Structural analysis of an endogenous 4-megadalton succinyl-CoA-generating metabolon.

The oxoglutarate dehydrogenase complex (OGDHc) participates in the tricarboxylic acid cycle and, in a multi-step reaction, decarboxylates α-ketoglutarate, transfers succinyl to CoA, and reduces NAD+. Due to its pivotal role in metabolism, OGDHc enzymatic components have been studied in isolation; however, their interactions within the endogenous OGDHc remain elusive. Here, we discern the organization of a thermophilic, eukaryotic, native OGDHc in its active state. By combining biochemical, biophysical, and bioinformatic methods, we resolve its composition, 3D architecture, and molecular function at 3.35 Å resolution. We further report the high-resolution cryo-EM structure of the OGDHc core (E2o), which displays various structural adaptations. These include hydrogen bonding patterns confining interactions of OGDHc participating enzymes (E1o-E2o-E3), electrostatic tunneling that drives inter-subunit communication, and the presence of a flexible subunit (E3BPo), connecting E2o and E3. This multi-scale analysis of a succinyl-CoA-producing native cell extract provides a blueprint for structure-function studies of complex mixtures of medical and biotechnological value.

Functional Analysis of Phospholipid Signaling and Actin Dynamics: The Use of Apical Growing Tobacco Pollen Tubes in a Case Study.

Signaling molecules are crucial to perceive and translate intra- and extracellular cues. Phosphoinositides and the proteins responsible for their biosynthesis (e.g., lipid kinases) are known to influence the (re)organization of cytoskeletal elements, namely, through interaction with actin and actin-binding proteins. Here we describe methods to functionally characterize lipid kinases and their phosphoinositide metabolites in relation to actin dynamics. These methods include GFP-tagged protein expression followed by time-resolved live imaging and quantitative image analysis. When combined with biochemical and interaction studies, these methods can be used to correlate signaling with actin dynamics, microfilament assembly, and intracellular trafficking, linking structure and function.

The pollen-specific class VIII-myosin ATM2 from Arabidopsis thaliana associates with the plasma membrane through a polybasic region binding anionic phospholipids.

Pollen tubes display polarized tip-growth and are a model to study the coordination of vesicular trafficking and cytoskeletal control. The molecular details of how dynamic actin filaments associate with the plasma membrane are currently unclear. In Arabidopsis thaliana, plasma membrane attachment of actin filaments may be mediated by four myosins representing the plant-specific myosin-subclass VIII, which localize to the plasma membrane and display only minor motor-activity. Here we explore the mode of membrane attachment of the pollen-expressed class VIII-myosins ATM2 and VIII-B through interaction with anionic membrane phospholipids. A fluorescent mCherry-ATM2-fusion decorated plasma membrane-peripheral actin filaments when expressed in tobacco pollen tubes, consistent with a role of class VIII-myosins at the membrane-cytoskeleton interface. As recombinant proteins, class VIII-myosins are prone to aggregation and to proteolysis, creating a challenge for their biochemical characterization. We describe a purification scheme for guanidinium chloride (GdmCl)-denatured recombinant proteins, followed by a renaturation protocol to obtain pure, soluble protein fragments of ATM2 and VIII-B. The fragments represent the C-terminal tail and coiled-coil-regions and lack the N-terminal actin-binding regions, IQ or motor domains. Based on lipid-overlays and liposome-sedimentation assays, the fragments of ATM2 and VIII-B bind anionic phospholipids. Small polybasic regions at the extreme C-termini were sufficient for lipid-binding of the respective protein fragments. When expressed in tobacco pollen tubes, a fluorescence-tagged variant of ATM2 lacking its lipid-binding region displayed substantially reduced plasma membrane association. The data indicate that class VIII-myosins may facilitate actin-plasma membrane attachment through interaction with anionic phospholipids, mediated by polybasic C-terminal lipid-binding domains.

Direct Observation of "Elongated" Conformational States in α-Synuclein upon Liquid-Liquid Phase Separation.

α-Synuclein (α-syn) is an intrinsically disordered protein (IDP) that undergoes liquid-liquid phase separation (LLPS), fibrillation, and forms insoluble intracellular Lewy bodies in neurons, which are the hallmark of Parkinson's Disease (PD). Neurotoxicity precedes the formation of aggregates and might be related to α-syn LLPS. The molecular mechanisms underlying the early stages of LLPS are still elusive. To obtain structural insights into α-syn upon LLPS, we take advantage of cross-linking/mass spectrometry (XL-MS) and introduce an innovative approach, termed COMPASS (COMPetitive PAiring StatisticS). In this work, we show that the conformational ensemble of α-syn shifts from a "hairpin-like" structure towards more "elongated" conformational states upon LLPS. We obtain insights into the critical initial stages of LLPS and establish a novel mass spectrometry-based approach that will aid to solve open questions in LLPS structural biology.

