PLUNC Is a Novel Airway Surfactant Protein with Anti-Biofilm Activity

Background

The PLUNC (“Palate, lung, nasal epithelium clone”) protein is an abundant secretory product of epithelia present throughout the conducting airways of humans and other mammals, which is evolutionarily related to the lipid transfer/lipopolysaccharide binding protein (LT/LBP) family. Two members of this family - the bactericidal/permeability increasing protein (BPI) and the lipopolysaccharide binding protein (LBP) - are innate immune molecules with recognized roles in sensing and responding to Gram negative bacteria, leading many to propose that PLUNC may play a host defense role in the human airways.

Methodology/Principal Findings

Based on its marked hydrophobicity, we hypothesized that PLUNC may be an airway surfactant. We found that purified recombinant human PLUNC greatly enhanced the ability of aqueous solutions to spread on a hydrophobic surface. Furthermore, we discovered that PLUNC significantly reduced surface tension at the air-liquid interface in aqueous solutions, indicating novel and biologically relevant surfactant properties. Of note, surface tensions achieved by adding PLUNC to solutions are very similar to measurements of the surface tension in tracheobronchial secretions from humans and animal models. Because surfactants of microbial origin can disperse matrix-encased bacterial clusters known as biofilms [1], we hypothesized that PLUNC may also have anti-biofilm activity. We found that, at a physiologically relevant concentration, PLUNC inhibited biofilm formation by the airway pathogen Pseudomonas aeruginosa in an in vitro model.

Conclusions/Significance

Our data suggest that the PLUNC protein contributes to the surfactant properties of airway secretions, and that this activity may interfere with biofilm formation by an airway pathogen.     

DeepQuanTR: MALDI‐MS‐based label‐free quantification of proteins in complex biological samples

The quantification of changes in protein abundance in complex biological specimens is essential for proteomic studies in basic and applied research. Here we report on the development and validation of the DeepQuanTR software for identification and quantification of differentially expressed proteins using LC‐MALDI‐MS. Following enzymatic digestion, HPLC peptide separation and normalization of MALDI‐MS signal intensities to the ones of internal standards, the software extracts peptide features, adjusts differences in HPLC retention times and performs a relative quantification of features. The annotation of multiple peptides to the corresponding parent protein allows the definition of a Protein Quant Value, which is related to protein abundance and which allows inter‐sample comparisons. The performance of DeepQuanTR was evaluated by analyzing 24 samples deriving from human serum spiked with different amounts of four proteins and eight complex samples of vascular proteins, derived from surgically resected human kidneys with cancer following ex vivo perfusion with a reactive ester biotin derivative. The identification and experimental validation of proteins, which were differentially regulated in cancerous lesions as compared with normal kidney, was used to demonstrate the power of DeepQuanTR. This software, which can easily be used with established proteomic methodologies, facilitates the relative quantification of proteins derived from a wide variety of different samples.     

Gene expression profiling of U12-type spliceosome mutant Drosophila reveals widespread changes in metabolic pathways

Background

The U12-type spliceosome is responsible for the removal of a subset of introns from eukaryotic mRNAs. U12-type introns are spliced less efficiently than normal U2-type introns, which suggests a rate-limiting role in gene expression. The Drosophila genome contains about 20 U12-type introns, many of them in essential genes, and the U12-type spliceosome has previously been shown to be essential in the fly.

Methodology/Principal Findings

We have used a Drosophila line with a P-element insertion in U6atac snRNA, an essential component of the U12-type spliceosome, to investigate the impact of U12-type introns on gene expression at the organismal level during fly development. This line exhibits progressive accumulation of unspliced U12-type introns during larval development and the death of larvae at the third instar stage. Surprisingly, microarray and RT-PCR analyses revealed that most genes containing U12-type introns showed only mild perturbations in the splicing of U12-type introns. In contrast, we detected widespread downstream effects on genes that do not contain U12-type introns, with genes related to various metabolic pathways constituting the largest group.

