Subunit‐selective proteasome activity profiling uncovers uncoupled proteasome subunit activities during bacterial infections

The proteasome is a nuclear‐cytoplasmic proteolytic complex involved in nearly all regulatory pathways in plant cells. The three different catalytic activities of the proteasome can have different functions, but tools to monitor and control these subunits selectively are not yet available in plant science. Here, we introduce subunit‐selective inhibitors and dual‐color fluorescent activity‐based probes for studying two of the three active catalytic subunits of the plant proteasome. We validate these tools in two model plants and use this to study the proteasome during plant–microbe interactions. Our data reveal that Nicotiana benthamiana incorporates two different paralogs of each catalytic subunit into active proteasomes. Interestingly, both β1 and β5 activities are significantly increased upon infection with pathogenic Pseudomonas syringae pv. tomato DC3000 lacking hopQ1‐1 [PtoDC3000(ΔhQ)] whilst the activity profile of the β1 subunit changes. Infection with wild‐type PtoDC3000 causes proteasome activities that range from strongly induced β1 and β5 activities to strongly suppressed β5 activities, revealing that β1 and β5 activities can be uncoupled during bacterial infection. These selective probes and inhibitors are now available to the plant science community, and can be widely and easily applied to study the activity and role of the different catalytic subunits of the proteasome in different plant species.     

Proteasome activity profiling: a simple,robust and versatile method revealing subunit‐selective inhibitors and cytoplasmic,defense‐induced proteasome activities

The proteasome plays essential roles in nearly all biological processes in plant defense and development, yet simple methods for displaying proteasome activities in extracts and living tissues are not available to plant science. Here, we introduce an easy and robust method to simultaneously display the activities of all three catalytic proteasome subunits in plant extracts or living plant tissues. The method is based on a membrane‐permeable, small‐molecule fluorescent probe that irreversibly reacts with the catalytic site of the proteasome catalytic subunits in an activity‐dependent manner. Activities can be quantified from fluorescent protein gels and used to study proteasome activities in vitro and in vivo. We demonstrate that proteasome catalytic subunits can be selectively inhibited by aldehyde‐based inhibitors, including the notorious caspase‐3 inhibitor DEVD. Furthermore, we show that the proteasome activity, but not its abundance, is significantly increased in Arabidopsis upon treatment with benzothiadiazole (BTH). This upregulation of proteasome activity depends on NPR1, and occurs mostly in the cytoplasm. The simplicity, robustness and versatility of this method will make this method widely applicable in plant science.     

Tissue- and developmental stage-specific changes in the subcellular localization of the 26S proteasome in the ovary of Drosophila melanogaster

The intracellular localization of the 26S proteasome in the different ovarian cell types of Drosophila melanogaster was studied by means of immunofluorescence staining and laser scanning microscopy, with the use of antibodies specific for regulatory complex subunits or the catalytic core of the 26S proteasome. During the previtellogenic phase of oogenesis (stages 1-6), strong cytoplasmic staining was observed in the nurse cells and follicular epithelial cells, but the proteasome was not detected in the nuclei of these cell types. The subcellular distribution of the 26S proteasome was completely different in the oocyte. Besides a constant, very faint cytoplasmic staining, there was a gradual nuclear accumulation of proteasomes during the previtellogenic phase of oogenesis. A characteristic subcellular redistribution of the 26S proteasome occurred in the ovarian cells during the vitellogenic phase of oogenesis. There was a gradual decline in the concentration of the 26S proteasome in the nucleus of the oocyte, and in the stage 10 oocyte the proteasome could barely be detected in the nucleus. This was accompanied by a massive nuclear accumulation of proteasomes in the follicular epithelial cells. These results demonstrate that the subcellular distribution of the 26S proteasome in higher eukaryotes is strictly tissue- and developmental stage-specific.     