Plasma membrane nano-organization specifies phosphoinositide effects on Rho-GTPases and actin dynamics in tobacco pollen tubes.

Pollen tube growth requires coordination of cytoskeletal dynamics and apical secretion. The regulatory phospholipid phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) is enriched in the subapical plasma membrane of pollen tubes of Arabidopsis thaliana and tobacco (Nicotiana tabacum) and can influence both actin dynamics and secretion. How alternative PtdIns(4,5)P2 effects are specified is unclear. In tobacco pollen tubes, spinning disc microscopy (SD) reveals dual distribution of a fluorescent PtdIns(4,5)P2-reporter in dynamic plasma membrane nanodomains vs. apparent diffuse membrane labeling, consistent with spatially distinct coexisting pools of PtdIns(4,5)P2. Several PI4P 5-kinases (PIP5Ks) can generate PtdIns(4,5)P2 in pollen tubes. Despite localizing to one membrane region, the PIP5Ks AtPIP5K2-EYFP and NtPIP5K6-EYFP display distinctive overexpression effects on cell morphologies, respectively related to altered actin dynamics or membrane trafficking. When analyzed by SD, AtPIP5K2-EYFP associated with nanodomains, whereas NtPIP5K6-EYFP localized diffusely. Chimeric AtPIP5K2-EYFP and NtPIP5K6-EYFP variants with reciprocally swapped membrane-associating domains evoked reciprocally shifted effects on cell morphology upon overexpression. Overall, active PI4P 5-kinase variants stabilized actin when targeted to nanodomains, suggesting a role of nanodomain-associated PtdIns(4,5)P2 in actin regulation. This notion is further supported by interaction and proximity of nanodomain-associated AtPIP5K2 with the Rho-GTPase NtRac5, and by its functional interplay with elements of Rho of plants signaling. Plasma membrane nano-organization may thus aid the specification of PtdIns(4,5)P2 functions to coordinate cytoskeletal dynamics and secretion.

A phosphoinositide 5-phosphatase from Solanum tuberosum is activated by PAMP-treatment and may antagonize phosphatidylinositol 4,5-bisphosphate at Phytophthora infestans infection sites.

Potato (Solanum tuberosum) plants susceptible to late blight disease caused by the oomycete Phytophthora infestans display enhanced resistance upon infiltration with the pathogen-associated molecular pattern (PAMP), Pep-13. Here, we characterize a potato gene similar to Arabidopsis 5-phosphatases which was identified in transcript arrays performed to identify Pep-13 regulated genes, and termed StIPP. Recombinant StIPP protein specifically dephosphorylated the D5-position of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2 ) in vitro. Other phosphoinositides or soluble inositolpolyphosphates were not converted. When transiently expressed in tobacco (Nicotiana tabacum) pollen tubes, a StIPP-YFP fusion localized to the subapical plasma membrane and antagonized PtdIns(4,5)P2 -dependent effects on cell morphology, indicating in vivo functionality. Phytophthora infestans-infection of N. benthamiana leaf epidermis cells resulted in relocalization of StIPP-GFP from the plasma membrane to the extra-haustorial membrane (EHM). Colocalizion with the effector protein RFP-AvrBlb2 at infection sites is consistent with a role of StIPP in the plant-oomycete interaction. Correlation analysis of fluorescence distributions of StIPP-GFP and biosensors for PtdIns(4,5)P2 or phosphatidylinositol 4-phosphate (PtdIns4P) indicate StIPP activity predominantly at the EHM. In Arabidopsis protoplasts, expression of StIPP resulted in the stabilization of the PAMP receptor, FLAGELLIN-SENSITIVE 2, indicating that StIPP may act as a PAMP-induced and localized antagonist of PtdIns(4,5)P2 -dependent processes during plant immunity.

Coordinated Localization and Antagonistic Function of NtPLC3 and PI4P 5-Kinases in the Subapical Plasma Membrane of Tobacco Pollen Tubes.

