Subcellular localization of proteasomes and their regulatory complexes in mammalian cells

Proteasomes can exist in several different molecular forms in mammalian cells. The core 20S proteasome, containing the proteolytic sites, binds regulatory complexes at the ends of its cylindrical structure. Together with two 19S ATPase regulatory complexes it forms the 26S proteasome, which is involved in ubiquitin-dependent proteolysis. The 20S proteasome can also bind 11S regulatory complexes (REG, PA28) which play a role in antigen processing, as do the three variable c-interferoninducible catalytic b-subunits (e.g. LMP7). In the present study, we have investigated the subcellular distribution of the different forms of proteasomes using subunit speci®c antibodies. Both 20S proteasomes and their 19S regulatory complexes are found in nuclear, cytosolic and microsomal preparations isolated from rat liver. LMP7 was enriched approximately two-fold compared with core a-type proteasome subunits in the microsomal preparations. 20S proteasomes were more abundant than 26S proteasomes, both in liver and cultured cell lines. Interestingly, some signi®cant differences were observed in the distribution of different subunits of the 19S regulatory complexes. S12, and to a lesser extent p45, were found to be relatively enriched in nuclear fractions from rat liver, and immuno¯uorescent labelling of cultured cells with anti-p45 antibodies showed stronger labelling in the nucleus than in the cytoplasm. The REG was found to be localized predominantly in the cytoplasm. Three- to six-fold increases in the level of REG were observed following cinterferon treatment of cultured cells but c-interferon had no obvious effect on its subcellular distribution. These results demonstrate that different regulatory complexes and subpopulations of proteasomes have different distributions within mammalian cells and, therefore, that the distribution is more complex than has been reported for yeast proteasomes.

High levels of BACH2 associated with lower levels of BCL2 transcript abundance in t(14;18)(q21;q34) translocation positive non-Hodgkin’s lymphoma

The t(14;18)(q21;q34) BCL2 translocation is a common genetic alteration in follicular and diffuse large B-cell lymphoma. However, it is not invariably associated with BCL2 gene overexpression due to undefined mechanisms that regulate expression from the proximal immunoglobulin heavy-chain (IgH) promoter. The BACH2 transcriptional repressor is able to modulate activity of this promoter. Here we have shown that, in tumor samples with BCL2 translocation, those with high levels of BACH2 had significantly lower BCL2 transcript abundance compared to those with low levels of BACH2. This indicates that BACH2 may be partially responsible for regulation of BCL2 expression from the t(14;18)(q21;q34) translocation.

Quantitative proteomic approach to study subcellular localization of membrane proteins

As proteins within cells are spatially organized according to their role, knowledge about protein localization gives insight into protein function. Here, we describe the LOPIT technique (localization of organelle proteins by isotope tagging) developed for the simultaneous and confident determination of the steady-state distribution of hundreds of integral membrane proteins within organelles. The technique uses a partial membrane fractionation strategy in conjunction with quantitative proteomics. Localization of proteins is achieved by measuring their distribution pattern across the density gradient using amine-reactive isotope tagging and comparing these patterns with those of known organelle residents. LOPIT relies on the assumption that proteins belonging to the same organelle will co-fractionate. Multivariate statistical tools are then used to group proteins according to the similarities in their distributions, and hence localization without complete centrifugal separation is achieved. The protocol requires approximately 3 weeks to complete and can be applied in a high-throughput manner to material from many varied sources.

Bcl-X(L) translocation in renal tubular epithelial cells in vitro protects distal cells from oxidative stress