Activity-Based Imaging Probes of the Proteasome

Over the years, the proteasome has been extensively investigated due to its crucial roles in many important signaling pathways and its implications in diseases. Two proteasome inhibitors—bortezomib and carfilzomib—have received FDA approval for the treatment of multiple myeloma, thereby validating the proteasome as a chemotherapeutic target. As a result, further research efforts have been focused on dissecting the complex biology of the proteasome to gain the insight required for developing next-generation proteasome inhibitors. It is clear that chemical probes have made significant contributions to these efforts, mostly by functioning as inhibitors that selectively block the catalytic activity of proteasomes. Analogues of these inhibitors are now providing additional tools for visualization of catalytically active proteasome subunits, several of which allow real-time monitoring of proteasome activity in living cells as well as in in vivo settings. These imaging probes will provide powerful tools for assessing the efficacy of proteasome inhibitors in clinical settings. In this review, we will focus on the recent efforts towards developing imaging probes of proteasomes, including the latest developments in immunoproteasome-selective imaging probes.     

Cell cycle-dependent change of proteasome distribution during embryonic development of the ascidian Halocynthia roretzi.

The proteasome is a multicatalytic proteinase complex composed of nonidentical subunits. By immunocytochemical analysis using monoclonal antibody raised against the egg proteasome, we demonstrate that the proteasome undergoes changes in its subcellular distribution, depending on the cell division cycle during embryonic development of the ascidian Halocynthia roretzi. During interphase, the proteasome is localized in the nucleus, i.e., in the nucleoplasm and along the nuclear membrane. The proteasome disappears from the nucleoplasm in prophase and from the nuclear envelope in prometaphase. During early metaphase, the proteasome is detectable in the chromosomes and, at late stages of metaphase, the immunoreactivity also occurs in the peripheral region of each spindle pole and at the mitotic spindle. In anaphase, however, the staining disappears in the mitotic apparatus. In telophase, the proteasome is again localized in the newly formed nucleus. In addition to the localization in the nucleus and around the mitotic apparatus, the proteasome shows cytoplasmic localization throughout the cell division cycle. Such a change of subcellular distribution of the proteasome is clearly demonstrated in the synchronously dividing blastomeres and also is believed to occur in the postcleavage embryos. These observations suggest that the proteasome may play a key role in the progression of cell division cycle.     

Movement of the free catalytic subunit of cAMP-dependent protein kinase into and out of the nucleus can be explained by diffusion.

The catalytic (C) subunit of cyclic AMP (cAMP) dependent protein kinase (PKA) has previously been shown to enter and exit the nucleus of cells when intracellular cAMP is raised and lowered, respectively. To determine the mechanism of nuclear translocation, fluorescently labeled C subunit was injected into living REF52 fibroblasts either as free C subunit or in the form of holoenzyme (PKA) in which the catalytic and regulatory subunits were labeled with fluorescein and rhodamine, respectively. Quantification of nuclear and cytoplasmic fluorescence intensities revealed that free C subunit nuclear accumulation was most similar to that of macromolecules that diffuse into the nucleus. A glutathione S-transferase-C subunit fusion protein did not enter the nucleus following cytoplasmic microinjection. Puncturing the nuclear membrane did not decrease the nuclear concentration of C subunit, and C subunit entry into the nucleus did not appear to be saturable. Cooling or depleting cells of energy failed to block movement of C subunit into the nucleus. Photobleaching experiments showed that even after reaching equilibrium at high [cAMP], individual molecules of C subunit continued to leave the nucleus at approximately the same rate that they had originally entered. These results indicate that diffusion is sufficient to explain most aspects of C subunit subcellular localization.     

Entamoeba histolytica: intracellular distribution of the proteasome

We have studied the intracellular distribution of proteasome subunits, corresponding to the catalytic (20S) core and the regulatory (19S) cap, in the extracellular protozoan parasite Entamoeba histolytica. Contrary to all cell types described to date, notably mammalian and yeast, in which the proteasome is found in the nucleus and actively imported into it, microscopic analysis and subcellular fractionation of E. histolytica trophozoites show that the proteasome is absent from the nucleus of these cells. We speculate that, given the relative abundance of mono- and multinucleated trophozoites in culture, a relationship may exist between this unusual distribution of the proteasome and the frequent lack of synchrony between karyo- and cytokinesis in this primitive eukaryote.     

