Dual targeted mitochondrial proteins are characterized by lower MTS parameters and total net charge

Background

In eukaryotic cells, identical proteins can be located in different subcellular compartments (termed dual-targeted proteins).

Methodology/Principal Findings

We divided a reference set of mitochondrial proteins (published single gene studies) into two groups: i) Dual targeted mitochondrial proteins and ii) Exclusive mitochondrial proteins. Mitochondrial proteins were considered dual-targeted if they were also found or predicted to be localized to the cytosol, the nucleus, the endoplasmic reticulum (ER) or the peroxisome. We found that dual localized mitochondrial proteins have i) A weaker mitochondrial targeting sequence (MitoProtII score, hydrophobic moment and number of basic residues) and ii) a lower whole-protein net charge, when compared to exclusive mitochondrial proteins. We have also generated an annotation list of dual-targeted proteins within the predicted yeast mitochondrial proteome. This considerably large group of dual-localized proteins comprises approximately one quarter of the predicted mitochondrial proteome. We supported this prediction by experimental verification of a subgroup of the predicted dual targeted proteins.

Conclusions/Significance

Taken together, these results establish dual targeting as a widely abundant phenomenon that should affect our concepts of gene expression and protein function. Possible relationships between the MTS/mature sequence traits and protein dual targeting are discussed.     

Analysis of the key elements of FFAT-like motifs identifies new proteins that potentially bind VAP on the ER, including two AKAPs and FAPP2

Background

Two phenylalanines (FF) in an acidic tract (FFAT)-motifs were originally described as having seven elements: an acidic flanking region followed by 6 residues (EFFDA–E). Such motifs are found in several lipid transfer protein (LTP) families, and they interact with a protein on the cytosolic face of the ER called vesicle-associated membrane protein-associated protein (VAP). Mutation of which causes ER stress and motor neuron disease, making it important to determine which proteins bind VAP. Among other proteins that bind VAP, some contain FFAT-like motifs that are missing one or more of the seven elements. Defining how much variation is tolerated in FFAT-like motifs is a preliminary step prior to the identification of the full range of VAP interactors.

Results

We used a quantifiable in vivo system that measured ER targeting in a reporter yeast strain that over-expressed VAP to study the effect of substituting different elements of FFAT-like motifs in turn. By defining FFAT-like motifs more widely than before, we found them in novel proteins the functions of which had not previously been directly linked to the ER, including: two PKA anchoring proteins, AKAP220 and AKAP110; a family of plant LTPs; and the glycolipid LTP phosphatidylinositol-four-phosphate adaptor-protein-2 (FAPP-2).

Conclusion

All of the seven essential elements of a FFAT motif tolerate variation, and weak targeting to the ER via VAP is still detected if two elements are substituted. In addition to the strong FFAT motifs already known, there are additional proteins with weaker FFAT-like motifs, which might be functionally important VAP interactors.     

The Distribution of GYR- and YLP-Like Motifs in Drosophila Suggests a General Role in Cuticle Assembly and Other Protein-Protein Interactions

Background

Arthropod cuticle is composed predominantly of a self-assembling matrix of chitin and protein. Genes encoding structural cuticular proteins are remarkably abundant in arthropod genomes, yet there has been no systematic survey of conserved motifs across cuticular protein families.

Methodology/Principal Findings

Two short sequence motifs with conserved tyrosines were identified in Drosophila cuticular proteins that were similar to the GYR and YLP Interpro domains. These motifs were found in members of the CPR, Tweedle, CPF/CPFL, and (in Anopheles gambiae) CPLCG cuticular protein families, and the Dusky/Miniature family of cuticle-associated proteins. Tweedle proteins have a characteristic motif architecture that is shared with the Drosophila protein GCR1 and its orthologs in other species, suggesting that GCR1 is also cuticular. A resilin repeat, which has been shown to confer elasticity, matched one of the motifs; a number of other Drosophila proteins of unknown function exhibit a motif architecture similar to that of resilin. The motifs were also present in some proteins of the peritrophic matrix and the eggshell, suggesting molecular convergence among distinct extracellular matrices. More surprisingly, gene regulation, development, and proteolysis were statistically over-represented ontology terms for all non-cuticular matches in Drosophila. Searches against other arthropod genomes indicate that the motifs are taxonomically widespread.