Polar tip growth of pollen tubes is regulated by the membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2), which localizes in a well-defined region of the subapical plasma membrane. How the PtdIns(4,5)P2 region is maintained is currently unclear. In principle, the formation of PtdIns(4,5)P2 by PI4P 5-kinases can be counteracted by phospholipase C (PLC), which hydrolyzes PtdIns(4,5)P2. Here, we show that fluorescence-tagged tobacco NtPLC3 displays a subapical plasma membrane distribution which frames that of fluorescence-tagged PI4P 5-kinases, suggesting that NtPLC3 may modulate PtdIns(4,5)P2-mediated processes in pollen tubes. The expression of a dominant negative NtPLC3 variant resulted in pollen tube tip swelling, consistent with a delimiting effect on PtdIns(4,5)P2 production. When pollen tube morphologies were assessed as a quantitative read-out for PtdIns(4,5)P2 function, NtPLC3 reverted the effects of a coexpressed PI4P 5-kinase, demonstrating that NtPLC3-mediated breakdown of PtdIns(4,5)P2 antagonizes the effects of PtdIns(4,5)P2 overproduction in vivo. When analyzed by spinning disc microscopy, fluorescence-tagged NtPLC3 displayed discontinuous membrane distribution omitting punctate areas of the membrane, suggesting that NtPLC3 is involved in the spatial restriction of plasma membrane domains also at the nanodomain scale. Together, the data indicate that NtPLC3 may contribute to the spatial restriction of PtdIns(4,5)P2 in the subapical plasma membrane of pollen tubes.

Forces during cellular uptake of viruses and nanoparticles at the ventral side.

Many intracellular pathogens, such as mammalian reovirus, mimic extracellular matrix motifs to specifically interact with the host membrane. Whether and how cell-matrix interactions influence virus particle uptake is unknown, as it is usually studied from the dorsal side. Here we show that the forces exerted at the ventral side of adherent cells during reovirus uptake exceed the binding strength of biotin-neutravidin anchoring viruses to a biofunctionalized substrate. Analysis of virus dissociation kinetics using the Bell model revealed mean forces higher than 30 pN per virus, preferentially applied in the cell periphery where close matrix contacts form. Utilizing 100 nm-sized nanoparticles decorated with integrin adhesion motifs, we demonstrate that the uptake forces scale with the adhesion energy, while actin/myosin inhibitions strongly reduce the uptake frequency, but not uptake kinetics. We hypothesize that particle adhesion and the push by the substrate provide the main driving forces for uptake.

Surface Immobilization of Viruses and Nanoparticles Elucidates Early Events in Clathrin-Mediated Endocytosis.

Clathrin-mediated endocytosis (CME) is an important entry pathway for viruses. Here, we applied click chemistry to covalently immobilize reovirus on surfaces to study CME during early host-pathogen interactions. To uncouple chemical and physical properties of viruses and determine their impact on CME initiation, we used the same strategy to covalently immobilize nanoparticles of different sizes. Using fluorescence live microscopy and electron microscopy, we confirmed that clathrin recruitment depends on particle size and discovered that the maturation into clathrin-coated vesicles (CCVs) is independent from cargo internalization. Surprisingly, we found that the final size of CCVs appears to be imprinted on the clathrin coat at early stages of cargo-cell interactions. Our approach has allowed us to unravel novel aspects of early interactions between viruses and the clathrin machinery that influence late stages of CME and CCVs formation. This method can be easily and broadly applied to the field of nanotechnology, endocytosis, and virology.

Molecular characterisation of human hepatitis E virus from Italy: comparative analysis of five reverse transcription-PCR assays.

BACKGROUND: Hepatitis E (HEV) is an important public-health concern as a major cause of enterically transmitted hepatitis worldwide. In industrialised countries it is considered rare, and largely confined to travellers returning from endemic areas. However, autochthonous (locally acquired) HEV infection is also emerging in these regions. The infection is caused by different genotypes, depending on whether it is travel-related or autochthonous. Conventional RT-PCR followed by sequencing of PCR products can identify HEV genotype and, depending on the region, the subtype, thus helping in defining the origin of infection and tracing the source of contamination. METHODS: We re-analysed a collection of serum samples previously confirmed as hepatitis E positive by anti-HEV IgM and IgG assays as well as by Real-Time PCR, with the aim to compare the performances of five different broad range RT-PCR assays that could be provided for molecular characterisation of HEV. This approach is certainly valuable to investigate the molecular epidemiology of acute hepatitis E in countries where co-circulation of different genotypes occurs, like Italy. RESULTS: Samples were analyzed by five assays targeting the ORF1, ORF2, and ORF2/3 regions. The sensitivity of these assays varied significantly, depending on the target region. Only 46% of samples tested positive by nested PCR; moreover, no single method was able to detect all positive samples. Most sequences originated from patients who had travelled to endemic areas (genotype 1), while the minority originated from Italian patients with no travel history (genotype 3). CONCLUSION: Broad range methods for molecular characterization of HEV still need to be improved to detect all circulating strains.

Viral infections acquired indoors through airborne, droplet or contact transmission.