Sts1 plays a key role in targeting proteasomes to the nucleus

The evidence that nuclear proteins can be degraded by cytosolic proteasomes has received considerable experimental support. However, the presence of proteasome subunits in the nucleus also suggests that protein degradation could occur within this organelle. We determined that Sts1 can target proteasomes to the nucleus and facilitate the degradation of a nuclear protein. Specific sts1 mutants showed reduced nuclear proteasomes at the nonpermissive temperature. In contrast, high expression of Sts1 increased the levels of nuclear proteasomes. Sts1 targets proteasomes to the nucleus by interacting with Srp1, a nuclear import factor that binds nuclear localization signals. Deletion of the NLS in Sts1 prevented its interaction with Srp1 and caused proteasome mislocalization. In agreement with this observation, a mutation in Srp1 that weakened its interaction with Sts1 also reduced nuclear targeting of proteasomes. We reported that Sts1 could suppress growth and proteolytic defects of rad23Δ rpn10Δ. We show here that Sts1 suppresses a previously undetected proteasome localization defect in this mutant. Taken together, these findings explain the suppression of rad23Δ rpn10Δ by Sts1 and suggest that the degradation of nuclear substrates requires efficient proteasome localization.     

Nuclear proteasomal degradation of Saccharomyces cerevisiae inorganic pyrophosphatase Ipp1p,a nucleocytoplasmic protein whose stability depends on its subcellular localization

Inorganic pyrophosphate (PPi) is an abundant by-product of cellular metabolism. PPi-producing reactions take place in the nucleus concurrently with reactions that use PPi as a substrate. Saccharomyces cerevisiae possesses two soluble pyrophosphatases (sPPases): Ipp1p, an essential and allegedly cytosolic protein, and Ipp2p, a mitochondrial isoenzyme. However, no sPPase has yet been unambiguously described in the nucleus. In vivo studies with fluorescent fusions together with activity and immunodetection analyses demonstrated that Ipp1p is a nucleocytoplasmic protein. Mutagenesis analysis showed that this sPPase possesses a nuclear localization signal which participates in its nuclear targeting. Enforced nucleocytoplasmic targeting by fusion to heterologous nuclear import and export signals caused changes in polypeptide abundance and activity levels, indicating that Ipp1p is less stable in the nucleus that in the cytoplasm. Low nuclear levels of this sPPase are physiologically relevant and may be related to its catalytic activity, since cells expressing a functional nuclear-targeted chimaera showed impaired growth and reduced chronological lifespan, while a nuclear-targeted catalytically inactive protein was not degraded and accumulated in the nucleus. Moreover, nuclear proteasome inhibition stabilized Ipp1p whereas nuclear targeting promoted its ubiquitination and interaction with Ubp3p, a component of the ubiquitin-proteasome system. Overall, our results indicate that Ipp1p is nucleocytoplasmic, that its stability depends on its subcellular localization and that sPPase catalytic competence drives its nuclear degradation through the ubiquitin-proteasome system. This suggests a new scenario for PPi homeostasis where both nucleocytoplasmic transport and nuclear proteasome degradation of the sPPase should contribute to control nuclear levels of this ubiquitous metabolite.     

一个水稻β-肌动蛋白基因的克隆及其亚细胞定位

目的:克隆一个水稻β-肌动蛋白基因,并研究其蛋白的亚细胞分布。方法:利用RT-PCR技术从日本晴水稻中扩增得到β-肌动蛋白基因Os Actin,将其与拟南芥肌动蛋白基因家族进行分子进化分析;利用胶体金免疫电镜技术对水稻β-肌动蛋白基因进行定位分析。结果:核苷酸和氨基酸序列分析表明,Os Actin与水稻的Os RAc1基因有较高的同源性(98%);胶体金免疫电镜定位结果显示β-肌动蛋白在细胞核、细胞质中均有分布。结论:获得了水稻β-肌动蛋白基因Os Actin,并研究了其蛋白在水稻韧皮部内的分布,为进一步探讨该基因的功能及作用机理奠定了基础。     

Functional processing of nuclear Ca2+/calmodulin-dependent protein kinase phosphatase (CaMKP-N): evidence for a critical role of proteolytic processing in the regulation of its catalytic activity, subcellular localization and substrate targeting in vivo

