Improving Identification of In-organello Protein-Protein Interactions Using an Affinity-enrichable, Isotopically Coded, and Mass Spectrometry-cleavable Chemical Crosslinker.

An experimental and computational approach for identification of protein-protein interactions by ex vivo chemical crosslinking and mass spectrometry (CLMS) has been developed that takes advantage of the specific characteristics of cyanurbiotindipropionylsuccinimide (CBDPS), an affinity-tagged isotopically coded mass spectrometry (MS)-cleavable crosslinking reagent. Utilizing this reagent in combination with a crosslinker-specific data-dependent acquisition strategy based on MS2 scans, and a software pipeline designed for integrating crosslinker-specific mass spectral information led to demonstrated improvements in the application of the CLMS technique, in terms of the detection, acquisition, and identification of crosslinker-modified peptides. This approach was evaluated on intact yeast mitochondria, and the results showed that hundreds of unique protein-protein interactions could be identified on an organelle proteome-wide scale. Both known and previously unknown protein-protein interactions were identified. These interactions were assessed based on their known sub-compartmental localizations. Additionally, the identified crosslinking distance constraints are in good agreement with existing structural models of protein complexes involved in the mitochondrial electron transport chain.

LAMTOR/Ragulator is a negative regulator of Arl8b- and BORC-dependent late endosomal positioning.

Signaling from lysosomes controls cellular clearance and energy metabolism. Lysosomal malfunction has been implicated in several pathologies, including neurodegeneration, cancer, infection, immunodeficiency, and obesity. Interestingly, many functions are dependent on the organelle position. Lysosomal motility requires the integration of extracellular and intracellular signals that converge on a competition between motor proteins that ultimately control lysosomal movement on microtubules. Here, we identify a novel upstream control mechanism of Arl8b-dependent lysosomal movement toward the periphery of the cell. We show that the C-terminal domain of lyspersin, a subunit of BLOC-1-related complex (BORC), is essential and sufficient for BORC-dependent recruitment of Arl8b to lysosomes. In addition, we establish lyspersin as the linker between BORC and late endosomal/lysosomal adaptor and mitogen activated protein kinase and mechanistic target of rapamycin activator (LAMTOR) complexes and show that epidermal growth factor stimulation decreases LAMTOR/BORC association, thereby promoting BORC- and Arl8b-dependent lysosomal centrifugal transport.

Pharmacological correction of misfolding of ABC proteins.

The endoplasmic reticulum (ER) quality control system distinguishes between correctly and incorrectly folded proteins to prevent processing of aberrantly folded conformations along the secretory pathway. Non-synonymous mutations can lead to misfolding of ABC proteins and associated disease phenotypes. Specific phenotypes may at least partially be corrected by small molecules, so-called pharmacological chaperones. Screening for folding correctors is expected to open an avenue for treatment of diseases such as cystic fibrosis and intrahepatic cholestasis.

Comprehensive comparative and semiquantitative proteome of a very low number of native and matched epstein-barr-virus-transformed B lymphocytes infiltrating human melanoma.

Melanoma, the deadliest form of skin cancer, is highly immunogenic and frequently infiltrated with immune cells including B cells. The role of tumor-infiltrating B cells (TIBCs) in melanoma is as yet unresolved, possibly due to technical challenges in obtaining TIBCs in sufficient quantity for extensive studies and due to the limited life span of B cells in vitro. A comprehensive workflow has thus been developed for successful isolation and proteomic analysis of a low number of TIBCs from fresh, human melanoma tissue. In addition, we generated in vitro-proliferating TIBC cultures using simultaneous stimulation with Epstein-Barr virus (EBV) and the TLR9 ligand CpG-oligodesoxynucleotide (CpG ODN). The FASP method and iTRAQ labeling were utilized to obtain a comparative, semiquantitative proteome to assess EBV-induced changes in TIBCs. By using as few as 100 000 B cells (∼5 µg protein)/sample for our proteomic study, a total number of 6507 proteins were identified. EBV-induced changes in TIBCs are similar to those already reported for peripheral B cells and largely involve changes in cell cycle proliferation, apoptosis, and interferon response, while most of the proteins were not significantly altered. This study provides an essential, further step toward detailed characterization of TIBCs including functional in vitro analysis.

Combining filter-aided sample preparation and pseudoshotgun technology to profile the proteome of a low number of early passage human melanoma cells.

The performance of two proteomic sample preparation methods, "pseudoshotgun" (PSG) and filter-aided sample preparation (FASP) were compared in terms of the number of identified proteins, representation of cellular component GO (gene ontology) categories in the obtained list of proteins, and the efficiency of both methods in the proteomic analysis of a very low number of cells. Both methods were combined to obtain a proteomic profile of a short-term culture (passage 3) of melanoma cells, established in our laboratory from a human metastatic melanoma lesion. The data revealed that with FASP, usually more proteins are identified than with PSG when analyzing a higher number of cells (≥ 5000/injection), whereas PSG is favorable when analyzing only a very small amount of cells (250-500/injection). PSG and FASP, however, are complementary techniques, as combining both methods further increases the number of identified proteins. Moreover, we show that it is feasible to identify a substantial number of proteins from only 250 cells/injection that is equivalent to 60 ng of protein.

Phosphosite mapping of P-type plasma membrane H+-ATPase in homologous and heterologous environments.

Phosphorylation is an important posttranslational modification of proteins in living cells and primarily serves regulatory purposes. Several methods were employed for isolating phosphopeptides from proteolytically digested plasma membranes of Arabidopsis thaliana. After a mass spectrometric analysis of the resulting peptides we could identify 10 different phosphorylation sites in plasma membrane H(+)-ATPases AHA1, AHA2, AHA3, and AHA4/11, five of which have not been reported before, bringing the total number of phosphosites up to 11, which is substantially higher than reported so far for any other P-type ATPase. Phosphosites were almost exclusively (9 of 10) in the terminal regulatory domains of the pumps. The AHA2 isoform was subsequently expressed in the yeast Saccharomyces cerevisiae. The plant protein was phosphorylated at multiple sites in yeast, and surprisingly, seven of nine of the phosphosites identified in AHA2 were identical in the plant and fungal systems even though none of the target sequences in AHA2 show homology to proteins of the fungal host. These findings suggest an unexpected accessibility of the terminal regulatory domain of plasma membrane H(+)-ATPase to protein kinase action.

Potassium as an intrinsic uncoupler of the plasma membrane H+-ATPase.

The plant plasma membrane proton pump (H(+)-ATPase) is stimulated by potassium, but it has remained unclear whether potassium is actually transported by the pump or whether it serves other roles. We now show that K(+) is bound to the proton pump at a site involving Asp(617) in the cytoplasmic phosphorylation domain, from where it is unlikely to be transported. Binding of K(+) to this site can induce dephosphorylation of the phosphorylated E(1)P reaction cycle intermediate by a mechanism involving Glu(184) in the conserved TGES motif of the pump actuator domain. Our data identify K(+) as an intrinsic uncoupler of the proton pump and suggest a mechanism for control of the H(+)/ATP coupling ratio. K(+)-induced dephosphorylation of E(1)P may serve regulatory purposes and play a role in negative regulation of the transmembrane electrochemical gradient under cellular conditions where E(1)P is accumulating.