大鼠肝细胞过氧化物酶体的提取

:采用蔗糖密度梯度离心法 ( 950 0 0× g,2 h)提取大鼠肝细胞过氧化物酶体 ,所得过氧化物酶体形态完整 ,纯度与肝匀浆相比提高了 2 6倍 ,仅有少量 ( 0 .5%～ 0 .9%)的微粒体和线粒体污染 ,回收率为 1 2 %。为研究过氧化物酶体提供了有效的分离方法。此法还可将过氧化物酶体、微粒体、线粒体同时进行分离。     

Differential proteomic analysis of the endoplasmic reticulum from developing and germinating seeds of castor (Ricinus communis) identifies seed protein precursors as significant components of the endoplasmic reticulum

The endoplasmic reticulum is a major compartment of storage protein and lipid biosynthesis. Maximal synthesis of these storage compounds occurs during seed development with breakdown occurring during germination. In this study, we have isolated four independent preparations of ER from both developing and germinating seeds of castor bean (Ricinus communis) and used 2-D DIGE, and a combination of PMF and MS/MS sequencing, to quantify and identify differences in protein complement at both stages. Ninety protein spots in the developing seeds are up-regulated and 19 individual proteins were identified, the majority of these are intermediates of seed storage synthesis and protein folding. The detection of these transitory storage proteins in the ER is discussed in terms of protein trafficking and processing. In germinating seed ER 15 spots are elevated, 5 of which were identified, amongst them was malate synthetase which is a component of the glyoxysome which is believed to originate from the ER. Notably no proteins involved in complex lipid biosynthesis were identified in the urea soluble ER fraction indicating that they are probably all integral membrane proteins.     

70-kDa peroxisomal membrane protein related protein (P70R/ABCD4) localizes to endoplasmic reticulum not peroxisomes, and NH2-terminal hydrophobic property determines the subcellular localization of ABC subfamily D proteins

70-kDa peroxisomal membrane protein related protein (P70R/ABCD4) is a member of ATP-binding cassette (ABC) protein subfamily D. ABC subfamily D proteins are also known as peroxisomal ABC proteins. Therefore, P70R is thought to be a peroxisomal membrane protein. However, the subcellular localization of P70R is not extensively investigated. In this study, we transiently expressed P70R in fusion with HA (P70R-HA) in CHO cells and examined subcellular localization by immunofluorescence. Surprisingly, P70R-HA was localized to the endoplasmic reticulum (ER), not to peroxisomes. To examine the ER-targeting property of P70R, we expressed various NH₂-terminal deletion constructs of P70R. Among the NH₂-terminal deletion constructs, mutant proteins starting with hydrophobic transmembrane segment (TMS) were localized to ER, but the ones containing the NH₂-terminal hydrophilic cytosolic domain were not. ABC subfamily D proteins destined for peroxisomes have NH₂-terminal hydrophilic region adjacent to TMS1. However, only P70R lacks the region and is translated with NH₂-terminal hydrophobic TMS1. Furthermore, attachment of the NH₂-terminal hydrophilic domain to the NH₂-terminus of P70R excluded P70R from the ER-targeting pathway. These data suggest that P70R resides in the ER but not the peroxisomal membranes, and the hydrophobic property of NH₂-terminal region determines the subcellular localization of ABC subfamily D proteins.     

The Mx GTPase family of interferon-induced antiviral proteins

Mx proteins are interferon-induced members of the dynamin superfamily of large GTPases. They inhibit a wide range of viruses by blocking an early stage of the replication cycle. Studies in genetically defined mouse strains highlight their powerful action in early antiviral host defence.     

Identification and localization of calreticulin in plant cells

Summary In the present study we have investigated the presence and distribution of calreticulin in plant protoplasts. Calreticulin was purified from plant homogenates using a selective ammonium sulfate precipitation procedure developed for the purification of mammalian calreticulins and shown to bind calcium in⁴⁵Ca²⁺ overlay assays. The protein was localized to plant cell endoplasmic reticulum by the indirect immunofluorescence staining of protoplasts with anti-calreticulin antibodies. No calreticulin was observed within large vacuoles. We conclude that calreticulin is present in the endoplasmic reticulum of plant cells, where, by analogy to the mammalian endoplasmic reticulum, it may play a major role in Ca²⁺ binding and storage.Abbreviations ER endoplasmic reticulum - SR sarcoplasmic reticulum - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - PBS phosphate-buffered saline     

Widespread tissue distribution of rabbit calreticulin,a non-muscle functional analogue of calsequestrin

Summary Calreticulin was identified in a variety of rabbit tissues by Western blot analysis. Indirect immunofluorescence studies on cultured cells or frozen sections from the corresponding tissues revealed that the protein was distributed to the endoplasmic reticulum or sarcoplasmic reticulum. Calreticulin was found to be an abundant calcium-binding protein in non-muscle and smooth muscle cells and a constitutent calcium-binding protein in cardiac and skeletal muscle. From the immunoblot data, calreticulin may exist as an isoform in rabbit neural retina. The present study establishes the ubiquity of calreticulin in intracellular calcium binding.     

