Aliarcobacter butzleri species by 16S rRNA gene sequence analysis, MLST and genomic analyses. The genome-based taxonomic assessment of the Ab species brought to the light the possibility to define different subspecies reflecting the source of isolation, even though further genomes from different sources should be available to support this hypothesis. The strains isolated from vegetables in the same geographic area shared the same distribution of COGs with a prevalence of the cluster "inorganic ion transport and metabolism", consistent with the lithotrophic nature of Arcobacter spp. None of the Ab strains (from shellfish and from vegetables) metabolized carbohydrates but utilized organic acids and amino acids as carbon sources. The metabolic fingerprinting of Ab resulted less discriminatory than the genome-based approach. The Ab strains isolated from vegetables and those isolated from shellfish endowed multiple resistance to several antibiotics and heavy metals.

Novel insights into the enterotoxigenic potential and genomic background of Staphylococcus aureus isolated from raw milk.

In this study, 53 Staphylococcus (S.) aureus strains were typed by 16S-23S rDNA intergenic spacer region (ISR) typing and staphylococcal enterotoxin gene (SEg) typing for all the staphylococcal enterotoxin (se) and staphylococcal enterotoxin-like toxin (sel) genes known to date, revealing a higher discriminatory power than that of multi locus sequence typing. Six strains, one of each ISR- and SEg-type, were genome sequenced and the ability to produce some classical and new SEs when growing in milk was investigated. The manual analysis of the six genomes allowed us to confirm, correct and expand the results of common available genomic data pipelines such as VirulenceFinder. Moreover, it enabled us to (i) investigate the actual location of se and sel genes, even for genes such as selY, whose location (in the core genome) was so far unknown, (ii) find novel allelic variants of se and sel genes and pseudogenes, (iii) correctly annotate se and sel genes and pseudogenes, and (iv) discover a novel type of enterotoxin gene cluster (egc), i.e. the egc type 5 in strains 356P and 364P, while S. argenteus MSHR1132 harbored the egc type 6. Four of the six S. aureus strains produced sufficient amounts of SEA, SEC, SED and SEH in milk to cause staphylococcal food poisoning (SFP), with S. aureus 372 P being the highest producer of SED in milk found to date, producing as much as ca. 47,300 ng/mL and 49,200 ng/mL of SED, after 24 and 48 h of incubation in milk at 37 °C, respectively. S. aureus 372 P released a low amount of SER in milk, most likely because the seR gene was present as a pseudogene, putatively encoding only 51 amino acids. These findings confirm that not only the classical SEs, but also the new ones can represent a potential hazard for the consumers' health if produced in foods in sufficient amounts. Therefore, the detection of SEs in foods, especially if involved in SFP cases, should focus not only on classical, but also on all the new SEs and SEls known to date. Where reference methods are unavailable, the presence of the relevant genes, by using the conventional and real time PCR protocols we exhaustively provided herein, and their nucleotide sequences, should be investigated.

Arcobacter butzleri: Up-to-date taxonomy, ecology, and pathogenicity of an emerging pathogen.

Arcobacter butzleri, recently emended to the Aliarcobacter butzleri comb. nov., is an emerging pathogen causing enteritis, severe diarrhea, septicaemia, and bacteraemia in humans and enteritis, stillbirth, and abortion in animals. Since its recognition as emerging pathogen on 2002, advancements have been made in elucidating its pathogenicity and epidemiology, also thanks to advent of genomics, which, moreover, contributed in emending its taxonomy. In this review, we provide an overview of the up-to-date taxonomy, ecology, and pathogenicity of this emerging pathogen. Moreover, the implication of A. butzleri in the safety of foods is pinpointed, and culture-dependent and independent detection, identification, and typing methods as well as strategies to control and prevent the survival and growth of this pathogen are provided.

Microbial quality and safety of milk and milk products in the 21st century.

Milk and milk products have been utilized by humans for many thousands of years. With the advent of metagenomic studies, our knowledge on the microbiota of milk and milk products, especially as affected by the environment, production, and storage parameters, has increased. Milk quality depends on chemical parameters (fat and protein content and absence of inhibitory substances), as well as microbial and somatic cells counts, and affects the price of milk. The effects of hygiene and effective cooling on the spoilage microbiota have shown that proteolytic and lipolytic bacteria such as Pseudomonas or Acinetobacter spp. predominate the spoilage bacterial populations. These bacteria can produce heat-stable proteases and lipases, which remain active after pasteurization and thus can spoil the milk during prolonged storage. Additionally, milk can become contaminated after pasteurization and therefore there is still a high demand on developing better cleaning and sanitation regimes and equipment, as well as test systems to (quantitatively) detect relevant pathogenic or spoilage microorganisms. Raw milk and raw milk cheese consumption is also increasing worldwide with the growing demand of minimally processed, sustainable, healthy, and local foods. In this context, emerging and re-emerging pathogens once again represent a major food safety challenge. As a result of global warming, it is conceivable that not only microbiological risks but also chemical risks relating to presence of mycotoxins or plant toxins in milk will increase. Herein, we provide an overview of the major microbial hazards occurring in the 21st century.

