Mechanisms that determine the transmembrane disposition of proteins.

The final orientation that a protein assumes in the membrane of the endoplasmic reticulum is determined by a few types of signal sequences and their respective interactions with the membrane insertion complex. Membrane insertion occurs via a series of discrete steps, some of which are regulated by GTP- and ATP-binding proteins. Analysis of the protein components in proximity to nascent secretory and membrane proteins has revealed novel proteins in the endoplasmic reticulum that may form part of the membrane insertion complex.

Protein translocation across membranes: common themes in divergent organisms.

Specific signal sequences are required for the translocation of proteins into and across both the endoplasmic reticulum of eukaryotes and the plasma membrane of prokaryotes. The similar properties of these signals, together with their ability to function when transferred between systems, suggested that the mechanisms of translocation in the two cases may be fundamentally similar. Indeed, recent findings have revealed striking similarities between essential components of the prokaryotic and eukaryotic translocation systems, suggesting that both are derived from a common ancestor.

The signal sequence interacts with the methionine-rich domain of the 54-kD protein of signal recognition particle.

The signal sequence of nascent preprolactin interacts with the 54-kD protein of the signal recognition particle (SRP54). To identify the domain or site on SRP54 that interacts with the signal sequence we used a photocross-linking approach followed by limited proteolysis and immunoprecipitation using anti-peptide antibodies specific for defined regions of SRP54. We found that the previously identified methionine-rich RNA-binding domain of SRP54 (SRP54M domain) also interacts with the signal sequence. The smallest fragment that was found to be crosslinked to the signal sequence comprised the COOH-terminal 6-kD segment of the SRP54M domain. No cross-link to the putative GTP-binding domain of SRP54 (SRP54G domain) was found. Proteolytic cleavage between the SRP54M domain and SRP54G domain did not impair the subsequent interaction between the signal sequence and the SRP54M domain. Our results show that both the RNA binding and signal sequence binding functions of SRP54 are performed by the SRP54M domain.

Requirements for the membrane insertion of signal-anchor type proteins.

Proteins which are inserted and anchored in the membrane of the ER by an uncleaved signal-anchor sequence can assume two final orientations. Type I signal-anchor proteins translocate the NH2 terminus across the membrane while type II signal-anchor proteins translocate the COOH terminus. We investigated the requirements for cytosolic protein components and nucleotides for the membrane targeting and insertion of single-spanning type I signal-anchor proteins. Besides the ribosome, signal recognition particle (SRP), GTP, and rough microsomes (RMs) no other components were found to be required. The GTP analogue GMPPNP could substitute for GTP in supporting the membrane insertion of IMC-CAT. By using a photocrosslinking assay we show that for secreted, type I and type II signal-anchor proteins the presence of both GTP and RMs is required for the release of the nascent chain from the 54-kD subunit of SRP. For two of the proteins studied the release of the nascent chain from SRP54 was accompanied by a new interaction with components of the ER. We conclude that the GTP-dependent release of the nascent chain from SRP54 occurs in an identical manner for each of the proteins studied.

The identification of proteins in the proximity of signal-anchor sequences during their targeting to and insertion into the membrane of the ER.

Using a photocross-linking approach we have investigated the cytosolic and membrane components involved in the targeting and insertion of signal-anchor proteins into the membrane of the ER. The nascent chains of both type I and type II signal-anchor proteins can be cross-linked to the 54-kD subunit of the signal recognition particle. Upon addition of rough microsomes the type I and type II signal-anchor proteins interact with a number of components. Both types of protein interact with an integral membrane protein, the signal sequence receptor, previously identified by its proximity to preprolactin during its translocation (Wiedmann, M., T.V. Kurzchalia, E. Hartmann, and T.A. Rapoport. 1987. Nature [Lond.] 328:830-833). Three proteins, previously unidentified, were found to be cross-linked to the nascent chains of the signal-anchor proteins. Among them was a 37-kD protein that was found to be the main component interacting with the type I SA protein used. These proteins were not seen in the absence of membranes suggesting they are components of the ER. The ability of the nascent chains to be cross-linked to these identified proteins was shown to be abolished by prior treatment with agents known to disrupt translocation intermediates or ribosomes. We propose that the newly identified proteins function either in the membrane insertion of only a subset of proteins or only at a specific stage of insertion.

Sec61p is adjacent to nascent type I and type II signal-anchor proteins during their membrane insertion.