Carbonylation of ER chaperone proteins in aged mouse liver

Progressive accumulation of oxidative damage to macromolecules in aged tissues is thought to contribute to the decline in tissue function characteristic of the aged phenotype. Mitochondria are a major intracellular source of reactive oxygen species (ROS); however, other organelles are also endogenous sources of oxyradicals and oxidants, which can damage macromolecules. We, therefore, sought to examine the relationship between aging and oxidative damage to ER resident proteins, which exist in a strongly oxidizing environment necessary for disulfide bond formation. In these studies, we have fractionated young and aged liver homogenates, resolved the proteins by 2D gel electrophoresis, assayed for oxidative damage as indicated by protein carbonylation, and identified BiP/Grp78, protein disulfide isomerase (PDI), and calreticulin as exhibiting an age-associated increase in oxidative damage. Increased carbonylation of these key proteins in aged liver suggests an age-associated impairment in protein folding, disulfide crosslinking, and glycosylation in the aged mouse liver.     

Chemogenomic and bioinformatic profiling of ERdj paralogs underpins their unique roles in cancer

The ER-resident Hsp70 paralog BiP is important in cellular homeostasis as well as in cancer cell progression. Although several BiP inhibitors have been developed, they have not succeeded in clinical trials due to toxicity issues. ER-resident co-chaperones (ERdjs) tailor the activity and specificity of BiP. Here, we report multiple-cancer analyses of BiP and ERdj genomic alterations including mRNA expression from cancer patients using available data from The Cancer Genome Atlas (TCGA). We examine the individual roles of BiP co-chaperones ERdj1-8 in mediating anticancer drug resistance through chemogenomic screening of ERdj1-8 CRISPR KO cells. In keeping with the idea that ERdjs regulate distinct facets of proteostasis, we find that each ERdj KO displays a unique signature of drug resistance. Taken together, our results demonstrate a novel way to understand functional specificity of ERdjs, suggesting a future personalized medicine approach, whereby ERdj mutation status is assessed to design an effective anticancer treatment plan.     

Treatment combinations targeting apoptosis to improve immunotherapy of melanoma

Immunotherapy based on T cell responses to the tumor is believed to involve killing of cancer cells by induction of apoptosis. The predominant mechanisms are death ligand-induced signaling mainly by TNF-related apoptosis-inducing ligand (TRAIL) mediated by CD4 T cells, monocytes and dendritic cells, and perforin/granzyme mediated apoptosis mediated by CD8 T cells and NK cells. Resistance against TRAIL involves loss of TRAIL death receptors and/or activation of the MEK and/or Akt signal pathways. Resistance to CD8 CTL responses also involves activation of the MEK and/or Akt pathways. Apoptosis induced by immune responses is regulated by the Bcl-2 family of proteins. Many reagents have been developed against the Bcl-2 antiapoptotic proteins and clinical trials combining them with immunotherapy are awaited. The second group of agents that regulate the Bcl-2 family of proteins are the signal pathway inhibitors. Clinical trials with inhibitors of RAS, RAF or MEK are in progress and would appear an exciting combination with immunotherapy. One of the main drivers of resistance to apoptosis are adaptive mechanisms that allow cancer cells to overcome endoplasmic reticulum (ER) stress. These adaptive mechanisms inhibit practically all known apoptotic pathways and create an acidic environment that may reduce infiltration of lymphocytes against the tumor. The signal pathway inhibitors may be effective against these adaptive processes but additional agents that target ER stress pathways are in development. In conclusion, combination of immunotherapy with agents that target antiapoptotic mechanisms in cancer cells offers a new approach that requires evaluation in clinical trials.     

The plant Golgi apparatus: Last 10 years of answered and open questions

Plant Golgi bodies possess unique morphological and functional characteristics that are key to several biological and biotechnological processes, such as transport of the cell’s building blocks to energy-rich compartments, including chloroplasts, storage vacuoles and a cellulosic cell wall. During the last decade it has become apparent that the plant Golgi apparatus has features that are remarkably different from other systems. Here we summarize the most recent findings on this organelle and we highlight pressing questions that are likely to drive the next 10 years of research on the plant Golgi apparatus.     