Interconnecting Flexibility, Structural Communication, and Function in RhoGEF Oncoproteins.

Dbl family Rho guanine nucleotide exchange factors (RhoGEFs) play a central role in cell biology by catalyzing the exchange of guanosine 5'-triphosphate for guanosine 5'-diphosphate (GDP) on RhoA. Insights into the oncogenic constitutive activity of the Lbc RhoGEF were gained by analyzing the structure and dynamics of the protein in different functional states and in comparison with a close homologue, leukemia-associated RhoGEF. Higher intrinsic flexibility, less dense and extended structure network, and less stable allosteric communication pathways in Lbc, compared to the nonconstitutively active homologue, emerged as major determinants of the constitutive activity. Independent of the state, the essential dynamics of the two RhoGEFs is contributed by the last 10 amino acids of Dbl homology (DH) and the whole pleckstrin homology (PH) domains and tends to be equalized by the presence of RhoA. The catalytic activity of the RhoGEF relies on the scaffolding action of the DH domain that primarily turns the switch I (SWI) of RhoA on itself through highly conserved amino acids participating in the stability core and essential for function. Changes in the conformation of SWI and disorganization of the RhoA regions deputed to nucleotide binding are among the major RhoGEF effects leading to GDP release. Binding of RhoA reorganizes the allosteric communication on RhoGEF, strengthening the communication among the canonical RhoA binding site on DH, a secondary RhoA binding site on PH, and the binding site for heterotrimeric G proteins, suggesting dual roles for RhoA as a catalysis substrate and as a regulatory protein. The structure network-based analysis tool employed in this study proved to be useful for predicting potentially druggable regulatory sites in protein structures.

Proteomic analysis of the food spoiler Pseudomonas fluorescens ITEM 17298 reveals the antibiofilm activity of the pepsin-digested bovine lactoferrin.

Pseudomonas fluorescens is implicated in food spoilage especially under cold storage. Due to its ability to form biofilm P. fluorescens resists to common disinfection strategies increasing its persistance especially across fresh food chain. Biofilm formation is promoted by several environmental stimuli, but gene expression and protein changes involved in this lifestyle are poorly investigated in this species. In this work a comparative proteomic analysis was performed to investigate metabolic pathways of underlying biofilm formation of the blue cheese pigmenting P. fluorescens ITEM 17298 after incubation at 15 and 30 °C; the same methodology was also applied to reveal the effects of the bovine lactoferrin hydrolysate (HLF) used as antibiofilm agent. At 15 °C biofilm biomass and motility increased, putatively sustained by the induction of regulators (PleD, AlgB, CsrA/RsmA) involved in these phenotypic traits. In addition, for the first time, TycC and GbrS, correlated to indigoidine synthesis (blue pigment), were detected and identified. An increase of virulence factors amounts (leukotoxin and PROKKA_04561) were instead found at 30 °C. HLF caused a significant reduction in biofilm biomass; indeed, at 15 °C HLF repressed PleD, TycC and GbrS and induced the negative regulators of alginate biosynthesis; at both temperatures induced the cyclic-di-GMP-binding biofilm dispersal mediator (PROKKA_02061). In conclusion, in this work protein determinats of biofilm formation were revelead in ITEM 17298 under the low temperature; the synthesis of these latter were inhibited by HLF confirming its possible exploitation as antibiofilm agent for biotechnological applications in cold stored foods.

Genomic characterization of Trichoderma atrobrunneum (T. harzianum species complex) ITEM 908: insight into the genetic endowment of a multi-target biocontrol strain.

