Formation of Disulfide Bridges Drives Oligomerization,Membrane Pore Formation,and Translocation of Fibroblast Growth Factor 2 to Cell Surfaces

Fibroblast growth factor 2 (FGF2) is a key signaling molecule in tumor-induced angiogenesis. FGF2 is secreted by an unconventional secretory mechanism that involves phosphatidylinositol 4,5-bisphosphate-dependent insertion of FGF2 oligomers into the plasma membrane. This process is regulated by Tec kinase-mediated tyrosine phosphorylation of FGF2. Molecular interactions driving FGF2 monomers into membrane-inserted FGF2 oligomers are unknown. Here we identify two surface cysteines that are critical for efficient unconventional secretion of FGF2. They represent unique features of FGF2 as they are absent from all signal-peptide-containing members of the FGF protein family. We show that phosphatidylinositol 4,5-bisphosphate-dependent FGF2 oligomerization concomitant with the generation of membrane pores depends on FGF2 surface cysteines as either chemical alkylation or substitution with alanines impairs these processes. We further demonstrate that the FGF2 variant forms lacking the two surface cysteines are not secreted from cells. These findings were corroborated by experiments redirecting a signal-peptide-containing FGF family member from the endoplasmic reticulum/Golgi-dependent secretory pathway into the unconventional secretory pathway of FGF2. Cis elements known to be required for unconventional secretion of FGF2, including the two surface cysteines, were transplanted into a variant form of FGF4 without signal peptide. The resulting FGF4/2 hybrid protein was secreted by unconventional means. We propose that the formation of disulfide bridges drives membrane insertion of FGF2 oligomers as intermediates in unconventional secretion of FGF2.     

Mechanistic Insights into PTS2-mediated Peroxisomal Protein Import: THE CO-RECEPTOR PEX5L DRASTICALLY INCREASES THE INTERACTION STRENGTH BETWEEN THE CARGO PROTEIN AND THE RECEPTOR PEX7*

The destination of peroxisomal matrix proteins is encoded by short peptide sequences, which have been characterized as peroxisomal targeting signals (PTS) residing either at the C terminus (PTS1) or close to the N terminus (PTS2). PTS2-carrying proteins interact with their cognate receptor protein PEX7 that mediates their transport to peroxisomes by a concerted action with a co-receptor protein, which in mammals is the PTS1 receptor PEX5L. Using a modified version of the mammalian two-hybrid assay, we demonstrate that the interaction strength between cargo and PEX7 is drastically increased in the presence of the co-receptor PEX5L. In addition, cargo binding is a prerequisite for the interaction between PEX7 and PEX5L and ectopic overexpression of PTS2-carrying cargo protein drastically increases the formation of PEX7-PEX5L complexes in this assay. Consistently, we find that the peroxisomal transfer of PEX7 depends on cargo binding and that ectopic overexpression of cargo protein stimulates this process. Thus, the sequential formation of a highly stable trimeric complex involving cargo protein, PEX7 and PEX5L stabilizes cargo binding and is a prerequisite for PTS2-mediated peroxisomal import.     

Spatially Restricted G Protein-coupled Receptor Activity via Divergent Endocytic Compartments