Conclusions

This survey suggests a more general definition for GYR and YLP motifs and reveals their contribution to several types of extracellular matrix. They may define sites of protein interaction with DNA or other proteins, based on ontology analysis. These results can help guide experimental studies on the biochemistry of cuticle assembly.     

Maltose-binding protein enhances secretion of recombinant human granzyme B accompanied by in vivo processing of a precursor MBP fusion protein

Background

The apoptosis-inducing serine protease granzyme B (GrB) is an important factor contributing to lysis of target cells by cytotoxic lymphocytes. Expression of enzymatically active GrB in recombinant form is a prerequisite for functional analysis and application of GrB for therapeutic purposes.

Methods and Findings

We investigated the influence of bacterial maltose-binding protein (MBP) fused to GrB via a synthetic furin recognition motif on the expression of the MBP fusion protein also containing an N-terminal α-factor signal peptide in the yeast Pichia pastoris. MBP markedly enhanced the amount of GrB secreted into culture supernatant, which was not the case when GrB was fused to GST. MBP-GrB fusion protein was cleaved during secretion by an endogenous furin-like proteolytic activity in vivo, liberating enzymatically active GrB without the need of subsequent in vitro processing. Similar results were obtained upon expression of a recombinant fragment of the ErbB2/HER2 receptor protein or GST as MBP fusions.

Conclusions

Our results demonstrate that combination of MBP as a solubility enhancer with specific in vivo cleavage augments secretion of processed and functionally active proteins from yeast. This strategy may be generally applicable to improve folding and increase yields of recombinant proteins.     

Peroxynitrite (ONOO?) is endogenously produced in arabidopsis peroxisomes and is overproduced under cadmium stress

Background and Aims

Peroxisomes are subcellular compartments involved in multiple cellular metabolic pathways. Peroxynitrite (ONOO⁻) is a nitric oxide-derived molecule which is a nitrating species that causes nitration of proteins. This study used cell biology techniques to explore the potential presence of peroxynitrite in peroxisomes and evaluated its content under stress conditions (excess cadmium).

Methods

Peroxynitrite, nitric oxide and superoxide anion were studied using cell-permeable specific fluorescent probes by confocal laser scanning microscopy in Arabidopsis thaliana transgenic plants expressing cyan fluorescent protein through the addition of peroxisomal targeting signal 1 (PTS1), which enables peroxisomes to be visualized in vivo.

Key Results

When no stress was applied, peroxynitrite was clearly localized in the peroxisomes of roots and stomatal guard cells. Under cadmium (150 μm) stress, the generation of peroxynitrite, nitric oxide and the superoxide anion (O₂^·–) increased and was localized in peroxisomes and the cytosol, participating in the generation of nitro-oxidative stress.

Conclusions

The results show that peroxisomes are an endogenous source of peroxynitrite, which is over-produced under cadmium stress, suggesting that the metabolism of reactive nitrogen species in peroxisomes could participate in the mechanism of the response to this heavy metal.     

No Need for Labels: The Autofluorescence of Leishmania tarentolae Mitochondria and the Necessity of Negative Controls

Background

Fluorescence microscopy is a powerful tool to study the morphology and function of subcellular compartments or to determine the localization of proteins. The method is also regularly used for the analysis of parasitic protists including kinetoplastida.

Results

Here, we report a significant autofluorescence of Leishmania tarentolae mitochondria. The autofluorescence, presumably caused by flavoproteins, was detectable using a variety of cell fixation protocols and had a maximum emission at approximately 538 nm. Stable signals were obtained with xenon lamps as a light source and filter sets that are commonly used for the detection of green fluorescent protein.