BACKGROUND: Indoor human environments, including homes, offices, schools, workplaces, transport systems and other settings, often harbor potentially unsafe microorganisms. Most previous studies of bioaerosols in indoor environments have addressed contamination with bacteria or fungi. Reports on the presence of viral aerosols in indoor air are scarce, however, despite the fact that viruses are probably the most common cause of infection acquired indoor. OBJECTIVE: This review discusses the most common respiratory (influenza viruses, rhinoviruses, coronaviruses, adenoviruses, respiratory syncytial viruses, and enteroviruses) and gastrointestinal (noroviruses) viral pathogens which can be easily transmitted in indoor environments. RESULTS: The vast majority of studies reviewed here concern hospital and other health facilities where viruses are a well-known cause of occupational and nosocomial infections. Studies on other indoor environments, on the other hand, including homes, nonindustrial workplaces and public buildings, are scarce. CONCLUSIONS: The lack of regulations, threshold values and standardized detection methods for viruses in indoor environments, make both research and interpretation of results difficult in this field, hampering infection control efforts. Further research will be needed to achieve a better understanding of virus survival in aerosols and on surfaces, and to elucidate the relationship between viruses and indoor environmental characteristics.

Mucosal and cutaneous human papillomaviruses detected in raw sewages.

Epitheliotropic viruses can find their way into sewage. The aim of the present study was to investigate the occurrence, distribution, and genetic diversity of Human Papillomaviruses (HPVs) in urban wastewaters. Sewage samples were collected from treatment plants distributed throughout Italy. The DNA extracted from these samples was analyzed by PCR using five PV-specific sets of primers targeting the L1 (GP5/GP6, MY09/MY11, FAP59/64, SKF/SKR) and E1 regions (PM-A/PM-B), according to the protocols previously validated for the detection of mucosal and cutaneous HPV genotypes. PCR products underwent sequencing analysis and the sequences were aligned to reference genomes from the Papillomavirus Episteme database. Phylogenetic analysis was then performed to assess the genetic relationships among the different sequences and between the sequences of the samples and those of the prototype strains. A broad spectrum of sequences related to mucosal and cutaneous HPV types was detected in 81% of the sewage samples analyzed. Surprisingly, sequences related to the anogenital HPV6 and 11 were detected in 19% of the samples, and sequences related to the "high risk" oncogenic HPV16 were identified in two samples. Sequences related to HPV9, HPV20, HPV25, HPV76, HPV80, HPV104, HPV110, HPV111, HPV120 and HPV145 beta Papillomaviruses were detected in 76% of the samples. In addition, similarity searches and phylogenetic analysis of some sequences suggest that they could belong to putative new genotypes of the beta genus. In this study, for the first time, the presence of HPV viruses strongly related to human cancer is reported in sewage samples. Our data increases the knowledge of HPV genomic diversity and suggests that virological analysis of urban sewage can provide key information useful in supporting epidemiological studies.

Emerging and potentially emerging viruses in water environments.

Among microorganisms, viruses are best fit to become emerging pathogens since they are able to adapt not only by mutation but also through recombination and reassortment and can thus become able to infect new hosts and to adjust to new environments. Enteric viruses are among the commonest and most hazardous waterborne pathogens, causing both sporadic and outbreak-related illness. The main health effect associated with enteric viruses is gastrointestinal illness, but they can also cause respiratory symptoms, conjunctivitis, hepatitis, central nervous system infections, and chronic diseases. Non-enteric viruses, such as respiratory and epitheliotrophic viruses are not considered waterborne, as they are not readily transmitted to water sources from infected individuals. The present review will focus on viral pathogens shown to be transmitted through water. It will also provide an overview of viruses that had not been a concern for waterborne transmission in the past, but that may represent potentially emerging waterborne pathogens due to their occurrence and persistence in water environments.

GIV noroviruses and other enteric viruses in bivalves: a preliminary study.

We evaluated the presence of the enteric viruses: norovirus, adenovirus, enterovirus, astrovirus, hepatitis A virus, and hepatitis E virus in bivalves using nested PCR methods and cell culture assays. Noroviruses GII.4 and GIV.1, adenoviruses types 1 and 2, hepatitis A, and echovirus type 7 were detected in the shellfish tested, which were often co-infected. This is the first study to detect such a high level of viral contamination in Italian mussels (up to four different viral groups in a single sample), and the first to document the presence of GIV NoV in shellfish.

Molecular detection and genetic diversity of norovirus genogroup IV: a yearlong monitoring of sewage throughout Italy.

Noroviruses cause acute viral gastroenteritis worldwide. They are classified in five genogroups, of which GI, GII, and GIV infect humans. Little information is available on the prevalence and clinical effects of GIV noroviruses. We conducted a large-scale molecular-epidemiological investigation, a yearlong monitoring of 11 wastewater treatment plants throughout Italy, with the aim of studying the circulation of GIV NoV, as well as its genetic diversity. Eight percent of samples tested positive, and sequence analysis showed a considerable degree of genetic variability. These results imply the need for further studies to elucidate the role of this virus as a gastroenteritis-causing pathogen.