Angiotensinogen localization and secretion in the rat pancreas

Renin and angiotensinogen have been previously found in the rat pancreas, and angiotensin receptors have been located in the apical domain of duct cells. To evaluate the possibility that angiotensin II could be generated within the duct system, we decided to determine whether angiotensinogen is present in rat pancreatic juice and the angiotensinogen-immunoreactive pancreatic cell types that could be responsible for its production. Angiotensinogen was detected in significant amounts by Western blotting in pancreatic juice collected from several individual rats. Different isoforms between plasma and pancreatic juice angiotensinogens were demonstrated by isoelectric focusing. Immunocytochemical experiments revealed angiotensinogen-immunoreactive cells at the periphery of the islets of Langerhans, and confocal microscopy demonstrated that most angiotensinogen-immunoreactive cells were glucagon-secreting cells. Secretion of angiotensinogen did not follow the regulated secretory pathway since it was absent from the glucagon-containing granules. This was confirmed by electron microscopy immunocytochemistry. Duct and acinar cells did not express angiotensinogen at an immunocytochemical detectable level. The present findings indicated an exocrine secretion of angiotensinogen by glucagon-secreting cells and suggest that one of the final targets of the local pancreatic renin-angiotensin system may be the duct epithelium.

Efficient developmental mis-targeting by the sporamin NTPP vacuolar signal to plastids in young leaves of sugarcane and Arabidopsis

Plant vacuoles are multi-functional, developmentally varied and can occupy up to 90% of plant cells. The N-terminal propeptide (NTPP) of sweet potato sporamin and the C-terminal propeptide (CTPP) of tobacco chitinase have been developed as models to target some heterologous proteins to vacuoles but so far tested on only a few plant species, vacuole types and payload proteins. Most studies have focused on lytic and protein-storage vacuoles, which may differ substantially from the sugar-storage vacuoles in crops like sugarcane. Our results extend the evidence that NTPP of sporamin can direct heterologous proteins to vacuoles in diverse plant species and indicate that sugarcane sucrose-storage vacuoles (like the lytic vacuoles in other plant species) are hostile to heterologous proteins. A low level of cytosolic NTPP-GFP (green fluorescent protein) was detectable in most cell types in sugarcane and Arabidopsis, but only Arabidopsis mature leaf mesophyll cells accumulated NTPP-GFP to detectable levels in vacuoles. Unexpectedly, efficient developmental mis-trafficking of NTPP-GFP to chloroplasts was found in young leaf mesophyll cells of both species. Vacuolar targeting by tobacco chitinase CTPP was inefficient in sugarcane, leaving substantial cytoplasmic activity of rat lysosomal beta-glucuronidase (GUS) [ER (endoplasmic reticulum)-RGUS-CTPP]. Sporamin NTPP is a promising targeting signal for studies of vacuolar function and for metabolic engineering. Such applications must take account of the efficient developmental mis-targeting by the signal and the instability of most introduced proteins, even in storage vacuoles.

Domains of the TGN: Coats, tethers and G proteins

The trans-Golgi network is the major sorting compartment of the secretory pathway for protein, lipid and membrane traffic. There is a constant flow of membrane and cargo to and from this compartment. Evidence is emerging that the trans-Golgi network has multiple biochemically and functionally distinct subdomains, each of which contributes to the combined sorting and transport requirements of this dynamic compartment. The recruitment of distinct arrays of protein complexes to trans-Golgi network membranes is likely to produce the diversity of structure and biochemistry observed amongst subdomains that serve to generate different carriers or maintain resident trans-Golgi network components. This review discusses how these subdomains may be formed and examines the molecular players involved, including G proteins, clathrin adaptors and golgin tethers. Diversity within these protein families is highlighted and shown to be critical for the functionality of the trans-Golgi network, as a mediator of protein sorting and membrane transport, and for the maintenance of Golgi structure.