Proteomic analysis of the role of S-nitrosoglutathione reductase in lipopolysaccharide-challenged mice

S-Nitrosoglutathione reductase (GSNOR) is a key regulator of protein S-nitrosylation, the covalent modification of cysteine residues by nitric oxide that can affect activities of many proteins. We recently discovered that excessive S-nitrosylation from GSNOR deficiency in mice under inflammation inactivates the key DNA repair protein O(6) -alkylguanine-DNA alkyltransferase and promotes both spontaneous and carcinogen-induced hepatocellular carcinoma. To explore further the mechanism of tumorigenesis due to GSNOR deficiency, we compared the protein expression profiles in the livers of wild-type and GSNOR-deficient (GSNOR(-/-) ) mice that were challenged with lipopolysaccharide to induce inflammation and expression of inducible nitric oxide synthase (iNOS). Two-dimensional difference gel electrophoresis analysis identified 38 protein spots of significantly increased intensity and 31 protein spots of significantly decreased intensity in the GSNOR(-/-) mice compared to those in the wild-type mice. We subsequently identified 19 upregulated and 19 downregulated proteins in GSNOR(-/-) mice using mass spectrometry. Immunoblot analysis confirmed in GSNOR(-/-) mice a large increase in the expression of the pro-inflammatory mediator S100A9, a protein previously implicated in human liver carcinogenesis. We also found a decrease in the expression of multiple members of the protein disulfide-isomerase (PDI) family and an alteration in the expression pattern of the endoplasmic reticulum (ER) chaperones in GSNOR(-/-) mice. Furthermore, altered expression of these proteins from GSNOR deficiency was prevented in mice lacking both GSNOR and iNOS. In addition, we detected S-nitrosylation of two members of the PDI protein family. These results suggest that S-nitrosylation resulting from GSNOR deficiency may promote carcinogenesis under inflammatory conditions in part through the disruption of inflammatory and ER stress responses.     

The role of the unfolded protein response in cancer progression: From oncogenesis to chemoresistance

Tumour cells endure both oncogenic and environmental stresses during cancer progression. Transformed cells must meet increased demands for protein and lipid production needed for rapid proliferation and must adapt to exist in an oxygen‐ and nutrient‐deprived environment. To overcome such challenges, cancer cells exploit intrinsic adaptive mechanisms such as the unfolded protein response (UPR). The UPR is a pro‐survival mechanism triggered by accumulation of unfolded or misfolded proteins in the endoplasmic reticulum (ER), a condition referred to as ER stress. IRE1, PERK and ATF6 are three ER anchored transmembrane receptors. Upon induction of ER stress, they signal in a coordinated fashion to re‐establish ER homoeostasis, thus aiding cell survival. Over the past decade, evidence has emerged supporting a role for the UPR in the establishment and progression of several cancers, including breast cancer, prostate cancer and glioblastoma multiforme. This review discusses our current knowledge of the UPR during oncogenesis, tumour growth, metastasis and chemoresistance.     

The endoplasmic reticulum network in the ascidian egg: localization and calcium content

In contrast with most other eggs, where the endoplasmic reticulum is mixed with many other organelles, in ascidians, continuous sheets and tubes of endoplasmic reticulum constitute the only prominent organelle in the immediate layer (0.5-1μm) beneath the plasma membrane, and occupies 16–20% of the cortical volume. We took advantage of this unusual stratification of the organelles in the ascidian egg, to carry X-ray microanalysis. Our measurements provide the first estimate of the calcium content of the endoplasmic reticulum network in an egg, and show it is the main calcium store.     

UFMylation:A Unique & Fashionable Modification for Life

Ubiquitin-fold modifier 1 (UFM1) is one of the newly-identified ubiquitin-like proteins. Similar to ubiquitin, UFM1 is conjugated to its target proteins by a three-step enzymatic reaction. The UFM1-activating enzyme, ubiquitin-like modifier-activating enzyme 5 (UBA5), serves as the E1 to activate UFM1;UFM1-conjugating enzyme 1 (UFC1) acts as the E2 to transfer the activated UFM1 to the active site of the E2;and the UFM1-specific ligase 1 (UFL1) acts as the E3 to recog-nize its substrate, transfer, and ligate the UFM1 from E2 to the substrate. This process is called ufmylation. UFM1 chains can be cleaved from its target proteins by UFM1-specific proteases (UfSPs), suggesting that the ufmylation modification is reversible. UFM1 cascade is conserved among nearly all of the eukaryotic organisms, but not in yeast, and associated with several cellular activities including the endoplasmic reticulum stress response and hematopoiesis. Furthermore, the UFM1 cascade is closely related to a series of human diseases. In this review, we summarize the molecular details of this reversible modification process, the recent progress of its functional studies, as well as its implication in tumorigenesis and potential therapeutic targets for cancer.     