Postendocytic sorting of G protein-coupled receptors (GPCRs) is driven by their interactions between highly diverse receptor sequence motifs with their interacting proteins, such as postsynaptic density protein (PSD95), Drosophila disc large tumor suppressor (Dlg1), zonula occludens-1 protein (zo-1) (PDZ) domain proteins. However, whether these diverse interactions provide an underlying functional specificity, in addition to driving sorting, is unknown. Here we identify GPCRs that recycle via distinct PDZ ligand/PDZ protein pairs that exploit their recycling machinery primarily for targeted endosomal localization and signaling specificity. The luteinizing hormone receptor (LHR) and β2-adrenergic receptor (B2AR), two GPCRs sorted to the regulated recycling pathway, underwent divergent trafficking to distinct endosomal compartments. Unlike B2AR, which traffics to early endosomes (EE), LHR internalizes to distinct pre-early endosomes (pre-EEs) for its recycling. Pre-EE localization required interactions of the LHR C-terminal tail with the PDZ protein GAIP-interacting protein C terminus, inhibiting its traffic to EEs. Rerouting the LHR to EEs, or EE-localized GPCRs to pre-EEs, spatially reprograms MAPK signaling. Furthermore, LHR-mediated activation of MAPK signaling requires internalization and is maintained upon loss of the EE compartment. We propose that combinatorial specificity between GPCR sorting sequences and interacting proteins dictates an unprecedented spatiotemporal control in GPCR signal activity.     

Myosin Vc Interacts with Rab32 and Rab38 Proteins and Works in the Biogenesis and Secretion of Melanosomes

Class V myosins are actin-based motors with conserved functions in vesicle and organelle trafficking. Herein we report the discovery of a function for Myosin Vc in melanosome biogenesis as an effector of melanosome-associated Rab GTPases. We isolated Myosin Vc in a yeast two-hybrid screening for proteins that interact with Rab38, a Rab protein involved in the biogenesis of melanosomes and other lysosome-related organelles. Rab38 and its close homolog Rab32 bind to Myosin Vc but not to Myosin Va or Myosin Vb. Binding depends on residues in the switch II region of Rab32 and Rab38 and regions of the Myosin Vc coiled-coil tail domain. Myosin Vc also interacts with Rab7a and Rab8a but not with Rab11, Rab17, and Rab27. Although Myosin Vc is not particularly abundant on pigmented melanosomes, its knockdown in MNT-1 melanocytes caused defects in the trafficking of integral membrane proteins to melanosomes with substantially increased surface expression of Tyrp1, nearly complete loss of Tyrp2, and significant Vamp7 mislocalization. Knockdown of Myosin Vc in MNT-1 cells more than doubled the abundance of pigmented melanosomes but did not change the number of unpigmented melanosomes. Together the data demonstrate a novel role for Myosin Vc in melanosome biogenesis and secretion.     

Proteomic screening of glutamatergic mouse brain synaptosomes isolated by fluorescence activated sorting

For decades, neuroscientists have used enriched preparations of synaptic particles called synaptosomes to study synapse function. However, the interpretation of corresponding data is problematic as synaptosome preparations contain multiple types of synapses and non‐synaptic neuronal and glial contaminants. We established a novel Fluorescence Activated Synaptosome Sorting (FASS) method that substantially improves conventional synaptosome enrichment protocols and enables high‐resolution biochemical analyses of specific synapse subpopulations. Employing knock‐in mice with fluorescent glutamatergic synapses, we show that FASS isolates intact ultrapure synaptosomes composed of a resealed presynaptic terminal and a postsynaptic density as assessed by light and electron microscopy. FASS synaptosomes contain bona fide glutamatergic synapse proteins but are almost devoid of other synapse types and extrasynaptic or glial contaminants. We identified 163 enriched proteins in FASS samples, of which FXYD6 and Tpd52 were validated as new synaptic proteins. FASS purification thus enables high‐resolution biochemical analyses of specific synapse subpopulations in health and disease.     

Use of in silico tools for classification of novel missense mutations identified in dystrophin gene in developing countries

DMD gene which is composed of 79 exons is the largest known gene located on X chromosome (Xp21). Point mutations in the dystrophin gene are responsible for 30–35% of cases with DMD/BMD. Mutation analysis of all the exons of the DMD gene is costly in developing countries, therefore, a few of the exons are selected to be analyzed routinely in clinical laboratories. In this study, direct sequencing was used for detection of point mutations in 10 exons of dystrophin gene in patients affected with DMD without detectable large rearrangements. Freely available programs were used to predict the damaging effects of the mutations. Point mutations were successfully detected in three patients. Three novel mutations, two missense mutations located on nonconservative domains and a single nucleotide deletion, were detected. Missense mutations were predicted to change splicing efficiency. Detection of point mutations by DNA analysis followed by prediction of the pathogenecity by using bioinformatic tool might be an asset to provide proper diagnosis or genetic counseling to patients and their family.     