Conclusions

On the one hand, we present a methodological approach to examine mitochondrial morphology or to study the colocalization of mitochondrial proteins without additional staining or labeling procedures. On the other hand, under certain experimental conditions, mitochondrial autofluorescence can result in false positive signals, demonstrating the necessity to analyze unlabeled cells as negative controls.     

M153R mutation in a pH-sensitive green fluorescent protein stabilizes its fusion proteins

Background

Green fluorescent protein (GFP) and its fusion proteins have been used extensively to monitor and analyze a wide range of biological processes. However, proteolytic cleavage often removes GFP from its fusion proteins, not only causing a poor signal-to-noise ratio of the fluorescent images but also leading to wrong interpretations.

Methodology/Principal Findings

Here, we report that the M153R mutation in a ratiometric pH-sensitive GFP, pHluorin, significantly stabilizes its fusion products while the mutant protein still retaining a marked pH dependence of 410/470 nm excitation ratio of fluorescence intensity. The M153R mutation increases the brightness in vivo but does not affect the 410/470-nm excitation ratios at various pH values.

Conclusions/Significance

Since the pHluorin(M153R) probe can be directly fused to the target proteins, we suggest that it will be a potentially powerful tool for the measurement of local pH in living cells as well as for the analysis of subcellular localization of target proteins.     

A novel small molecule target in human airway smooth muscle for potential treatment of obstructive lung diseases: a staged high-throughput biophysical screening

Background

A newly identified mechanism of smooth muscle relaxation is the interaction between the small heat shock protein 20 (HSP20) and 14-3-3 proteins. Focusing upon this class of interactions, we describe here a novel drug target screening approach for treating airflow obstruction in asthma.

Methods

Using a high-throughput fluorescence polarization (FP) assay, we screened a library of compounds that could act as small molecule modulators of HSP20 signals. We then applied two quantitative, cell-based biophysical methods to assess the functional efficacy of these molecules and rank-ordered their abilities to relax isolated human airway smooth muscle (ASM). Scaling up to the level of an intact tissue, we confirmed in a concentration-responsive manner the potency of the cell-based hit compounds.

Results

Among 58,019 compound tested, 268 compounds caused 20% or more reduction of the polarized emission in the FP assay. A small subset of these primary screen hits, belonging to two scaffolds, caused relaxation of isolated ASM cell in vitro and attenuated active force development of intact tissue ex vivo.

Conclusions

This staged biophysical screening paradigm provides proof-of-principle for high-throughput and cost-effective discovery of new small molecule therapeutic agents for obstructive lung diseases.     

Dual Localized AtHscB Involved in Iron Sulfur Protein Biogenesis in Arabidopsis

Background

Iron-sulfur clusters are ubiquitous structures which act as prosthetic groups for numerous proteins involved in several fundamental biological processes including respiration and photosynthesis. Although simple in structure both the assembly and insertion of clusters into apoproteins requires complex biochemical pathways involving a diverse set of proteins. In yeast, the J-type chaperone Jac1 plays a key role in the biogenesis of iron sulfur clusters in mitochondria.

Methodology/Principal Findings

In this study we demonstrate that AtHscB from Arabidopsis can rescue the Jac1 yeast knockout mutant suggesting a role for AtHscB in iron sulfur protein biogenesis in plants. In contrast to mitochondrial Jac1, AtHscB localizes to both mitochondria and the cytosol. AtHscB interacts with AtIscU1, an Isu-like scaffold protein involved in iron-sulfur cluster biogenesis, and through this interaction AtIscU1 is most probably retained in the cytosol. The chaperone AtHscA can functionally complement the yeast Ssq1knockout mutant and its ATPase activity is enhanced by AtHscB and AtIscU1. Interestingly, AtHscA is also localized in both mitochondria and the cytosol. Furthermore, AtHscB is highly expressed in anthers and trichomes and an AtHscB T-DNA insertion mutant shows reduced seed set, a waxless phenotype and inappropriate trichome development as well as dramatically reduced activities of the iron-sulfur enzymes aconitase and succinate dehydrogenase.