Plant Peroxisomes: Molecular Basis of the Regulation of their Functions

β -oxidation, the glyoxylate cycle and the glycolate pathway for photorespiration. Recent molecular biological studies have revealed that most of these enzymes possess either one of two peroxisomal targeting signals (PTS) within their amino acid sequence. One of the signals, PTS1, is found at the carboxy-terminus, while the other, PTS2, is found within the amino-terminal presequence. Subsequent to the synthesis and folding of these enzymes in the cytosol, the targeting signal in the folded proteins may bind to the corresponding receptors. At present, only a receptor that recognizes PTS1 has been identified in higher plants. After the binding of the protein and the receptor, the protein complex may be recognized by docking proteins that exist in the peroxisomal membrane. The mechanisms responsible for the recognition of peroxisomal proteins are now under investigation. Genetic analyses of Arabidopsis mutants with defective peroxisomes may give us some clues to understanding the mechanisms of peroxisomal protein import. Received 18 November 1999/ Accepted in revised form 13 January 2000     

Mutual interaction between oxidative stress and endoplasmic reticulum stress in the pathogenesis of diseases specifically focusing on non-alcoholic fatty liver disease

Reactive oxygen species(ROS) are produced during normal physiologic processes with the consumption of oxygen. While ROS play signaling roles, when they are produced in excess beyond normal antioxidative capacity this can cause pathogenic damage to cells. The majority of such oxidation occurs in polyunsaturated fatty acids and sulfhydryl group in proteins, resulting in lipid peroxidation and protein misfolding, respectively. The accumulation of misfolded proteins in the endoplasmic reticulum(ER) is enhanced under conditions of oxidative stress and results in ER stress, which, together, leads to the malfunction of cellular homeostasis. Multiple types of defensive machinery are activated in unfolded protein response under ER stress to resolve this unfavorable situation. ER stress triggers the malfunction of protein secretion and is associated with a variety of pathogenic conditions including defective insulin secretion from pancreatic β-cells and accelerated lipid droplet formation in hepatocytes. Herein we use nonalcoholic fatty liver disease(NAFLD) as an illustration of such pathological liver conditions that result from ER stress in association with oxidative stress. Protecting the ER by eliminating excessive ROS viathe administration of antioxidants or by enhancing lipidmetabolizing capacity via the activation of peroxisome proliferator-activated receptors represent promising therapeutics for NAFLD.     

The fine structure of the corpus luteum in Hyrax

Summary Hyrax possess luteal cells with many ultrastructural features similar to those described in other mammalian species. The process of luteinization is gradual. Cells with increasing amounts of smooth endoplasmic reticulum become predominant in the developing gland. The luteal cells of Procavia and Heterohyrax show many ultrastructural similarities. In both species the corpus luteum persists throughout pregnancy.     

The Histone Database: a comprehensive resource for histones and histone fold-containing proteins

The Histone Database is a curated and searchable collection of full-length sequences and structures of histones and nonhistone proteins containing histone-like folds, compiled from major public databases. Several new histone fold-containing proteins have been identified, including the huntingtin-interacting protein HYPM. Additionally, based on the recent crystal structure of the Son of Sevenless protein, an interpretation of the sequence analysis of the histone fold domain is presented. The database contains an updated collection of multiple sequence alignments for the four core histones (H2A, H2B, H3, and H4) and the linker histones (H1/H5) from a total of 975 organisms. The database also contains information on the human histone gene complement and provides links to three-dimensional structures of histone and histone fold-containing proteins. The Histone Database is a comprehensive bioinformatics resource for the study of structure and function of histones and histone fold-containing proteins. The database is available at http://research.nhgri.nih.gov/histones/.     

Mass spectrometry‐based absolute quantification of microsomal cytochrome P450 2D6 in human liver

We describe the assay of the human cytochrome P450 2D6 in a set of 30 genotyped liver samples using the ‘absolute quantification’ (AQUA) technique. We found approximately 30 fmol CYP2D6 per μg of microsomal protein, with the values spanning from 0 to nearly 80 fmol/μg. This is greater by a factor of 5–10 from the compared to the currently accepted value, which was around 5 fmol/μg. Our results thus suggest that the amount of cytochrome P450 (CYP) enzymes in liver have to be reassessed. We used quantitative Western blotting, calibration standards and activity assays, to validate the results. Our results show, that using the AQUA technique a true assay of CYP2D6 in human liver was possible.