BACKGROUND: So far, biocontrol agent selection has been performed mainly by time consuming in vitro confrontation tests followed by extensive trials in greenhouse and field. An alternative approach is offered by application of high-throughput techniques, which allow extensive screening and comparison among strains for desired genetic traits. In the genus Trichoderma, the past assignments of particular features or strains to one species need to be reconsidered according to the recent taxonomic revisions. Here we present the genome of a biocontrol strain formerly known as Trichoderma harzianum ITEM 908, which exhibits both growth promoting capabilities and antagonism against different fungal pathogens, including Fusarium graminearum, Rhizoctonia solani, and the root-knot nematode Meloidogyne incognita. By genomic analysis of ITEM 908 we investigated the occurrence and the relevance of genes associated to biocontrol and stress tolerance, providing a basis for future investigation aiming to unravel the complex relationships between genomic endowment and exhibited activities of this strain. RESULTS: The MLST analysis of ITS-TEF1 concatenated datasets reclassified ITEM 908 as T. atrobrunneum, a species recently described within the T. harzianum species complex and phylogenetically close to T. afroharzianum and T. guizhouense. Genomic analysis revealed the presence of a broad range of genes encoding for carbohydrate active enzymes (CAZYmes), proteins involved in secondary metabolites production, peptaboils, epidithiodioxopiperazines and siderophores potentially involved in parasitism, saprophytic degradation as well as in biocontrol and antagonistic activities. This abundance is comparable to other Trichoderma spp. in the T. harzianum species complex, but broader than in other biocontrol species and in the species T. reesei, known for its industrial application in cellulase production. Comparative analysis also demonstrated similar genomic organization of major secondary metabolites clusters, as in other Trichoderma species. CONCLUSIONS: Reported data provide a contribution to a deeper understanding of the mode of action and identification of activity-specific genetic markers useful for selection and improvement of biocontrol strains. This work will also enlarge the availability of genomic data to perform comparative studies with the aim to correlate phenotypic differences with genetic diversity of Trichoderma species.

Gut Dysbiosis and Muscle Aging: Searching for Novel Targets against Sarcopenia.

Advanced age is characterized by several changes, one of which is the impairment of the homeostasis of intestinal microbiota. These alterations critically influence host health and have been associated with morbidity and mortality in older adults. "Inflammaging," an age-related chronic inflammatory process, is a common trait of several conditions, including sarcopenia. Interestingly, imbalanced intestinal microbial community has been suggested to contribute to inflammaging. Changes in gut microbiota accompanying sarcopenia may be attenuated by supplementation with pre- and probiotics. Although muscle aging has been increasingly recognized as a biomarker of aging, the pathophysiology of sarcopenia is to date only partially appreciated. Due to its development in the context of the age-related inflammatory milieu, several studies favor the hypothesis of a tight connection between sarcopenia and inflammaging. However, conclusive evidence describing the signaling pathways involved has not yet been produced. Here, we review the current knowledge of the changes in intestinal microbiota that occur in advanced age with a special emphasis on findings supporting the idea of a modulation of muscle physiology through alterations in gut microbial composition and activity.

Dissecting intrinsic and ligand-induced structural communication in the ß3 headpiece of integrins.

BACKGROUND: Graph theory is widely used to dissect structural communication in biomolecular systems. Here, graph theory-based approaches were applied to the headpiece of integrins, adhesion cell-surface receptors that transmit signals across the plasma membranes. METHODS: Protein Structure Network (PSN) analysis incorporating dynamic information either from molecular dynamics simulations or from Elastic Network Models was applied to the ß3 domains from integrins αVß3 and αIIbß3 in their apo and ligand-bound states. RESULTS: Closed and open states of the ß headpiece are characterized by distinct allosteric communication pathways involving highly conserved amino acids at the two different α/ß interfaces in the ßI domain, the closed state being prompted to the closed-to-open transition. In the closed state, pure antagonism is associated with the establishment of communication pathways that start from the ligand, pass through the ß1/α3,α4 interface, and end up in the hybrid domain by involving the Y110-Q82 link, which is weakened in the agonist-bound states. CONCLUSIONS: Allosteric communication in integrins relies on highly conserved and functionally relevant amino acid residues. The αßα-sandwich architecture of integrin ßI domain dictates the structural communication between ligand binding site and hybrid domain. Differently from agonists, pure antagonists are directly involved in allosteric communication pathways and exert long-distance strengthening of the ßI/hybrid interface. Release of the structure network in the ligand binding site is associated with the close-to-open transition accompanying the activation process. GENERAL SIGNIFICANCE: The study strengthens the power of graph-based analyses to decipher allosteric communication intrinsic to protein folds and modified by functionally different ligands.

Comparison of the isomerization mechanisms of human melanopsin and invertebrate and vertebrate rhodopsins.