Conclusions/Significance

U12-type intron-containing genes exhibited variable gene-specific responses to the splicing defect, with some genes showing up- or downregulation, while most did not change significantly. The observed residual U12-type splicing activity could be explained with the mutant U6atac allele having a low level of catalytic activity. Detailed analysis of all genes suggested that a defect in the splicing of the U12-type intron of the mitochondrial prohibitin gene may be the primary cause of the various downstream effects detected in the microarray analysis.     

PSMB8 encoding the β5i proteasome subunit is mutated in joint contractures, muscle atrophy, microcytic anemia, and panniculitis-induced lipodystrophy syndrome

We performed homozygosity mapping in two recently reported pedigrees from Portugal and Mexico with an autosomal-recessive autoinflammatory syndrome characterized by joint contractures, muscle atrophy, microcytic anemia, and panniculitis-induced lipodystrophy (JMP). This revealed only one homozygous region spanning 2.4 Mb (5818 SNPs) on chromosome 6p21 shared by all three affected individuals from both families. We directly sequenced genes involved in immune response located in this critical region, excluding the HLA complex genes. We found a homozygous missense mutation c.224C>T (p.Thr75Met) in the proteasome subunit, beta-type, 8 (PSMB8) gene in affected patients from both pedigrees. The mutation segregated in an autosomal-recessive fashion and was not detected in 275 unrelated ethnically matched healthy subjects. PSMB8 encodes a catalytic subunit of the 20S immunoproteasomes called β5i. Immunoproteasome-mediated proteolysis generates immunogenic epitopes presented by major histocompatibility complex (MHC) class I molecules. Threonine at position 75 is highly conserved and its substitution with methionine disrupts the tertiary structure of PSMB8. As compared to normal lymphoblasts, those from an affected patient showed significantly reduced chymotrypsin-like proteolytic activity mediated by immunoproteasomes. We conclude that mutations in PSMB8 cause JMP syndrome, most probably by affecting MHC class I antigen processing.     

Heme pocket structural properties of a bacterial truncated hemoglobin from Thermobifida fusca

An acidic surface variant (ASV) of the "truncated" hemoglobin from Thermobifida fusca was designed with the aim of creating a versatile globin scaffold endowed with thermostability and a high level of recombinant expression in its soluble form while keeping the active site unmodified. This engineered protein was obtained by mutating the surface-exposed residues Phe107 and Arg91 to Glu. Molecular dynamics simulations showed that the mutated residues remain solvent-exposed, not affecting the overall protein structure. Thus, the ASV was used in a combinatorial mutagenesis of the distal heme pocket residues in which one, two, or three of the conserved polar residues [TyrB10(54), TyrCD1(67), and TrpG8(119)] were substituted with Phe. Mutants were characterized by infrared and resonance Raman spectroscopy and compared with the wild-type protein. Similar Fe-proximal His stretching frequencies suggest that none of the mutations alters the proximal side of the heme cavity. Two conformers were observed in the spectra of the CO complexes of both wild-type and ASV protein: form 1 with ν(FeC) and ν(CO) at 509 and 1938 cm(-1) and form 2 with ν(FeC) and ν(CO) at 518 and 1920 cm(-1), respectively. Molecular dynamics simulations were performed for the wild-type and ASV forms, as well as for the TyrB10 mutant. The spectroscopic and computational results demonstrate that CO interacts with TrpG8 in form 1 and interacts with both TrpG8 and TyrCD1 in form 2. TyrB10 does not directly interact with the bound CO.     