Ca(2+)/calmodulin-dependent protein kinase phosphatase (CaMKP) and its nuclear homolog CaMKP-N are Ser/Thr protein phosphatases that belong to the PPM family. These phosphatases are highly specific for multifunctional CaM kinases and negatively regulate their activities. CaMKP-N is only expressed in the brain and specifically localized in the nucleus. In this study, we found that zebrafish CaMKP-N (zCaMKP-N) underwent proteolytic processing in both the zebrafish brain and Neuro2a cells. In Neuro2a cells, the proteolytic processing was effectively inhibited by the proteasome inhibitors MG-132, Epoxomicin, and Lactacystin, suggesting that the ubiquitin-proteasome pathway was involved in this processing. Using MG-132, we found that the proteolytic processing changed the subcellular localization of zCaMKP-N from the nucleus to the cytosol. Accompanying this change, the cellular targets of zCaMKP-N in Neuro2a cells were significantly altered. Furthermore, we obtained evidence that the zCaMKP-N activity was markedly activated when the C-terminal domain was removed by the processing. Thus, the proteolytic processing of zCaMKP-N at the C-terminal region regulates its catalytic activity, subcellular localization and substrate targeting in vivo.     

Nuclear translocation of caspase-3 is dependent on its proteolytic activation and recognition of a substrate-like protein(s)

Caspase-3 is thought to play an important role(s) in the nuclear morphological changes that occur in apoptotic cells and many nuclear substrates for caspase-3 have been identified despite the cytoplasmic localization of procaspase-3. Therefore, whether activated caspase-3 is localized in the nuclei and how active caspase-3 has access to its nuclear targets are important and unresolved questions. Here we confirmed nuclear localizations for both caspase-3-p17 and caspase-3-p12 subunits of active caspase in apoptotic cells using subcellular fractionation analysis. We also prepared polyclonal and monoclonal antibodies specific for active caspase-3 to define the subcellular localization of active caspase-3. Immunocytochemical observations using anti-active caspase-3 antibodies showed nuclear accumulation of active caspase-3 during apoptosis. In addition, caspase-3, but not caspase-7, translocated from the cytoplasm into the nucleus after induction of apoptosis. Mutations at the cleavage site between the p17 and p12 subunits and the substrate recognition site for the P3 amino acid of the DXXD substrate cleavage motif inhibited nuclear translocation of caspase-3, indicating that nuclear transport of active caspase-3 required proteolytic activation and substrate recognition. These results suggest that active caspase-3 is translocated in association with a substrate-like protein(s) from the cytoplasm into the nucleus during progression through apoptosis.     

Identification of cellulose synthase AtCesA7 (IRX3) in vivo phosphorylation sites—a potential role in regulating protein degradation

Cellulose is central to plant development and is synthesised at the plasma membrane by an organised protein complex that contains three different cellulose synthase proteins. The ordered assembly of these three catalytic subunits is essential for normal cellulose synthesis. The way in which the relative levels of these three proteins are regulated within the cell is currently unknown. In this work it is shown that one of the cellulose synthases essential for secondary cell wall cellulose synthesis in Arabidopsis thaliana, AtCesA7, is phosphorylated in vivo. Analysis of in vivo phosphorylation sites by mass spectrometry reveals that two serine residues are phosphorylated. These residues occur in a region of hyper-variability between the cellulose synthase catalytic subunits. The region of the protein containing these phosphorylation sites can be phosphorylated by a plant extract in vitro. Incubation of this region with plant extracts results in its degradation via a proteasome dependant pathway. Full length endogenous CesA7 is also degraded via a proteasome dependant pathway in whole plant extracts. This data suggests that phosphorylation of the catalytic subunits may target them for degradation via a proteasome dependant pathway. This is a possible mechanism by which plants regulate the relative levels of the three proteins whose specific interaction are required to form an active cellulose synthase complex. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Role of regulatory subunits and protein kinase inhibitor (PKI) in determining nuclear localization and activity of the catalytic subunit of protein kinase A