We have identified membrane components which are adjacent to type I and type II signal-anchor proteins during their insertion into the membrane of the ER. Using two different cross-linking approaches a 37-38-kD nonglycosylated protein, previously identified as P37 (High, S., D. Görlich, M. Wiedmann, T. A. Rapoport, and B. Dobberstein. 1991. J. Cell Biol. 113:35-44), was found adjacent to all the membrane inserted nascent chains used in this study. On the basis of immunoprecipitation, this ER protein was shown to be identical to the recently identified mammalian Sec61 protein. Thus, Sec61p is the principal cross-linking partner of both type I and type II signal-anchor proteins during their membrane insertion (this work), and of secretory proteins during their translocation (Görlich, D., S. Prehn, E. Hartmann, K.-U. Kalies, and T. A. Rapoport. 1992. Cell. 71:489-503). We propose that membrane proteins of both orientations, and secretory proteins employ the same ER translocation sites, and that Sec61p is a core component of these sites.

Interaction of the thiol-dependent reductase ERp57 with nascent glycoproteins.

Calnexin and calreticulin interact specifically with newly synthesized glycoproteins in the endoplasmic reticulum (ER) and function as molecular chaperones. The carbohydrate-specific interactions between ER components and glycoproteins synthesized in isolated canine pancreatic microsomes were analyzed using a cross-linking approach. A carbohydrate-dependent interaction between newly synthesized glycoproteins, the thiol-dependent reductase ERp57, and either calnexin or calreticulin was identified. The interaction between ERp57 and the newly synthesized glycoproteins required trimming of the N-linked oligosaccharide side chain. Thus, it is likely that ERp57 functions as part of the glycoprotein-specific quality control machinery operating in the lumen of the ER.

ERp57 functions as a subunit of specific complexes formed with the ER lectins calreticulin and calnexin.

ERp57 is a lumenal protein of the endoplasmic reticulum (ER) and a member of the protein disulfide isomerase (PDI) family. In contrast to archetypal PDI, ERp57 interacts specifically with newly synthesized glycoproteins. In this study we demonstrate that ERp57 forms discrete complexes with the ER lectins, calnexin and calreticulin. Specific ERp57/calreticulin complexes exist in canine pancreatic microsomes, as demonstrated by SDS-PAGE after cross-linking, and by native electrophoresis in the absence of cross-linking. After in vitro translation and import into microsomes, radiolabeled ERp57 can be cross-linked to endogenous calreticulin and calnexin while radiolabeled PDI cannot. Likewise, radiolabeled calreticulin is cross-linked to endogenous ERp57 but not PDI. Similar results were obtained in Lec23 cells, which lack the glucosidase I necessary to produce glycoprotein substrates capable of binding to calnexin and calreticulin. This observation indicates that ERp57 interacts with both of the ER lectins in the absence of their glycoprotein substrate. This result was confirmed by a specific interaction between in vitro synthesized calreticulin and ERp57 prepared in solution in the absence of other ER components. We conclude that ERp57 forms complexes with both calnexin and calreticulin and propose that it is these complexes that can specifically modulate glycoprotein folding within the ER lumen.

Formation and turnover of NSF- and SNAP-containing "fusion" complexes occur on undocked, clathrin-coated vesicle-derived membranes.

Specificity of vesicular transport is determined by pair-wise interaction between receptors (SNAP receptors or SNAREs) associated with a transport vesicle and its target membrane. Two additional factors, N-ethylmaleimide-sensitive fusion protein (NSF) and soluble NSF attachment protein (SNAP) are ubiquitous components of vesicular transport pathways. However, the precise role they play is not known. On the basis that NSF and SNAP can be recruited to preformed SNARE complexes, it has been proposed that NSF- and SNAP-containing complexes are formed after SNARE-dependent docking of transport vesicles. This would enable ATPase-dependent complex disassembly to be coupled directly to membrane fusion. Alternatively, binding and release of NSF/SNAP may occur before vesicle docking, and perhaps be involved in the activation of SNAREs. To gain more information about the point at which so-called 20S complexes form during the transport vesicle cycle, we have examined NSF/SNAP/SNARE complex turnover on clathrin-coated vesicle-derived membranes in situ. This has been achieved under conditions in which the extent of membrane docking can be precisely monitored. We demonstrate by UV-dependent cross-linking experiments, coupled to laser light-scattering analysis of membranes, that complexes containing NSF, SNAP, and SNAREs will form and dissociate on the surface of undocked transport vesicles.

Protein translocation at the membrane of the endoplasmic reticulum.

