Peter van der Geer

Phosphopeptide mapping and phosphoamino acid analysis by two-dimensional separation on thin-layer cellulose plates

Publisher Summary This chapter discusses the phosphopeptide mapping and phosphoamino acid analysis by two-dimensional separation on thin-layer cellulose plates. Peptide mapping is a powerful technique used to help determine peptide structure and composition of proteins. Peptide maps or fingerprints of proteolyzed proteins are usually obtained by resolution on either one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), reversed-phase high-performance liquid chromatography (HPLC), or by two-dimensional separation on thin-layer cellulose (TLC) plates. The most common applications of peptide mapping are (1) to compare proteins encoded by the same or related genes, (2) to prepare individual peptides for determining amino acid composition and sequence, and (3) to determine the precise location of amino acid residues that are posttranslationally modified by fatty acid acylation, glycosylation, methylation, acetylation, or phosphorylation.

MitoNEET is an iron-containing outer mitochondrial membrane protein that regulates oxidative capacity

Members of the thiazolidinedione (TZD) class of insulin-sensitizing drugs are extensively used in the treatment of type 2 diabetes. Pioglitazone, a member of the TZD family, has been shown to bind specifically to a protein named mitoNEET [Colca JR, McDonald WG, Waldon DJ, Leone JW, Lull JM, Bannow CA, Lund ET, Mathews WR (2004) Am J Physiol 286:E252–E260]. Bioinformatic analysis reveals that mitoNEET is a member of a small family of proteins containing a domain annotated as a CDGSH-type zinc finger. Although annotated as a zinc finger protein, mitoNEET contains no zinc, but instead contains 1.6 mol of Fe per mole of protein. The conserved sequence C-X-C-X2-(S/T)-X3-P-X-C-D-G-(S/A/T)-H is a defining feature of this unique family of proteins and is likely involved in iron binding. Localization studies demonstrate that mitoNEET is an integral protein present in the outer mitochondrial membrane. An amino-terminal anchor sequence tethers the protein to the outer membrane with the CDGSH domain oriented toward the cytoplasm. Cardiac mitochondria isolated from mitoNEET-null mice demonstrate a reduced oxidative capacity, suggesting that mito- NEET is an important iron-containing protein involved in the control of maximal mitochondrial respiratory rates.

The outer mitochondrial membrane protein mitoNEET contains a novel redox-active 2Fe-2S cluster

The outer mitochondrial membrane protein mitoNEET was discovered as a binding target of pioglitazone, an insulin-sensitizing drug of the thiazolidinedione class used to treat type 2 diabetes (Colca, J. R., McDonald, W. G., Waldon, D. J., Leone, J. W., Lull, J. M., Bannow, C. A., Lund, E. T., and Mathews, W. R. (2004) Am. J. Physiol. 286, E252–E260). We have shown that mitoNEET is a member of a small family of proteins containing a 39-amino-acid CDGSH domain. Although the CDGSH domain is annotated as a zinc finger motif, mitoNEET was shown to contain iron (Wiley, S. E., Murphy, A. N., Ross, S. A., van der Geer, P., and Dixon, J. E. (2007) Proc. Natl. Acad. Sci. U. S. A. 104, 5318–5323). Optical and electron paramagnetic resonance spectroscopy showed that it contained a redox-active pH-labile Fe-S cluster. Mass spectrometry showed the loss of 2Fe and 2S upon cofactor extrusion. Spectroscopic studies of recombinant proteins showed that the 2Fe-2S cluster was coordinated by Cys-3 and His-1. The His ligand was shown to be involved in the observed pH lability of the cluster, indicating that loss of this ligand via protonation triggered release of the cluster. mitoNEET is the first identified 2Fe-2S-containing protein located in the outer mitochondrial membrane. Based on the biophysical data and domain fusion analysis, mitoNEET may function in Fe-S cluster shuttling and/or in redox reactions.

v-Src induces Shc binding to tyrosine 63 in the cytoplasmic domain of the LDL receptor-related protein 1