Regulation of protein kinase A by subcellular localization may be critical to target catalytic subunits to specific substrates. We employed epitope-tagged catalytic subunit to correlate subcellular localization and gene-inducing activity in the presence of regulatory subunit or protein kinase inhibitor (PKI). Transiently expressed catalytic subunit distributed throughout the cell and induced gene expression. Co-expression of regulatory subunit or PKI blocked gene induction and prevented nuclear accumulation. A mutant PKI lacking the nuclear export signal blocked gene induction but not nuclear accumulation, demonstrating that nuclear export is not essential to inhibit gene induction. When the catalytic subunit was targeted to the nucleus with a nuclear localization signal, it was not sequestered in the cytoplasm by regulatory subunit, although its activity was completely inhibited. PKI redistributed the nuclear catalytic subunit to the cytoplasm and blocked gene induction, demonstrating that the nuclear export signal of PKI can override a strong nuclear localization signal. With increasing PKI, the export process appeared to saturate, resulting in the return of catalytic subunit to the nucleus. These results demonstrate that both the regulatory subunit and PKI are able to completely inhibit the gene-inducing activity of the catalytic subunit even when the catalytic subunit is forced to concentrate in the nuclear compartment.     

Eukaryotic cyclophilin as a molecular switch for effector activation

Gram-negative phytopathogenic bacteria, such as Pseudomonas syringae, deliver multiple effector proteins into plant cells during infection. It is hypothesized that certain plant and mammalian effector proteins need to traverse the type III secretion system unfolded and are delivered into host cells as inactive enzymes. We have previously identified cyclophilin as the Arabidopsis eukaryotic activator of AvrRpt2, a P. syringae effector that is a cysteine protease. Cyclophilins are general folding catalysts and possess peptidyl-prolyl cis/trans isomerase (PPIase) activity. In this paper, we demonstrate the mechanism of AvrRpt2 activation by the Arabidopsis cyclophilin ROC1. ROC1 mutants lacking PPIase enzymatic activity were unable to activate AvrRpt2. Furthermore, nuclear magnetic resonance spectroscopy revealed a structural change in AvrRpt2 from an unfolded to a folded state in the presence of ROC1. Using in vitro binding assays, ROC1's consensus binding sequence was identified as GPxL, a motif present at four sites within AvrRpt2. The GPxL motifs are located in close proximity to AvrRpt2's catalytic triad and are required for protease activity both in vitro and in planta. These data suggest that after delivery into the plant cell during infection, cyclophilin binds AvrRpt2 at four sites and properly folds the effector protein by peptidyl-prolyl cis/trans isomerization.     

An efficient Agrobacterium-mediated transient transformation of Arabidopsis

Agrobacterium tumefaciens-mediated transient transformation has been a useful procedure for characterization of proteins and their functions in plants, including analysis of protein-protein interactions. Agrobacterium-mediated transient transformation of Nicotiana benthamiana by leaf infiltration has been widely used due to its ease and high efficiency. However, in Arabidopsis this procedure has been challenging. Previous studies suggested that this difficulty was caused by plant immune responses triggered by perception of Agrobacterium. Here, we report a simple and robust method for Agrobacterium-mediated transient transformation in Arabidopsis. AvrPto is an effector protein from the bacterial plant pathogen Pseudomonas syringae that suppresses plant immunity by interfering with plant immune receptors. We used transgenic Arabidopsis plants that conditionally express AvrPto under the control of a dexamethasone (DEX)-inducible promoter. When the transgenic plants were pretreated with DEX prior to infection with Agrobacterium carrying a β-glucuronidase (GUS, uidA) gene with an artificial intron and driven by the CaMV 35S promoter, transient GUS expression was dramatically enhanced compared to that in mock-pretreated plants. This transient expression system was successfully applied to analysis of the subcellular localization of a cyan fluorescent protein (CFP) fusion and a protein-protein interaction in Arabidopsis. Our findings enable efficient use of Agrobacterium-mediated transient transformation in Arabidopsis thaliana.     

Live-cell fluorescence imaging reveals the dynamics of protein kinase CK2 individual subunits

Protein kinase CK2 is a multifunctional enzyme which has long been described as a stable heterotetrameric complex resulting from the association of two catalytic (alpha or alpha') and two regulatory (beta) subunits. To track the spatiotemporal dynamics of CK2 in living cells, we fused its catalytic alpha and regulatory beta subunits with green fluorescent protein (GFP). Both CK2 subunits contain nuclear localization domains that target them independently to the nucleus. Imaging of stable cell lines expressing low levels of GFP-CK2alpha or GFP-CK2beta revealed the existence of CK2 subunit subpopulations exhibiting differential dynamics. Once in the nucleus, they diffuse randomly at different rates. Unlike CK2beta, CK2alpha can shuttle, showing the dynamic nature of the nucleocytoplasmic trafficking of the kinase. When microinjected in the cytoplasm, the isolated CK2 subunits are rapidly translocated into the nucleus, whereas the holoenzyme complex remains in this cell compartment, suggesting an intramolecular masking of the nuclear localization sequences that suppresses nuclear accumulation. However, binding of FGF-2 to the holoenzyme triggers its nuclear translocation. Since the substrate specificity of CK2alpha is dramatically changed by its association with CK2beta, the control of the nucleocytoplasmic distribution of each subunit may represent a unique potential regulatory mechanism for CK2 activity.     