BACKGROUND: The molecular pathogenesis of different sensitivities of the renal proximal and distal tubular cell populations to ischemic injury, including ischemia-reperfusion (IR)-induced oxidative stress, is not well-defined. An in vitro model of oxidative stress was used to compare the survival of distal [Madin-Darby canine kidney (MDCK)] and proximal [human kidney-2 (HK-2)] renal tubular epithelial cells, and to analyze for links between induced cell death and expression and localization of selected members of the Bcl-2 gene family (anti-apoptotic Bcl-2 and Bcl-X(L), pro-apoptotic Bax and Bad). METHODS: Cells were treated with 1 mmol/L hydrogen peroxide (H2O2) or were grown in control medium for 24 hours. Cell death (apoptosis) was quantitated using defined morphological criteria. DNA gel electrophoresis was used for biochemical identification. Protein expression levels and cellular localization of the selected Bcl-2 family proteins were analyzed (Western immunoblots, densitometry, immunoelectron microscopy). RESULTS: Apoptosis was minimal in control cultures and was greatest in treated proximal cell cultures (16.93 +/- 4.18% apoptosis) compared with treated distal cell cultures (2.28 +/- 0.85% apoptosis, P < 0.001). Endogenous expression of Bcl-X(L) and Bax, but not Bcl-2 or Bad, was identified in control distal cells. Bcl-X(L) and Bax had nonsignificant increases (P> 0.05) in these cells. Bcl-2, Bax, and Bcl-X(L), but not Bad, were endogenously expressed in control proximal cells. Bcl-X(L) was significantly decreased in treated proximal cultures (P < 0.05), with Bax and Bcl-2 having nonsignificant increases (P> 0.05). Immunoelectron microscopy localization indicated that control and treated but surviving proximal cells had similar cytosolic and membrane localization of the Bcl-2 proteins. In comparison, surviving cells in the treated distal cultures showed translocation of Bcl-X(L) from cytosol to the mitochondria after treatment with H2O2, a result that was confirmed using cell fractionation and analysis of Bcl-X(L) expression levels of the membrane and cytosol proteins. Bax remained distributed evenly throughout the surviving distal cells, without particular attachment to any cellular organelle. CONCLUSION: The results indicate that in this in vitro model, the increased survival of distal compared with proximal tubular cells after oxidative stress is best explained by the decreased expression of anti-apoptotic Bcl-X(L) in proximal cells, as well as translocation of Bcl-X(L) protein to mitochondria within the surviving distal cells.

The complete mitochondrial genome of the yellow coaster, Acraea issoria (Lepidoptera: Nymphalidae: Heliconiinae: Acraeini): sequence, gene organization and a unique tRNA translocation event

In this paper, the complete mitochondrial genome of Acraea issoria (Lepidoptera: Nymphalidae: Heliconiinae: Acraeini) is reported; a circular molecule of 15,245 bp in size. For A. issoria, genes are arranged in the same order and orientation as the complete sequenced mitochondrial genomes of the other lepidopteran species, except for the presence of an extra copy of tRNAIle(AUR)b in the control region. All protein-coding genes of A. issoria mitogenome start with a typical ATN codon and terminate in the common stop codon TAA, except that COI gene uses TTG as its initial codon and terminates in a single T residue. All tRNA genes possess the typical clover leaf secondary structure except for tRNASer(AGN), which has a simple loop with the absence of the DHU stem. The sequence, organization and other features including nucleotide composition and codon usage of this mitochondrial genome were also reported and compared with those of other sequenced lepidopterans mitochondrial genomes. There are some short microsatellite-like repeat regions (e.g., (TA)9, polyA and polyT) scattered in the control region, however, the conspicuous macro-repeats units commonly found in other insect species are absent.

Differential subcellular and extracellular localisations of proteins required for insulin-like growth factor- and extracellular matrix-induced signalling events in breast cancer progression

Background: Cancer metastasis is the main contributor to breast cancer fatalities as women with the metastatic disease have poorer survival outcomes than women with localised breast cancers. There is an urgent need to develop appropriate prognostic methods to stratify patients based on the propensities of their cancers to metastasise. The insulin-like growth factor (IGF)-I:IGF binding protein (IGFBP):vitronectin complexes have been shown to stimulate changes in gene expression favouring increased breast cancer cell survival and a migratory phenotype. We therefore investigated the prognostic potential of these IGF- and extracellular matrix (ECM) interaction-induced proteins in the early identification of breast cancers with a propensity to metastasise using patient-derived tissue microarrays. Methods: Semiquantitative immunohistochemistry analyses were performed to compare the extracellular and subcellular distribution of IGF- and ECM-induced signalling proteins among matched normal, primary cancer and metastatic cancer formalin-fixed paraffin-embedded breast tissue samples. Results: The IGF- and ECM-induced signalling proteins were differentially expressed between subcellular and extracellular localisations. Vitronectin and IGFBP-5 immunoreactivity was lower while β1 integrin immunoreactivity was higher in the stroma surrounding metastatic cancer tissues, as compared to normal breast and primary cancer stromal tissues. Similarly, immunoreactive stratifin was found to be increased in the stroma of primary as well as metastatic breast tissues. Immunoreactive fibronectin and β1 integrin was found to be highly expressed at the leading edge of tumours. Based on the immunoreactivity it was apparent that the cell signalling proteins AKT1 and ERK1/2 shuffled from the nucleus to the cytoplasm with tumour progression. Conclusion: This is the first in-depth, compartmentalised analysis of the distribution of IGF- and ECM-induced signalling proteins in metastatic breast cancers. This study has provided insights into the changing pattern of cellular localisation and expression of IGF- and ECM-induced signalling proteins in different stages of breast cancer. The differential distribution of these biomarkers could provide important prognostic and predictive indicators that may assist the clinical management of breast disease, namely in the early identification of cancers with a propensity to metastasise, and/or recur following adjuvant therapy.