Conclusions

Our data suggest that AtHscB together with AtHscA and AtIscU1 plays an important role in the biogenesis of iron-sulfur proteins in both mitochondria and the cytosol.     

Network analysis identifies protein clusters of functional importance in juvenile idiopathic arthritis

Introduction

Our objective was to utilise network analysis to identify protein clusters of greatest potential functional relevance in the pathogenesis of oligoarticular and rheumatoid factor negative (RF-ve) polyarticular juvenile idiopathic arthritis (JIA).

Methods

JIA genetic association data were used to build an interactome network model in BioGRID 3.2.99. The top 10% of this protein:protein JIA Interactome was used to generate a minimal essential network (MEN). Reactome FI Cytoscape 2.83 Plugin and the Disease Association Protein-Protein Link Evaluator (Dapple) algorithm were used to assess the functionality of the biological pathways within the MEN and to statistically rank the proteins. JIA gene expression data were integrated with the MEN and clusters of functionally important proteins derived using MCODE.

Results

A JIA interactome of 2,479 proteins was built from 348 JIA associated genes. The MEN, representing the most functionally related components of the network, comprised of seven clusters, with distinct functional characteristics. Four gene expression datasets from peripheral blood mononuclear cells (PBMC), neutrophils and synovial fluid monocytes, were mapped onto the MEN and a list of genes enriched for functional significance identified. This analysis revealed the genes of greatest potential functional importance to be PTPN2 and STAT1 for oligoarticular JIA and KSR1 for RF-ve polyarticular JIA. Clusters of 23 and 14 related proteins were derived for oligoarticular and RF-ve polyarticular JIA respectively.

Conclusions

This first report of the application of network biology to JIA, integrating genetic association findings and gene expression data, has prioritised protein clusters for functional validation and identified new pathways for targeted pharmacological intervention.     

Discovery of a novel small secreted protein family with conserved N-terminal IGY motif in Dikarya fungi

Background

Small secreted proteins (SSPs) are employed by plant pathogenic fungi as essential strategic tools for their successful colonization. SSPs are often species-specific and so far only a few widely phylogenetically distributed SSPs have been identified.

Results

A novel fungal SSP family consisting of 107 members was identified in the poplar tree fungal pathogen Marssonina brunnea, which accounts for over 17% of its secretome. We named these proteins IGY proteins (IGYPs) based on the conserved three amino acids at the N-terminus. In spite of overall low sequence similarity among IGYPs; they showed conserved N- and C-terminal motifs and a unified gene structure. By RT-PCR-seq, we analyzed the IGYP gene models and validated their expressions as active genes during infection. IGYP homologues were also found in 25 other Dikarya fungal species, all of which shared conserved motifs and the same gene structure. Furthermore, 18 IGYPs from 11 fungi also shared similar genomic contexts. Real-time RT-PCR showed that 8 MbIGYPs were highly expressed in the biotrophic stage. Interestingly, transient assay of 12 MbIGYPs showed that the MbIGYP13 protein induced cell death in resistant poplar clones.

Conclusions

In total, 154 IGYPs in 26 fungi of the Dikarya subkingdom were discovered. Gene structure and genomic context analyses indicated that IGYPs originated from a common ancestor. In M. brunnea, the expansion of highly divergent MbIGYPs possibly is associated with plant-pathogen arms race.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1151) contains supplementary material, which is available to authorized users.     

Qualitative and quantitative multiplexed proteomic analysis of complex yeast protein fractions that modulate the assembly of the yeast prion Sup35p

Background

The aggregation of the baker''s yeast prion Sup35p is at the origin of the transmissible [PSI⁺] trait. We and others have shown that molecular chaperones modulate Sup35p aggregation. However, other protein classes might be involved in [PSI⁺] formation.