Design,synthesis and biological evaluation of triaryl compounds as novel 20S proteasome inhibitors

Thirty novel triaryl compounds were designed and synthesized based on the known proteasome inhibitor PI-1840. Most of them showed significant inhibition against the β5c subunit of human 20S proteasome, and five of them exhibited IC₅₀ values at the sub-micromolar level, which were comparable to or even more potent than PI-1840. The most active two (1c and 1d) showed IC₅₀ values of 0.12 and 0.18 μM against the β5c subunit, respectively, while they displayed no obvious inhibition against the β2c, β1c and β5i subunits. Molecular docking provided informative clues for the subunit selectivity. The potent and subunit selective proteasome inhibitors identified herein represent new chemical templates for further molecular optimization.     

The subcellular distribution of the Arabidopsis histidine phosphotransfer proteins is independent of cytokinin signaling

Cytokinins are a class of mitogenic plant hormones that play an important role in most aspects of plant development, including shoot and root growth, vascular and photomorphogenic development and leaf senescence. A model for cytokinin perception and signaling has emerged that is similar to bacterial two‐component phosphorelays. In this model, binding of cytokinin to the extracellular domain of the Arabidopsis histidine kinase (AHKs) receptors induces autophosphorylation within the intracellular histidine‐kinase domain. The phosphoryl group is subsequently transferred to cytosolic Arabidopsis histidine phosphotransfer proteins (AHPs), which have been suggested to translocate to the nucleus in response to cytokinin treatment, where they then transfer the phosphoryl group to nuclear‐localized response regulators (Type‐A and Type‐B ARRs). We examined the effects of cytokinin on AHP subcellular localization in Arabidopsis and, contrary to expectations, the AHPs maintained a constant nuclear/cytosolic distribution following cytokinin treatment. Furthermore, mutation of the conserved phosphoacceptor histidine residue of the AHP, as well as disruption of multiple cytokinin signaling elements, did not affect the subcellular localization of the AHP proteins. Finally, we present data indicating that AHPs maintain a nuclear/cytosolic distribution by balancing active transport into and out of the nucleus. Our findings suggest that the current models indicating relocalization of AHP protein into the nucleus in response to cytokinin are incorrect. Rather, AHPs actively maintain a consistent nuclear/cytosolic distribution regardless of the status of the cytokinin response pathway.     

Identification of the goldfish 20S proteasome beta6 subunit bound to nuclear matrix

Proteasomes are large, multisubunit particles that act as the proteolytic machinery for most of the regulated intracellular protein breakdown in eukaryotic cells. Proteasomes are present in both the nucleus and cytoplasm. When we analyzed the molecular composition of protein constituents of the nuclear matrix preparation of goldfish oocytes by two-dimensional polyacrylamide gel electrophoresis followed by sequence analysis, we found a 26 kDa spot identical in amino acid sequence to the beta6 subunits of the 20S proteasome. No spot of other subunits of 20S proteasome was detected. Here we describe the cloning, sequencing and expression analysis of Carassius auratus, beta6_ca, which encodes one of the proteasome beta subunits from goldfish ovary. From the screening of an ovarian cDNA library, two types of cDNA were obtained, one 941 bp and the other 884 bp long. The deduced amino acid sequences comprise 239 and 238 residues, respectively. These deduced amino acid sequences are highly homologous to those of beta6 subunits of other vertebrates. Immunoblot analysis of nuclear matrix using anti-proteasome antibodies showed only a spot of beta6_ca. These results suggest that the beta6 subunit of the goldfish 20S proteasome, beta6_ca, is responsible for anchoring proteasomes in the nucleus.