Lessons from tomographic studies of the mammalian Golgi

Basic structure studies of the biosynthetic machinery of the cell by electron microscopy (EM) have underpinned much of our fundamental knowledge in the areas of molecular cell biology and membrane traffic. Driven by our collective desire to understand how changes in the complex and dynamic structure of this enigmatic organelle relate to its pivotal roles in the cell, the comparatively high-resolution glimpses of the Golgi and other compartments of the secretory pathway offered to us through EM have helped to inspire the development and application of some of our most informative, complimentary (molecular, biochemical and genetic) approaches. Even so, no one has yet even come close to relating the basic molecular mechanisms of transport, through and from the Golgi, to its ultrastructure, to everybody's satisfaction. Over the past decade, EM tomography has afforded new insights into structure -function relationships of the Golgi and provoked a re-evaluation of older paradigms. By providing a set of tools for structurally dissecting cells at high-resolution in three-dimensions (3D), EM tomography has emerged as a method for studying molecular cell biology in situ. As we move rapidly toward the establishment of molecular atlases of organelles through advances in proteomics and genomics, tomographic studies of the Golgi offer the tantalizing possibility that one day, we will be able to map the spatio-temporal coordinates of Golgi-related proteins and lipids accurately in the context of 4D cellular space. (c) 2005 Elsevier B.V. All rights reserved.

Calcium transport and signaling in the mammary gland: Targets for breast cancer

The mammary gland is subjected to extensive calcium loads during lactation to support the requirements of milk calcium enrichment. Despite the indispensable nature of calcium homeostasis and signaling in regulating numerous biological functions, the mechanisms by which systemic calcium is transported into milk by the mammary gland are far from completely understood. Furthermore, the implications of calcium signaling in terms of reaulating proliferation, differentiation and apoptosis in the breast are currently uncertain. Deregulation of calcium homeostasis and signaling is associated with mammary gland pathophysiology and as such, calcium transporters, channels and binding proteins represent potential drug targets for the treatment of breast cancer. (c) 2005 Elsevier B.V. All rights reserved.

Interferon-induced, antiviral human MxA protein localizes to a distinct subcompartment of the smooth endoplasmic reticulum

Human MxA protein belongs to the superfamily of dynamin-like large GTPases that are involved in intracellular membrane trafficking. MxA is induced by interferons-alpha/beta (IFN-alpha/beta) and is a key component of the antiviral response against RNA viruses. Here, we show that MxA localizes to membranes that are positive for specific markers of the smooth endoplasmic reticulum, such as Syntaxin17, but is excluded from other membrane compartments. Overexpression of MxA leads to a characteristic reorganization of the associated membranes. Interestingly, Hook3, mannose-6-phosphate receptor, and Lamp-1, which normally accumulate in cis-Golgi, endosomes, and lysosomes, respectively, also colocalized with MxA, indicating that these markers were redistributed to the MxA-positive compartment. Functional assays, however, did not show any effect of MxA on endocytosis or the secretory pathway. The present results demonstrate that MxA is an IFN-induced antiviral effector protein that resembles the constitutively expressed large GTPase family members in its capacity to localize to and reorganize intracellular membranes.

Subcellular localization of mammalian type II membrane proteins

Application of a computational membrane organization prediction pipeline, MemO, identified putative type II membrane proteins as proteins predicted to encode a single alpha-helical transmembrane domain (TMD) and no signal peptides. MemO was applied to RIKEN's mouse isoform protein set to identify 1436 non-overlapping genomic regions or transcriptional units (TUs), which encode exclusively type II membrane proteins. Proteins with overlapping predicted InterPro and TMDs were reviewed to discard false positive predictions resulting in a dataset comprised of 1831 transcripts in 1408 TUs. This dataset was used to develop a systematic protocol to document subcellular localization of type II membrane proteins. This approach combines mining of published literature to identify subcellular localization data and a high-throughput, polymerase chain reaction (PCR)-based approach to experimentally characterize subcellular localization. These approaches have provided localization data for 244 and 169 proteins. Type II membrane proteins are localized to all major organelle compartments; however, some biases were observed towards the early secretory pathway and punctate structures. Collectively, this study reports the subcellular localization of 26% of the defined dataset. All reported localization data are presented in the LOCATE database (http://www.locate.imb.uq.edu.au).

