Dysregulated myosin in Hermansky-Pudlak syndrome lung fibroblasts is associated with increased cell motility.

Hermansky-Pudlak syndrome (HPS) is an autosomal recessive disorder characterized by improper biogenesis of lysosome-related organelles (LROs). Lung fibrosis is the leading cause of death among adults with HPS-1 and HPS-4 genetic types, which are associated with defects in the biogenesis of lysosome-related organelles complex-3 (BLOC-3), a guanine exchange factor (GEF) for a small GTPase, Rab32. LROs are not ubiquitously present in all cell types, and specific cells utilize LROs to accomplish dedicated functions. Fibroblasts are not known to contain LROs, and the function of BLOC-3 in fibroblasts is unclear. Here, we report that lung fibroblasts isolated from patients with HPS-1 have increased migration capacity. Silencing HPS-1 in normal lung fibroblasts similarly leads to increased migration. We also show that the increased migration is driven by elevated levels of Myosin IIB. Silencing HPS1 or RAB32 in normal lung fibroblasts leads to increased MYOSIN IIB levels. MYOSIN IIB is downstream of p38-MAPK, which is a known target of angiotensin receptor signaling. Treatment with losartan, an angiotensin receptor inhibitor, decreases MYOSIN IIB levels and impedes HPS lung fibroblast migration in vitro. Furthermore, pharmacologic inhibition of angiotensin receptor with losartan seemed to decrease migration of HPS lung fibroblasts in vivo in a zebrafish xenotransplantation model. Taken together, we demonstrate that BLOC-3 plays an important role in MYOSIN IIB regulation within lung fibroblasts and contributes to fibroblast migration.

Mutations in KIAA0753 cause Joubert syndrome associated with growth hormone deficiency.

Joubert syndrome and related disorders (JSRD) are a heterogeneous group of ciliopathies defined based on the mid-hindbrain abnormalities that result in the characteristic "molar tooth sign" on brain imaging. The core clinical findings of JSRD are hypotonia, developmental delay, abnormal eye movements and breathing abnormalities. To date, more than 30 JSRD genes that encode proteins important for structure and/or function of cilia have been identified. Here, we present 2 siblings with Joubert syndrome associated with growth hormone deficiency. Whole exome sequencing of the family identified compound heterozygous mutations in KIAA0753, i.e., a missense mutation (p.Arg257Gly) and an intronic mutation (c.2359-1G>C). The intronic mutation alters normal splicing by activating a cryptic acceptor splice site in exon 16. The novel acceptor site skips nine nucleotides, deleting three amino acids from the protein coding frame. KIAA0753 (OFIP) is a centrosome and pericentriolar satellite protein, previously not known to cause Joubert syndrome. We present comprehensive clinical descriptions of the Joubert syndrome patients as well as the cellular phenotype of defective ciliogenesis in the patients' fibroblasts.

Fully automated micro- and nanoscale one- or two-dimensional high-performance liquid chromatography system for liquid chromatography-mass spectrometry compatible with non-volatile salts for ion exchange chromatography.

A one- or two-dimensional high performance liquid chromatography system for electrospray ionization mass spectrometers has been developed that is optimized for ion exchange and reversed phase separations. A unique and simple valve configuration permits the use of a variety of non-volatile salts; ammonium sulfate was used in an example of strong cation exchange separations. The system was designed and evaluated for both micro- and nanoflow chromatography. The peptide detection limit was approximately 100 fmol for micro- and 20 fmol for nanoflow, demonstrating the concentration and mass sensitivity improvements expected with nanoelectrospray ionization. The 1D/2D-HPLC MS system is fully automated for routine peptide analyses, compatible with direct injection of proteolytic digests, and exhibits chromatographic reproducibility and sensitivity. Software permits operator selection of either a 1D or 2D configuration with corresponding system parameters as required for individual samples. The hardware elements and resulting performance are described in this paper.

Characterizing complex peptide mixtures using a multi-dimensional liquid chromatography-mass spectrometry system: Saccharomyces cerevisiae as a model system.

A rugged, reproducible, multi-dimensional LC-MS system was developed to identify and characterize proteins involved in protein-protein interactions and/or protein complexes. Our objective was to optimize chromatographic parameters for complex protein mixture analyses using automated peptide sequence recognition as an analytical end-point. The chromatographic system uses orthogonal separation mechanisms by employing strong cation exchange (SCX) in the first dimension and reversed phase (RP) in the second dimension. The system is fully automated and sufficiently robust to handle direct injections of protein digests. This system incorporates a streamlined post analysis results comparison, called DBParser, which permitted comprehensive evaluation of sample loading and chromatographic conditions to optimize the performance and reproducibility. Peptides obtained from trypsin digestion of a yeast soluble extract provided an open-ended model system containing a wide variety and dynamic range of components. Conditions are described that resulted in an average (n = 4) of 1489 unique peptide identifications, corresponding to 459 non-redundant protein sequence database records (SDRs) in the 20 microg soluble fraction digest.

