Taking organelles apart, putting them back together and creating new ones: lessons from the endoplasmic reticulum.

The endoplasmic reticulum (ER) is a highly dynamic organelle. It is composed of four subcompartments including nuclear envelope (NE), rough ER (rER), smooth ER (sER) and transitional ER (tER). The subcompartments are interconnected, can fragment and dissociate and are able to reassemble again. They coordinate with cell function by way of protein regulators in the surrounding cytosol. The activity of the many associated molecular machines of the ER as well as the fluid nature of the limiting membrane of the ER contribute extensively to the dynamics of the ER. This review examines the properties of the ER that permit its isolation and purification and the physiological conditions that permit reconstitution both in vitro and in vivo in normal and in disease conditions.

Proteomic analysis of the transitional endoplasmic reticulum in hepatocellular carcinoma: an organelle perspective on cancer.

The transitional endoplasmic reticulum (tER) is composed of both rough and smooth ER membranes and thus participates in functions attributed to both these two subcellular compartments. In this paper we have compared the protein composition of tER isolated from dissected liver tumor nodules of aflatoxin B1-treated rats with that of tER from control liver. Tandem mass spectrometry (MS), peptide counts and immunoblot validation were used to identify and determine the relative expression level of proteins. Inhibitors of apoptosis (i.e. PGRMC1, tripeptidyl peptidase II), proteins involved in ribosome biogenesis (i.e. nucleophosmin, nucleolin), proteins involved in translation (i.e. eEF-2, and subunits of eIF-3), proteins involved in ubiquitin metabolism (i.e. proteasome subunits, USP10) and proteins involved in membrane traffic (i.e. SEC13-like 1, SEC23B, dynactin 1) were found overexpressed in tumor tER. Transcription factors (i.e. Pur-beta, BTF3) and molecular targets for C-Myc and NF-kappa B were observed overexpressed in tER from tumor nodules. Down-regulated proteins included cytochrome P450 proteins and enzymes involved in fatty acid metabolism and in steroid metabolism. Unexpectedly expression of the protein folding machinery (i.e. calreticulin) and proteins of the MHC class I peptide-loading complex did not change. Proteins of unknown function were detected in association with the tER and the novel proteins showing differential expression are potential new tumor markers. In many cases differential expression of proteins in tumor tER was comparable to that of corresponding genes reported in the Oncomine human database. Thus the molecular profile of tumor tER is different and this may confer survival advantage to tumor cells in cancer.

Brain-specific proteins decline in the cerebrospinal fluid of humans with Huntington disease.

We integrated five sets of proteomics data profiling the constituents of cerebrospinal fluid (CSF) derived from Huntington disease (HD)-affected and -unaffected individuals with genomics data profiling various human and mouse tissues, including the human HD brain. Based on an integrated analysis, we found that brain-specific proteins are 1.8 times more likely to be observed in CSF than in plasma, that brain-specific proteins tend to decrease in HD CSF compared with unaffected CSF, and that 81% of brain-specific proteins have quantitative changes concordant with transcriptional changes identified in different regions of HD brain. The proteins found to increase in HD CSF tend to be liver-associated. These protein changes are consistent with neurodegeneration, microgliosis, and astrocytosis known to occur in HD. We also discuss concordance between laboratories and find that ratios of individual proteins can vary greatly, but the overall trends with respect to brain or liver specificity were consistent. Concordance is highest between the two laboratories observing the largest numbers of proteins.

Quantitative proteomics analysis of the secretory pathway.

We report more than 1400 proteins of the secretory-pathway proteome and provide spatial information on the relative presence of each protein in the rough and smooth ER Golgi cisternae and Golgi-derived COPI vesicles. The data support a role for COPI vesicles in recycling and cisternal maturation, showing that Golgi-resident proteins are present at a higher concentration than secretory cargo. Of the 1400 proteins, 345 were identified as previously uncharacterized. Of these, 230 had their subcellular location deduced by proteomics. This study provides a comprehensive catalog of the ER and Golgi proteomes with insight into their identity and function.

Early dysregulation of hippocampal proteins in transgenic rats with Alzheimer's disease-linked mutations in amyloid precursor protein and presenilin 1.

The response of the hippocampal proteome to expression of mutant proteins present in familial forms of Alzheimer's disease (AD) was studied using transgenic rats. These animals carry both the amyloid precursor protein Swedish and 717 mutation (APP(SW+717)) as well as the presenilin 1 Finnish mutation (PS1(FINN)). This transgenic rat model displays intracellular amyloid beta (Abeta) in neurons of the neocortex and the hippocampus (CA2 and CA3). The hippocampus was selected as it is one of the first brain regions affected in AD and is involved in the processing of short-term memory and spatial memory. Applying a proteomic approach, we demonstrate that the expression of APP(SW+717) and PS1(FINN) transgenes causes changes in expression of hippocampal proteins, some of which have been previously linked to learning and memory formation. The protein alterations documented here occur in the absence of plaque formation and prior to the onset of cognitive deficits later observed in these transgenic rats. This indicates that molecular changes take place in the hippocampal neurons in response to expression of mutant proteins APP(SW+717) and PS1(FINN), which precede the occurrence of overt extracellular accumulation of extracellular amyloid. The implications of these findings on our understanding of the early stages of AD are discussed.

Tandem MS analysis of brain clathrin-coated vesicles reveals their critical involvement in synaptic vesicle recycling.

Tandem MS has identified 209 proteins of clathrin-coated vesicles (CCVs) isolated from rat brain. An overwhelming abundance of peptides were assigned to the clathrin coat with a 1:1 stoichiometry observed for clathrin heavy and light chains and a 2:1 stoichiometry of clathrin heavy chain with clathrin adaptor protein heterotetramers. Thirty-two proteins representing many of the known components of synaptic vesicles (SVs) were identified, supporting that a main function for brain CCVs is to recapture SVs after exocytosis. A ratio of vesicle-N-ethylmaleimide-sensitive factor attachment protein receptors to target-N-ethylmaleimide-sensitive factor attachment protein receptors, similar to that previously detected on SVs, supports a single-step model for SV sorting during CCV-mediated recycling of SVs. The uncovering of eight previously undescribed proteins, four of which have to date been linked to clathrin-mediated trafficking, further attests to the value of the current organelle-based proteomics strategy.