The process of insertion into and translocation across the ER membrane is a significant step in the biosynthesis of a membrane or secretory protein. This commits the protein to a destination within the "secretory pathway" (Palade, 1975) and is part of a complex series of events involving protein targeting, translocation, maturation and sorting, which finally results in a biologically-active protein being delivered to its correct subcellular location. The focus for this review has been the initial events of this process. Proteins which constitute at least a part of the actual translocation site across the ER membrane have been identified and the minimum components required to reconstitute ER translocation in vitro have been defined. A detailed description of the architecture of the ER translocation site and the molecular events occurring during translocation and membrane insertion remain goals for the future. The process occurring in vivo may be more complex since (i) each translocation site may only promote a single round of translocation in vitro whereas in vivo the sites must operate catalytically and go through many cycles of translocation and insertion (see Gilmore, 1993) and (ii) the in vivo requirement for a translocation site which is impermeable to small molecules (in order not to dissipate chemical gradients and the redox potential) is unlikely to be important for in vitro assays. Thus, other components which play a vital role in protein translocation and membrane insertion in vivo may remain to be identified. Our future aim must be to place a detailed understanding of the molecular events of the translocation process into the context of the normal cellular environment.

Protein targeting and translocation at the endoplasmic reticulum membrane--through the eye of a needle?

SRP-dependent and SRP-independent targeting routes deliver precursor proteins to the ER membrane translocon. These precursors are translocated into (for membrane proteins) and across (for secretory protein) the ER membrane via aqueous channels composed of oligomers of the Sec61 complex. Both ends of the ER translocon are 'gated' and the opening and closing of these gates are closely regulated. The lateral exit of hydrophobic polypeptide regions into the phospholipid bilayer also appears to be a carefully controlled process. Accessory components are transiently associated with active ER translocation sites and modify the nascent polypeptide as it appears on the luminal side of the membrane.

The Sec61 complex is essential for the insertion of proteins into the membrane of the endoplasmic reticulum.

Cross-linking studies have implicated Sec61 alpha as the principal component adjacent to newly synthesised membrane proteins during insertion into the endoplasmic reticulum. Using proteoliposomes which have been reconstituted from purified components of the endoplasmic reticulum [Görlich, D and Rapoport, T.A., Cell 75 (1993) 615-630] we have found that the Sec61 complex, consisting of three subunits, is essential for the insertion of single-spanning membrane proteins. This is true for signal-anchor proteins of both orientations, and for proteins with a cleavable signal sequence. These results support the view that Sec61 alpha is a major component of the ER translocation site and promotes both the insertion of membrane proteins and the translocation of secretory proteins.

Glycoprotein folding in the endoplasmic reticulum: a tale of three chaperones?

The endoplasmic reticulum (ER) is a major site of protein synthesis and its inside, or lumen, is a major site of protein folding. The lumen of the ER contains many folding factors and molecular chaperones, which facilitate protein folding by increasing both the rate and the efficiency of this process. Amongst the many ER folding factors, there are three components that specifically modulate the folding glycoproteins bearing N-linked carbohydrate side chains. These components are calnexin, calreticulin and ERp57, and this review focuses on the molecular basis for their capacity to influence glycoprotein folding.

The ABC's of staff retention.

The purpose of this article is to aid management in recognizing the key components to staff retention. Preparation for recruitment efforts, evaluating compensation, and establishing and maintaining good channels of communication are worthy undertakings for the purposes of reducing turnover. Combating turnover is really as easy as Appreciating your staff, rewarding the Behavior you want, and Continuing to ensure a work environment that is conducive to a place employees want to work. One of the key factors is listening. Do you hear what your staff is saying? Can you address their concerns? Do you communicate regularly with line staff? Are there ideas they have that can lead to improvements? Lead by example. If your staff sees your passion for the work, they will respect you and work hard to deliver what you have agreed are the goals on an individual and overall business objective level. Once you have established the aggregate levels that exist within your practice, you can move on to evaluating where each employee falls within the range.

Alzheimer's disease: early alterations in brain DNA methylation at ANK1, BIN1, RHBDF2 and other loci.

We used a collection of 708 prospectively collected autopsied brains to assess the methylation state of the brain's DNA in relation to Alzheimer's disease (AD). We found that the level of methylation at 71 of the 415,848 interrogated CpGs was significantly associated with the burden of AD pathology, including CpGs in the ABCA7 and BIN1 regions, which harbor known AD susceptibility variants. We validated 11 of the differentially methylated regions in an independent set of 117 subjects. Furthermore, we functionally validated these CpG associations and identified the nearby genes whose RNA expression was altered in AD: ANK1, CDH23, DIP2A, RHBDF2, RPL13, SERPINF1 and SERPINF2. Our analyses suggest that these DNA methylation changes may have a role in the onset of AD given that we observed them in presymptomatic subjects and that six of the validated genes connect to a known AD susceptibility gene network.