A simple peak detection and label-free quantitation algorithm for chromatography-mass spectrometry

Background

Label-free quantitation of mass spectrometric data is one of the simplest and least expensive methods for differential expression profiling of proteins and metabolites. The need for high accuracy and performance computational label-free quantitation methods is still high in the biomarker and drug discovery research field. However, recent most advanced types of LC-MS generate huge amounts of analytical data with high scan speed, high accuracy and resolution, which is often impossible to interpret manually. Moreover, there are still issues to be improved for recent label-free methods, such as how to reduce false positive/negatives of the candidate peaks, how to expand scalability and how to enhance and automate data processing. AB3D (A simple label-free quantitation algorithm for Biomarker Discovery in Diagnostics and Drug discovery using LC-MS) has addressed these issues and has the capability to perform label-free quantitation using MS1 for proteomics study.

Results

We developed an algorithm called AB3D, a label free peak detection and quantitative algorithm using MS1 spectral data. To test our algorithm, practical applications of AB3D for LC-MS data sets were evaluated using 3 datasets. Comparisons were then carried out between widely used software tools such as MZmine 2, MSight, SuperHirn, OpenMS and our algorithm AB3D, using the same LC-MS datasets. All quantitative results were confirmed manually, and we found that AB3D could properly identify and quantify known peptides with fewer false positives and false negatives compared to four other existing software tools using either the standard peptide mixture or the real complex biological samples of Bartonella quintana (strain JK31). Moreover, AB3D showed the best reliability by comparing the variability between two technical replicates using a complex peptide mixture of HeLa and BSA samples. For performance, the AB3D algorithm is about 1.2 - 15 times faster than the four other existing software tools.

Conclusions

AB3D is a simple and fast algorithm for label-free quantitation using MS1 mass spectrometry data for large scale LC-MS data analysis with higher true positive and reasonable false positive rates. Furthermore, AB3D demonstrated the best reproducibility and is about 1.2- 15 times faster than those of existing 4 software tools.

Electronic supplementary material

The online version of this article (doi:10.1186/s12859-014-0376-0) contains supplementary material, which is available to authorized users.     

In silico analysis of Single Nucleotide Polymorphisms (SNPs) in human BRAF gene

BRAF gene mutations are frequently seen in both inherited and somatic diseases. However, the harmful mutations for BRAF gene have not been predicted in silico. Owing to the importance of BRAF gene in cell division, differentiation and secretion processes, the functional analysis was carried out to explore the possible association between genetic mutations and phenotypic variations. Genomic analysis of BRAF was initiated with SIFT followed by PolyPhen and SNPs&GO servers to retrieve the 85 deleterious non-synonymous SNPs (nsSNPs) from dbSNP. A total of 5 mutations i.e. c.406T>G (S136A), c.1446G>T (R462I), c.1556 A>G (K499E), c.1860 T>A (V600E) and c.2352 C>T (P764L) that are found to exert benign effects on the BRAF protein structure and function were chosen for further analysis. Protein structural analysis with these amino acid variants was performed by using I-Mutant, FOLD-X, HOPE, NetSurfP, Swiss PDB viewer, Chimera and NOMAD-Ref servers to check their solvent accessibility, molecular dynamics and energy minimization calculations. Our in silico analysis suggested that S136A and P764L variants of BRAF could directly or indirectly destabilize the amino acid interactions and hydrogen bond networks thus explain the functional deviations of protein to some extent. Screening for BRAF, S136A and P764Lvariants may be useful for disease molecular diagnosis and also to design the molecular inhibitors of BRAF pathways.