The abundance of short proteins in the mammalian proteome

Short proteins play key roles in cell signalling and other processes, but their abundance in the mammalian proteome is unknown. Current catalogues of mammalian proteins exhibit an artefactual discontinuity at a length of 100 aa, so that protein abundance peaks just above this length and falls off sharply below it. To clarify the abundance of short proteins, we identify proteins in the FANTOM collection of mouse cDNAs by analysing synonymous and nonsynonymous substitutions with the computer program CRITICA. This analysis confirms that there is no real discontinuity at length 100. Roughly 10% of mouse proteins are shorter than 100 aa, although the majority of these are variants of proteins longer than 100 aa. We identify many novel short proteins, including a dark matter'' subset containing ones that lack detectable homology to other known proteins. Translation assays confirm that some of these novel proteins can be translated and localised to the secretory pathway.

Evaluation and comparison of mammalian subcellular localization prediction methods

Background: Determination of the subcellular location of a protein is essential to understanding its biochemical function. This information can provide insight into the function of hypothetical or novel proteins. These data are difficult to obtain experimentally but have become especially important since many whole genome sequencing projects have been finished and many resulting protein sequences are still lacking detailed functional information. In order to address this paucity of data, many computational prediction methods have been developed. However, these methods have varying levels of accuracy and perform differently based on the sequences that are presented to the underlying algorithm. It is therefore useful to compare these methods and monitor their performance. Results: In order to perform a comprehensive survey of prediction methods, we selected only methods that accepted large batches of protein sequences, were publicly available, and were able to predict localization to at least nine of the major subcellular locations (nucleus, cytosol, mitochondrion, extracellular region, plasma membrane, Golgi apparatus, endoplasmic reticulum (ER), peroxisome, and lysosome). The selected methods were CELLO, MultiLoc, Proteome Analyst, pTarget and WoLF PSORT. These methods were evaluated using 3763 mouse proteins from SwissProt that represent the source of the training sets used in development of the individual methods. In addition, an independent evaluation set of 2145 mouse proteins from LOCATE with a bias towards the subcellular localization underrepresented in SwissProt was used. The sensitivity and specificity were calculated for each method and compared to a theoretical value based on what might be observed by random chance. Conclusion: No individual method had a sufficient level of sensitivity across both evaluation sets that would enable reliable application to hypothetical proteins. All methods showed lower performance on the LOCATE dataset and variable performance on individual subcellular localizations was observed. Proteins localized to the secretory pathway were the most difficult to predict, while nuclear and extracellular proteins were predicted with the highest sensitivity.

Inhibitors of cyclo-oxygenase-2 and secretory phospholipase A2 preserve bone architecture following ovariectomy in adult rats

Epidemiological evidence and in vitro data suggest that COX-2 is a key regulator of accelerated remodeling. Accelerated states of osteoblast and osteoclast activity are regulated by prostaglandins in vitro, but experimental evidence for specific roles of cyclooxygenase-2 (COX-2) and secretory phospholipase A(2) (sPLA(2)) in activated states of remodeling in vivo is lacking. The aim of this study was to determine the effect of specific inhibitors of sPLA(2)-IIa and COX-2 on bone remodeling activated by estrogen deficiency in adult female rats. One hundred and twenty-four adult female Wistar rats were ovariectomized (OVX) or sham-operated. Rats commenced treatment 14 days after surgery with either vehicle, a COX-2 inhibitor (DFU at 0.02 mg/kg/day and 2.0 mg/kg/day) or a sPLA(2)-group-IIa inhibitor (KH064 at 0.4 mg/kg/day and 4.0 mg/kg/day). Treatment continued daily until rats were sacrificed at 70 days or 98 days post-OVX. The right tibiae were harvested, fixed and embedded in methylmethacrylate for structural histomorphometric bone analysis at the proximal tibial metaphysis. The specific COX-2 or sPLA(2) inhibitors prevented ovariectomy-induced (OVX-induced) decreases in trabecular connectivity (P < 0.05); suppressed the acceleration of bone resorption; and maintained bone turnover at SHAM levels following OVX in the rat. The sPLA2 inhibitor significantly suppressed increases in osteoclast surface induced by OVX (P < 0.05), while the effect of COX-2 inhibition was less marked. These findings demonstrate that inhibitors of COX-2 and sPLA(2)-IIa can effectively suppress OVX-induced bone loss in the adult rat by conserving trabecular bone mass and architecture through reduced bone remodeling and decreased resorptive activity. Moreover, we report an important role of sPLA(2)-IIa in osteoclastogenesis that may be independent of the COX-2 metabolic pathway in the OVX rat in vivo. (c) 2006 Elsevier Inc. All rights reserved.