Germline predictors of androgen deprivation therapy response in advanced prostate cancer.

OBJECTIVE: To evaluate whether germline variations in genes involved in sex steroid biosynthesis and metabolic pathways predict time to treatment failure for patients with advanced prostate cancer undergoing androgen deprivation therapy (ADT), because there are few known clinical predictors of response. PATIENTS AND METHODS: In a cohort of 304 patients with advanced prostate cancer undergoing ADT, we genotyped 746 single-nucleotide polymorphisms (SNPs) from 72 genes from germline DNA (680 tagSNPs from 58 genes and 66 candidate SNPs from 20 genes [6 genes common in both]). Association with the primary end point of time to ADT failure was assessed using proportional hazards regression models at the gene level (for genes with tagging SNPs) and at the SNP level. False discovery rates (FDRs) of 0.10 or less were considered noteworthy to account for multiple testing. RESULTS: At the gene level, TRMT11 showed the strongest association with time to ADT failure (P<.001; FDR=0.008). Two of 4 TRMT11 tagSNPs were associated with time to ADT failure. Median time to ADT failure for rs1268121 (A>G) was 3.05 years for the AA, 4.27 years for the AG, and 6.22 years for the GG genotypes (P=.002), and for rs6900796 (G>A), it was 2.42 years for the GG, 3.52 years for the AG, and 4.18 years for the AA genotypes (P<.001). No other gene level or SNP level tests had an FDR of 0.10 or less. CONCLUSION: Genetic variation in TRMT11 was associated with time to ADT failure. Confirmation of these preliminary findings in an independent cohort is needed.

Association of TNFSF8 polymorphisms with peripheral neutrophil count.

OBJECTIVE: To investigate the association between 347 single-nucleotide polymorphisms within candidate genes of the tumor necrosis factor, interleukin 1 and interleukin 6 families with neutrophil count. PATIENTS AND METHODS: Four hundred cases with heart failure after myocardial infarction (MI) were matched by age, sex, and date of incident MI to 694 controls (MI without post-MI heart failure). Both genotypes and neutrophil count at admission for incident MI were available in 314 cases and 515 controls. RESULTS: We found significant associations between the TNFSF8 poly morphisms rs927374 (P=5.1 x 10(-5)) and rs2295800 (P=1.3 x 10(-4)) and neutrophil count; these single-nucleotide polymorphisms are in high linkage disequilibrium (r(2)=0.97). Associations persisted after controlling for clinical characteristics and were unchanged after adjusting for case-control status. For rs927374, the neutrophil count of GG homozygotes (7.6±5.1) was 16% lower than that of CC homozygotes (9.0±5.2). CONCLUSION: The TNFSF8 polymorphisms rs927374 and rs2295800 were associated with neutrophil count. This finding suggests that post-MI inflammatory response is genetically modulated.

Efficacy of omega-3 fatty acids in mood disorders - a systematic review and metaanalysis.

OBJECTIVES: Existing efficacy trials of Omega-3 (omega-3) fatty acids in mood disorders have yielded inconsistent results. The current paper is an effort to provide a systematic review and meta-analysis to evaluate efficacy of omega-3 fatty acids in treatment of mood disorders. DESIGN: We searched Medline, Embase, PsychInfo, and the Cochrane Controlled Trials registry up to June 2008 for randomized trials investigating efficacy of omega-3 fatty acids in mood disorders.We conducted random effects meta-analyses.We used the I2 statistic to quantify between-study inconsistency, and conducted pre-specified subgroup analyses to explore potential explanations for inconsistency. OBSERVATIONS: We included 21 trials in our systematic review and found 13 trials to be eligible for meta-analysis. The pooled standardized mean difference in depressed mood states (n = 554 in 12 trials) was -0.47 (95% CI:-0.92,-0.02; I2 = 82.7; p = 0.07) and in manic mood states (n = 126 in 4 trials) was 0.22 (95% CI: -0.21, 0.65; I2 = 40.5; p = 0.31).We did not identify any treatment- subgroup interaction across forms of omega-3 fatty acids preparations (P = 0.99) or patient diagnosis (bipolar vs. unipolar depressive disorder; P = 0.96); there was a significant correlation between omega-3 fatty acids dose and treatment effect on depressive symptoms (r = 0.5, p = 0.04), but not on manic symptoms (P = 0.3). CONCLUSIONS: The available evidence suggests that omega-3 fatty acids are a potential treatment of depressive disorders, but not mania. The unexplained between-study inconsistency and imprecision of the pooled estimates mitigate this suggestion. Large randomized placebo-controlled trials are needed to better estimate the value of this intervention for patients with depression.