Results

We designed a functional proteomic study that combines two techniques to identify modulators of Sup35p aggregation and describe the changes associated to [PSI⁺] formation. The first allows measuring the effect of fractionated Saccharomyces cerevisiae cytosolic extracts from [PSI⁺] and [psi⁻] yeast cells on Sup35p assembly. The second is a multiplex qualitative and quantitative comparison of protein composition of active and inactive fractions using a gel-free and label-free LC-MS approach. We identify changes in proteins involved in translation, folding, degradation, oxido-reduction and metabolic processes.

Conclusion

Our functional proteomic study provides the first inventory list of over 300 proteins that directly or indirectly affect Sup35p aggregation and [PSI⁺] formation. Our results highlight the complexity of the cellular changes accompanying [PSI⁺] formation and pave the way for in vitro studies aimed to document the effect of individual and/or combinations of proteins identified here, susceptible of affecting Sup35p assembly.     

Extension of Yeast Chronological Lifespan by Methylamine

Background

Chronological aging of yeast cells is commonly used as a model for aging of human post-mitotic cells. The yeast Saccharomyces cerevisiae grown on glucose in the presence of ammonium sulphate is mainly used in yeast aging research. We have analyzed chronological aging of the yeast Hansenula polymorpha grown at conditions that require primary peroxisome metabolism for growth.

Methodology/Principal Findings

The chronological lifespan of H. polymorpha is strongly enhanced when cells are grown on methanol or ethanol, metabolized by peroxisome enzymes, relative to growth on glucose that does not require peroxisomes. The short lifespan of H. polymorpha on glucose is mainly due to medium acidification, whereas most likely ROS do not play an important role. Growth of cells on methanol/methylamine instead of methanol/ammonium sulphate resulted in further lifespan enhancement. This was unrelated to medium acidification. We show that oxidation of methylamine by peroxisomal amine oxidase at carbon starvation conditions is responsible for lifespan extension. The methylamine oxidation product formaldehyde is further oxidized resulting in NADH generation, which contributes to increased ATP generation and reduction of ROS levels in the stationary phase.

Conclusion/Significance

We conclude that primary peroxisome metabolism enhanced chronological lifespan of H. polymorpha. Moreover, the possibility to generate NADH at carbon starvation conditions by an organic nitrogen source supports further extension of the lifespan of the cell. Consequently, the interpretation of CLS analyses in yeast should include possible effects on the energy status of the cell.     

Mutational Profiling of Kinases in Human Tumours of Pancreatic Origin Identifies Candidate Cancer Genes in Ductal and Ampulla of Vater Carcinomas

Background

Protein kinases are key regulators of cellular processes (such as proliferation, apoptosis and invasion) that are often deregulated in human cancers. Accordingly, kinase genes have been the first to be systematically analyzed in human tumors leading to the discovery that many oncogenes correspond to mutated kinases. In most cases the genetic alterations translate in constitutively active kinase proteins, which are amenable of therapeutic targeting. Tumours of the pancreas are aggressive neoplasms for which no effective therapeutic strategy is currently available.

Methodology/Principal Findings

We conducted a DNA-sequence analysis of a selected set of 35 kinase genes in a panel of 52 pancreatic exocrine neoplasms, including 36 pancreatic ductal adenocarcinoma, and 16 ampulla of Vater cancer. Among other changes we found somatic mutations in ATM, EGFR, EPHA3, EPHB2, and KIT, none of which was previously described in cancers.

Conclusions/Significance

Although the alterations identified require further experimental evaluation, the localization within defined protein domains indicates functional relevance for most of them. Some of the mutated genes, including the tyrosine kinases EPHA3 and EPHB2, are clearly amenable to pharmacological intervention and could represent novel therapeutic targets for these incurable cancers.