Association between serum free IGF-I and IGFBP-3 levels in type-I diabetes patients affected with associated autoimmune diseases or diabetic complications

BACKGROUND: Patients with type 1 diabetes (T1DM) present lower serum free IGF and IGFBP-3 values than healthy people. T1DM patients often present with associated autoimmune diseases such as thyroiditis or coeliac disease, and over time they frequently develop proliferative retinopathy, neuropathy or nephropathy in different combinations. OBJECTIVE: The aim of this study was to evaluate the effect of two associated autoimmune diseases or three diabetic complications on the serum free IGF-I or IGFBP-3 levels in T1DM patients, who also have a family history of T1DM. Design. 246 T1DM patients were enrolled, and then subdivided into groups according to diabetic complications or associated autoimmune diseases. Demographic and clinical data were recorded. Serum free IGF-I and IGFBP-3 levels were determined by IRMA. RESULTS: IGF-I and IGFBP-3 generally present correlated serum values as confirmed in this study. Those patients with autoimmune thyroiditis and coeliac disease presented with significantly lower serum values of IGFBP-3, whereas free IGF-I was significantly lower in patients with the different diabetic complications. Retinopathy presented a slightly significant reduction in serum free IGF-I, while neuropathy and nephropathy showed a more pronounced fall. The number of complications was related to progressively decreasing free IGF-I levels. T1DM family history was associated with lower serum free IGF-I concentrations. These findings were confirmed after correction for age, glycosylated haemoglobin levels, insulin treatment protocol, body mass index, serum creatinine and sex. CONCLUSION: Despite a direct correlation between serum free IGF-I and IGFBP-3, the correlation between the two molecules in patients with associated autoimmune diseases was lost, possibly due to different mechanisms of metabolic regulation.     

Temperature affects longevity and age-related locomotor and cognitive decay in the short-lived fish Nothobranchius furzeri

Temperature variations are known to modulate aging and life-history traits in poikilotherms as different as worms, flies and fish. In invertebrates, temperature affects lifespan by modulating the slope of age-dependent acceleration in death rate, which is thought to reflect the rate of age-related damage accumulation. Here, we studied the effects of temperature on aging kinetics, aging-related behavioural deficits, and age-associated histological markers of senescence in the short-lived fish Nothobranchius furzeri. This species shows a maximum captive lifespan of only 3 months, which is tied with acceleration in growth and expression of aging biomarkers. These biological peculiarities make it a very convenient animal model for testing the effects of experimental manipulations on life-history traits in vertebrates. Here, we show that (i) lowering temperature from 25 degrees C to 22 degrees C increases both median and maximum lifespan; (ii) life extension is due to reduction in the slope of the age-dependent acceleration in death rate; (iii) lowering temperature from 25 degrees C to 22 degrees C retards the onset of age-related locomotor and learning deficits; and (iv) lowering temperature from 25 degrees C to 22 degrees C reduces the accumulation of the age-related marker lipofuscin. We conclude that lowering water temperature is a simple experimental manipulation which retards the rate of age-related damage accumulation in this short-lived species.     

Proteoglycan mechanics studied by single-molecule force spectroscopy of allotypic cell adhesion glycans

Early Metazoans had to evolve the first cell adhesion system addressed to maintaining stable interactions between cells constituting different individuals. As the oldest extant multicellular animals, sponges are good candidates to have remnants of the molecules responsible for that crucial innovation. Sponge cells associate in a species-specific process through multivalent calcium-dependent interactions of carbohydrate structures on an extracellular membrane-bound proteoglycan termed aggregation factor. Single-molecule force spectroscopy studies of the mechanics of aggregation factor self-binding indicate the existence of intermolecular carbohydrate adhesion domains. A 200-kDa aggregation factor glycan (g200) involved in cell adhesion exhibits interindividual differences in size and epitope content which suggest the existence of allelic variants. We have purified two of these g200 distinct forms from two individuals of the same sponge species. Comparison of allotypic versus isotypic g200 binding forces reveals significant differences. Surface plasmon resonance measurements show that g200 self-adhesion is much stronger than its binding to other unrelated glycans such as chondroitin sulfate. This adhesive specificity through multiple carbohydrate binding domains is a type of cooperative interaction that can contribute to explain some functions of modular proteoglycans in general. From our results it can be deduced that the binding strength/surface area between two aggregation factor molecules is comparable with that of focal contacts in vertebrate cells, indicating that strong carbohydrate-based cell adhesions evolved at the very start of Metazoan history.