GABAB receptors expressed in human aortic endothelial cells mediate intracellular calcium concentration regulation and endothelial nitric oxide synthase translocation

GABAB receptors regulate the intracellular Ca2+ concentration ([Ca2+]i) in a number of cells (e.g., retina, airway epithelium and smooth muscle), but whether they are expressed in vascular endothelial cells and similarly regulate the [Ca2+]i is not known. The purpose of this study was to investigate the expression of GABAB receptors, a subclass of receptors to the inhibitory neurotransmitter γ-aminobutyric acid (GABA), in cultured human aortic endothelial cells (HAECs), and to explore if altering receptor activation modified [Ca2+]i and endothelial nitric oxide synthase (eNOS) translocation. Real-time PCR, western blots and immunofluorescence were used to determine the expression of GABAB1 and GABAB2 in cultured HAECs. The effects of GABAB receptors on [Ca2+]i in cultured HAECs were demonstrated using fluo-3. The influence of GABAB receptors on eNOS translocation was assessed by immunocytochemistry. Both GABAB1 and GABAB2 mRNA and protein were expressed in cultured HAECs, and the GABAB1 and GABAB2 proteins were colocated in the cell membrane and cytoplasm. One hundred μM baclofen caused a transient increase of [Ca2+]i and eNOS translocation in cultured HAECs, and the effects were attenuated by pretreatment with the selective GABAB receptor antagonists CGP46381 and CGP55845. GABAB receptors are expressed in HAECs and regulate the [Ca2+]i and eNOS translocation. Cultures of HAECs may be a useful in vitro model for the study of GABAB receptors and vascular biology.

Assessment of the short-term success of a translocation of lesser short-tailed bats Mystacina tuberculata

Translocation is a powerful tool that has been used in the conservation of a wide range of taxa. However, few translocations of bats have been attempted and we know of no successes. The few translocations which have been attempted have either failed due to dispersal from the release site or have not been monitored sufficiently to determine the cause of failure. We assessed the short-term success of a translocation of lesser short-tailed bats Mystacina tuberculata by the New Zealand Department of Conservation, where 3 release methods were used to minimise dispersal or mortality: bats were juveniles, were maintained in captivity at the release site, and were provided with supplementary food and roosts following release. Success was assessed by determining if founders remained at the release site and maintained condition (weight). Recapture showed that at least 9 of the 20 bats remained at the release site 232 d after release. There was weak evidence that bats lost weight, although final weights were comparable to those of bats from a natural population. However, all bats caputred 8 mo after release had damaged, infected ears and some were balding. The problem was treated but recurred, and bats were returned to captivity. Our results are th first to demonstrate that translocated bats can remain at their release site and survive. However, disease may be an issue in future translocations.

Translocation of bats as a conservation strategy : previous attempts and potential problems

Translocation is an increasingly popular conservation tool from which a wide range of taxa have benefited. However, to our knowledge, bats have not been translocated successfully. Bats differ behaviourally, morphologically and physiologically from the taxa for which translocation the- ory has been developed, so existing guidelines may not be directly transferable. We review previous translocations of bats and discuss characteristics of bats that may require special consideration dur- ing translocation. Their vagility and homing ability, coloniality, roost requirements, potential ability to transmit diseases, susceptibility to anthropomorphic impacts, and cryptic nature have implications for establishing populations, effects of these populations on the release site, and ability to monitor translocation success following release. We hope that our discussion of potential problems will be able to supplement the existing, more generic guidelines to provide a starting point for the planning of bat translocations.