We recently observed that the LDL receptor-related protein 1 (LRP-1) is tyrosine phosphorylated in v-Src-transformed cells. Using a GST-fusion protein containing the cytoplasmic domain of LRP-1, we show that LRP-1 is a direct substrate for v-Src in vitro. To study LRP-1 phosphorylation in vivo, we constructed an LRP-1 minireceptor composed of the β chain linked at the amino-terminus to a Myc epitope (Myc-LRPβ). When expressed together with v-Src, Myc-LRPβ becomes phosphorylated on tyrosine. Of the four tyrosine residues present in the cytoplasmic domain of LRP-1, only Tyr 63 is phosphorylated by v-Src in vivo or in vitro. Using fibroblasts deficient in Src, Yes and Fyn, we were able to show that there are multiple kinases present in the cell that can phosphorylate LRP-1. Tyrosine-phosphorylated LRP-1 associates with Shc, a PTB and SH2 domain containing signaling protein that is involved in the activation of Ras. Binding of the purified Shc PTB domain to Tyr 63 containing peptides shows that the interaction between LRP-1 and Shc is direct. We found that DAB, a PTB domain containing signaling protein that is involved in signaling by LDL receptor-related proteins in the nervous system, did not bind to full-length LRP-1. Our observations suggest that LRP-1 may be involved in normal and malignant signal transduction through a direct interaction with Shc adaptor proteins.

Phosphopeptide Mapping and Phosphoamino Acid Analysis on Cellulose Thin-Layer Plates

Publisher Summary This chapter discusses the use of phosphopeptide mapping and phosphoamino acid analysis on thin-layer cellulose. Phosphopeptide mapping is an important technique in the study of protein phosphorylation. It is used to determine the number and precise identity of sites of phosphorylation, to estimate the stoichiometry of phosphorylation at particular sites, and to deduce the identity of protein kinases responsible for their phosphorylation. Additionally, comparative phosphopeptide mapping is an invaluable tool for determining the identity, or lack of identity of phosphoproteins obtainable only in trace amounts. Phosphopeptide mapping has the advantage that it is extremely sensitive. In addition, individual phosphopeptides can be isolated from the inert cellulose coating of the plate, and used for further characterization including phosphoamino acid determination, N-terminal sequencing, and secondary digestion with additional proteases and chemicals.

Wolfram Syndrome protein, Miner1, regulates sulphydryl redox status, the unfolded protein response, and Ca2+ homeostasis

Miner1 is a redox‐active 2Fe2S cluster protein. Mutations in Miner1 result in Wolfram Syndrome, a metabolic disease associated with diabetes, blindness, deafness, and a shortened lifespan. Embryonic fibroblasts from Miner1−/− mice displayed ER stress and showed hallmarks of the unfolded protein response. In addition, loss of Miner1 caused a depletion of ER Ca2+ stores, a dramatic increase in mitochondrial Ca2+ load, increased reactive oxygen and nitrogen species, an increase in the GSSG/GSH and NAD+/NADH ratios, and an increase in the ADP/ATP ratio consistent with enhanced ATP utilization. Furthermore, mitochondria in fibroblasts lacking Miner1 displayed ultrastructural alterations, such as increased cristae density and punctate morphology, and an increase in O2 consumption. Treatment with the sulphydryl anti‐oxidant N‐acetylcysteine reversed the abnormalities in the Miner1 deficient cells, suggesting that sulphydryl reducing agents should be explored as a treatment for this rare genetic disease.

Interactions of the NPXY microdomains of the low density lipoprotein receptor-related protein 1.

The low density lipoprotein receptor‐related protein 1 (LRP1) mediates internalization of a large number of proteins and protein–lipid complexes and is widely implicated in Alzheimers disease. The cytoplasmic domain of LRP1 (LRP1‐CT) can be phosphorylated by activated protein‐tyrosine kinases at two NPXY motifs in LRP1‐CT; Tyr 4507 is readily phosphorylated and must be phosphorylated before phosphorylation of Tyr 4473 occurs. Pull‐down experiments from brain lysate revealed numerous proteins binding to LRP1‐CT, but the results were highly variable. To separate which proteins bind to each NPXY motif and their phosphorylation dependence, each NPXY motif microdomain was prepared in both phosphorylated and non‐phosphorylated forms and used to probe rodent brain extracts for binding proteins. Proteins that bound specifically to the microdomains were identified by LC‐MS/MS, and confirmed by Western blot. Recombinant proteins were then tested for binding to each NPXY motif. The NPXY4507 (membrane distal) was found to interact with a large number of proteins, many of which only bound the tyrosine‐phosphorylated form. This microdomain also bound a significant number of other proteins in the unphosphorylated state. Many of the interactions were later confirmed to be direct with recombinant proteins. The NPXY4473 (membrane proximal) bound many fewer proteins and only to the phosphorylated form.