Homozygosity mapping and whole-exome sequencing to detect SLC45A2 and G6PC3 mutations in a single patient with oculocutaneous albinism and neutropenia.

We evaluated a 32-year-old woman whose oculocutaneous albinism (OCA), bleeding diathesis, neutropenia, and history of recurrent infections prompted consideration of the diagnosis of Hermansky-Pudlak syndrome type 2. This was ruled out because of the presence of platelet δ-granules and absence of AP3B1 mutations. As parental consanguinity suggested an autosomal recessive mode of inheritance, we employed homozygosity mapping, followed by whole-exome sequencing, to identify two candidate disease-causing genes, SLC45A2 and G6PC3. Conventional dideoxy sequencing confirmed pathogenic mutations in SLC45A2, associated with OCA type 4 (OCA-4), and G6PC3, associated with neutropenia. The substantial reduction of SLC45A2 protein in the patient's melanocytes caused the mislocalization of tyrosinase from melanosomes to the plasma membrane and also led to the incorporation of tyrosinase into exosomes and secretion into the culture medium, explaining the hypopigmentation in OCA-4. Our patient's G6PC3 mRNA expression level was also reduced, leading to increased apoptosis of her fibroblasts under endoplasmic reticulum stress. To our knowledge, this report describes the first North American patient with OCA-4, the first culture of human OCA-4 melanocytes, and the use of homozygosity mapping, followed by whole-exome sequencing, to identify disease-causing mutations in multiple genes in a single affected individual.

Assessing data quality of peptide mass spectra obtained by quadrupole ion trap mass spectrometry.

An algorithm is introduced to assess spectral quality for peptide CID spectra acquired by a quadrupole ion trap mass spectrometer. The method employs a quadratic discriminant function calibrated with manually classified 'bad' and 'good' quality spectra, producing a single 'spectral quality' score. Many spectra examined that do not have significant matches are assessed to have good spectral quality, indicating that advances in search methods may yield substantial improvements in results.

Comparative enantioseparations with native beta-cyclodextrin and heptakis-(2-O-methyl- 3,6-di-O-sulfo)-beta-cyclodextrin in capillary electrophoresis.

Twenty-three cationic chiral analytes were resolved in capillary electrophoresis using native beta-cyclodextrin and single isomer heptakis-(2-O-methyl-3,6-di-O-sulfo)-beta-cyclodextrin as chiral selectors. For 12 of 16 chiral analytes resolved with both chiral selectors the enantiomer migration order was opposite. In selected cases the structure of cyclodextrin-analyte complexes in aqueous solution was investigated using one-dimensional transverse rotating frame nuclear Overhauser and exchange spectroscopy. It was found that in contrast to mainly inclusion-type complexes between chiral analytes and beta-cyclodextrin, external complexes are formed between the chiral analytes and structurally crowded, highly charged heptakis-(2-O-methyl-3,6-di-O-sulfo)-beta-cyclodextrin.

Open mass spectrometry search algorithm.

Large numbers of MS/MS peptide spectra generated in proteomics experiments require efficient, sensitive and specific algorithms for peptide identification. In the Open Mass Spectrometry Search Algorithm (OMSSA), specificity is calculated by a classic probability score using an explicit model for matching experimental spectra to sequences. At default thresholds, OMSSA matches more spectra from a standard protein cocktail than a comparable algorithm. OMSSA is designed to be faster than published algorithms in searching large MS/MS datasets.

DBParser: web-based software for shotgun proteomic data analyses.

We describe a web-based program called 'DBParser' for rapidly culling, merging, and comparing sequence search engine results from multiple LC-MS/MS peptide analyses. DBParser employs the principle of parsimony to consolidate redundant protein assignments and derive the most concise set of proteins consistent with all of the assigned peptide sequences observed in an experiment or series of experiments. The resulting reports summarize peptide and protein identifications from multidimensional experiments that may contain a single data set or combine data from a group of data sets, all related to a single analytical sample. Additionally, the results of multiple experiments, each of which may contain several data sets, can be compared in reports that identify features that are common or different. DBParser actively links to the primary mass spectral data and to public online databases such as NCBI, GO, and Swiss-Prot in order to structure contextually specific reports for biologists and biochemists.