The potential availability of roosting sites for lesser short-tailed bats (Mystacina tuberculata) on Kapiti Island, New Zealand: Implications for a translocation

Lesser short-tailed bats (Mystacina tuberculata) have recently been translocated to Kapiti Island in an attempt to form a new population of this threatened species. However, the island's vegetation is regenerating, and there was doubt that the forests provided enough large trees with cavities for bats to roost in. This study measured the availability of tree-trunk cavities of the right size for potential roost sites on Kapiti Island, and assessed if habitat restoration would be required to increase the translocation's chance of success. First, trees with cavities accessible to us were sampled in six of Kapiti Island's forest types. Size variables known to affect roost site selection by lesser short-tailed bats at the tree and cavity level were measured. Trees were classified as containing cavities that could potentially provide suitable roosts if their values for all variables measured fell within the range of roosts used by lesser short-tailed bats in natural populations. Roosts were classified as suitably sized for solitary bats or for colonies, using measurements from both types of roosts in natural populations. Second, the density of these potential roost cavities was calculated. Cavities of a size potentially suitable for colonies were found in four of the six forest types at densities ranging from 3.2 +/- 3.2 SE to 52.4 +/- 14.0 trees per ha. Density of potential solitary roosts was much higher. Not all potential cavities will be suitable because they may be damp, poorly insulated, or have an unsuitable microclimate. Nevertheless, our estimates indicated that the two most extensive forest types each contained thousands of potential cavities of a size suitable for colonies of lesser short-tailed bats. In addition, there were tens of thousands of cavities large enough to shelter solitary bats. Roost habitat restoration appears unnecessary to assist translocated Mystacina tuberculata on Kapiti Island.

Evidence of homing following translocation of long‐tailed bats (Chalinolobus tuberculatus) at Grand Canyon Cave, New Zealand

Homing behaviour in the New Zealand long-tailed bat (Chalinolobus tuberculatus), a temperate insectivorous species, was investigated at Grand Canyon Cave, central North Island. A pilot study of nine adult male bats was conducted to determine whether use of the cave was regular enough for a homing study. Eight bats returned to the cave over the 3 week monitoring period, six on the night of the following release. Nine additional bats carrying radio transmitters were then released at three sites (three at each site) c.5, 10 and 20km due east of the border of, and outside the population's known familiar area respectively. All but one of these nine was subsequently detected at the cave. Results suggest that adult long-tailed bats are able to return home following displacement both inside and outside their familiar area. Implications of these findings for translocations of bats and the possessions of a potential long distance navigation system by this species are discussed.

Mechanistic details of the membrane perforation and passive translocation of TAT peptides

The trans-activator of transcription (TAT) peptide is regarded as the “gold standard” for cell-penetrating peptides, capable of traversing a mammalian membrane passively into the cytosolic space. This characteristic has been exploited through conjugation of TAT for applications such as drug delivery. However, the process by which TAT achieves membrane penetration remains ambiguous and unresolved. Mechanistic details of TAT peptide action are revealed herein by using three complementary methods: quartz crystal microbalance with dissipation (QCM-D), scanning electrochemical microscopy (SECM) and atomic force microscopy (AFM). When combined, these three scales of measurement define that the membrane uptake of the TAT peptide is by trans-membrane insertion using a “worm-hole” pore that leads to ion permeability across the membrane layer. AFM data provided nanometre-scale visualisation of TAT punctuation using a mammalian-mimetic membrane bilayer. The TAT peptide does not show the same specificity towards a bacterial mimetic membrane and QCM-D and SECM showed that the TAT peptide demonstrates a disruptive action towards these membranes. This investigation supports the energy-independent uptake of the cationic TAT peptide and provides empirical data that clarify the mechanism by which the TAT peptide achieves its membrane activity. The novel use of these three biophysical techniques provides valuable insight into the mechanism for TAT peptide translocation, which is essential for improvements in the cellular delivery of TAT-conjugated cargoes including therapeutic agents required to target specific intracellular locations.