Structural and functional consequences of tyrosine phosphorylation in the LRP1 cytoplasmic domain.

The cytoplasmic domain of LRP1 contains two NPXY motifs that have been shown to interact with signaling proteins. In previous work, we showed that Tyr4507 in the distal NPXY motif is phosphorylated by v-Src, whereas denaturation of the protein was required for phosphorylation of Tyr4473 in the membraneproximal NPXY motif. Amide H/D exchange studies reveal that the distal NPXY motif is fully solvent-exposed, whereas the proximal one is not. Phosphopeptide mapping combined with in vitro and in vivo kinase experiments show that Tyr4473 can be phosphorylated, but only if Tyr4507 is phosphorylated or substituted with glutamic acid. Amide H/D exchange experiments indicate that solvent accessibility increases across the entire LRP1 cytoplasmic region upon phosphorylation at Tyr4507; in particular the NPXY4473 motif becomes much more exposed. This differential phosphorylation is functionally relevant: binding of Snx17, which is known to bind at the proximal NPXY motif, is inhibited by phosphorylation at Tyr4473. Conversely, Shp2 binds most strongly when both of the NPXY motifs in LRP1 are phosphorylated.

Protein instability following transport or storage on dry ice

properties of transcription2,6 (Supplementary Note 4). To validate these methods, we first used simulations (Supplementary Note 5). For transcription sites smaller than the optical resolution, all methods yielded accurate estimates. However, for larger transcription sites, the simple methods led to gross underestimates, whereas FISH-quant gave accurate results (Fig. 1e). For elongated transcription sites, only the PSF superposition approach worked reliably. For experimental validation, we used an artificial reporter with transcription sites frequently exceeding the resolution. An RNase protection assay provided a rough, but independent, estimate of the ratio of mature versus nascent mRNA. The assay yielded ratios in the same range as the FISH-based quantifications, confirming their general validity (Supplementary Note 6 and Supplementary Methods). For a more accurate assessment of simple methods and FISH-quant, we then compared the nascent transcript counts. Much as for the large simulated transcription sites, the simple methods led to underestimated counts (Fig. 1f). Thus, FISH-quant accurately quantified nascent mRNA even when simple approaches did not. Finally, we used FISH-quant to analyze b-actin mRNA after serum induction and measured more than twice the amount of nascent mRNA than we did with simple methods, which illustrates the importance of accurate quantification even for endogenous genes (Supplementary Note 6 and Supplementary Methods). FISH-quant could also be applied to other structures with a dense accumulation of mRNA, such as processing (P)-bodies or stress granules. FISH-quant is controlled via graphical user interfaces in Matlab and requires no computational expertise. A batch mode allows users to automatically process multiple images. FISH-quant is available at http://code.google.com/p/fish-quant/ with a detailed manual and test data.

Toll-like receptors stimulate regulated intramembrane proteolysis of the CSF-1 receptor through Erk activation.

The CSF‐1 receptor is a protein‐tyrosine kinase that regulates the renewal, differentiation and activation of monocytes and macrophages. We have recently shown that the CSF‐1 receptor undergoes regulated intramembrane proteolysis, or RIPping. Here, we report that RIPping can be observed in response to pathogen‐associated molecules, which act through Toll‐like receptors (TLRs). TLR‐induced CSF‐1 receptor RIPping is largely independent of protein kinase C, while maximal RIPping depends on Erk activation. Our studies show that CSF‐1 receptor RIPping can be activated by various intracellular signal transduction pathways and that RIPping is likely to play an important role during macrophage activation.