Comparative modeling and ab initio multiconfigurational quantum chemistry are combined to investigate the reactivity of the human nonvisual photoreceptor melanopsin. It is found that both the thermal and photochemical isomerization of the melanopsin 11-cis retinal chromophore occur via a space-saving mechanism involving the unidirectional, counterclockwise twisting of the =C11H-C12H= moiety with respect to its Lys340-linked frame as proposed by Warshel for visual pigments [Warshel A (1976) Nature 260(5553):679-683]. A comparison with the mechanisms documented for vertebrate (bovine) and invertebrate (squid) visual photoreceptors shows that such a mechanism is not affected by the diversity of the three chromophore cavities. Despite such invariance, trajectory computations indicate that although all receptors display less than 100 fs excited state dynamics, human melanopsin decays from the excited state ∼40 fs earlier than bovine rhodopsin. Some diversity is also found in the energy barriers controlling thermal isomerization. Human melanopsin features the highest computed barrier which appears to be ∼2.5 kcal mol(-1) higher than that of bovine rhodopsin. When assuming the validity of both the reaction speed/quantum yield correlation discussed by Warshel, Mathies and coworkers [Weiss RM, Warshel A (1979) J Am Chem Soc 101:6131-6133; Schoenlein RW, Peteanu LA, Mathies RA, Shank CV (1991) Science 254(5030):412-415] and of a relationship between thermal isomerization rate and thermal activation of the photocycle, melanopsin turns out to be a highly sensitive pigment consistent with the low number of melanopsin-containing cells found in the retina and with the extraretina location of melanopsin in nonmammalian vertebrates.

Single molecule analysis of functionally asymmetric G protein-coupled receptor (GPCR) oligomers reveals diverse spatial and structural assemblies.

Formation of G protein-coupled receptors (GPCRs) into dimers and higher order oligomers represents a key mechanism in pleiotropic signaling, yet how individual protomers function within oligomers remains poorly understood. We present a super-resolution imaging approach, resolving single GPCR molecules to ∼ 8 nm resolution in functional asymmetric dimers and oligomers using dual-color photoactivatable dyes and localization microscopy (PD-PALM). PD-PALM of two functionally defined mutant luteinizing hormone receptors (LHRs), a ligand-binding deficient receptor (LHR(B-)) and a signaling-deficient (LHR(S-)) receptor, which only function via intermolecular cooperation, favored oligomeric over dimeric formation. PD-PALM imaging of trimers and tetramers revealed specific spatial organizations of individual protomers in complexes where the ratiometric composition of LHR(B-) to LHR(S-) modulated ligand-induced signal sensitivity. Structural modeling of asymmetric LHR oligomers strongly aligned with PD-PALM-imaged spatial arrangements, identifying multiple possible helix interfaces mediating inter-protomer associations. Our findings reveal that diverse spatial and structural assemblies mediating GPCR oligomerization may acutely fine-tune the cellular signaling profile.

The KDEL receptor couples to Gαq/11 to activate Src kinases and regulate transport through the Golgi.

Membrane trafficking involves large fluxes of cargo and membrane across separate compartments. These fluxes must be regulated by control systems to maintain homoeostasis. While control systems for other key functions such as protein folding or the cell cycle are well known, the mechanisms that control secretory transport are poorly understood. We have previously described a signalling circuit operating at the Golgi complex that regulates intra-Golgi trafficking and is initiated by the KDEL receptor (KDEL-R), a protein previously known to mediate protein recycling from the Golgi to the endoplasmic reticulum (ER). Here, we investigated the KDEL-R signalling mechanism. We show that the KDEL-R is predicted to fold like a G-protein-coupled receptor (GPCR), and that it binds and activates the heterotrimeric signalling G-protein Gα(q/11) which, in turn, regulates transport through the Golgi complex. These findings reveal an unexpected GPCR-like mode of action of the KDEL-R and shed light on a core molecular control mechanism of intra-Golgi traffic.

WebPSN: a web server for high-throughput investigation of structural communication in biomacromolecules.

UNLABELLED: We developed a mixed Protein Structure Network (PSN) and Elastic Network Model-Normal Mode Analysis (ENM-NMA)-based strategy (i.e. PSN-ENM) to investigate structural communication in biomacromolecules. The approach starts from a Protein Structure Graph and searches for all shortest communication pathways between user-specified residues. The graph is computed on a single preferably high-resolution structure. Information on system's dynamics is supplied by ENM-NMA. The PSN-ENM methodology is made of multiple steps both in the setup and analysis stages, which may discourage inexperienced users. To facilitate its usage, we implemented WebPSN, a freely available web server that allows the user to easily setup the calculation, perform post-processing analyses and both visualize and download numerical and 3D representations of the output. Speed and accuracy make this server suitable to investigate structural communication, including allosterism, in large sets of bio-macromolecular systems. AVAILABILITY AND IMPLEMENTATION: The WebPSN server is freely available at http://webpsn.hpc.unimore.it.