Caspase-like Activity Research Articles

Routes to cell death in animal and plant kingdoms: from classic apoptosis to alternative ways to die—a review

Programmed cell death is fundamental for multicellular organisms either in animal or plant kingdom. Classic apoptosis, which represents the best studied form of cell death, is dependent on caspase protease activity in animals. These proteases are not present in plants, where caspase-like activities, including metacaspases, are involved in the execution of plant cell death. Beyond apoptosis, various non-apoptotic forms of cell death also exist, including autophagy, necroptosis, pyroptosis, and ferroptosis. These types of cell death can be activated independently of apoptosis and sometimes occur when apoptosis is inhibited. Non-apoptotic forms of cell death are best characterized in animals, whereas, in plants, the literature is less extensive, but the molecular executioners of plant cell death are becoming clearer. The aim of this review is to describe different types of cell death and the mechanisms involved in animals and plants, highlighting similarities and differences in the two kingdoms. Moreover, implications of cell death pathways in cancer are discussed.

Gender Differences on the Effects of Ibuprofen on Proteasome Function in Mice Heart

Introduction and objectivesIbuprofen, a cyclo‐oxygenase inhibitor, is a commonly used non‐steroidal anti‐inflammatory (NSAID) agent used in the treatment of pain, fever and inflammation. The long‐term use of ibuprofen is associated with increased risks for cardiovascular events and stroke. The effect of ibuprofen on cardiac function as well as if gender differences exist with respect to ibuprofen's effect on cardiac function are not well understood. Accordingly, the aim of this study was to explore the effects of ibuprofen on protein homeostasis in mice cardiac tissue and to determine if ibuprofen affects male and female cardiac proteasome activity in a similar manner.Method/ResultsAged matched male and female C57BL/6J mice were randomly assigned to two groups: (A) control (B) group treated with ibuprofen. Mice were treated with ibuprofen (100 mg/kg) dissolved in drinking water for 6 weeks.Since impaired proteasome function is associated with cardiovascular disorders, β1 caspase‐like activity, β2 trypsin‐like activity, and β5 chymotrypsin‐like proteasome activities were measured using bortezomib as a specific inhibitor of proteasome in cardiac tissue of both male and female mice. In female mice chronic treatment with ibuprofen showed decreased proteasome function as decreased β1, β2, and β5 activity was observed in ibuprofen treated female animals. However, proteasome activity was not affected in male animals treated with ibuprofen. Excess accumulation of misfolded and oxidized proteins alters proteasome function, therefore levels of oxidized proteins in heart lysates was measured by oxyblot method using dinitrophenylhydrazine (DNPH). The levels of oxidized proteins increased in only female animals treated with ibuprofen. The levels of polyubiquitinated proteins was similar in male and female ibuprofen treated hearts compared to control hearts. An increase in free ubiquitin levels was only observed in the hearts of female animals treated with ibuprofen when compared to control animals and ibuprofen treated male animals.ConclusionOur findings suggest, for the first time, that gender differences on proteasome function occur in the heart of ibuprofen treated animals. These data show that chronic treatment of ibuprofen decreased proteasome activities in females as well as increased the levels of oxidized proteins and free ubiquitin levels in only ibuprofen treated female mice hearts.Support or Funding InformationAmerican Heart Association Award #16GRNT31350040This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Ibuprofen cases Proteasome dysfunction in mice liver

Introduction and objectivesPrevious clinical studies have suggested that nonsteroidal anti‐inflammatory drugs (NSAIDs) are associated with increased risk of stroke and cardiovascular disease. Acetaminophen, the active ingredient in Tylenol, is not an NSAID and is associated with increased risk of liver toxicity. The common use of NSAIDs for pain and fever relief is a concern but it is unclear if NSAIDs is also causing adverse effects on other tissues such as the liver, kidney and brain. Previous research in our laboratory has suggested that NSAIDs cause proteasome dysfunction in cardiac tissue. To investigate the possibility that NSAIDs also influence liver function, ibuprofen treated mice were used to determine if this NSAID affects the ubiquitin proteasome system (UPS). The UPS is critical for cell function as it is responsible for degradation of 60–80% of intracellular proteins.Method/ResultsEight‐week‐old male and female C57BL/6J mice were randomly assigned to one of 4 groups: (A) control (B) Group treated with ibuprofen (C) Group treated with ibuprofen + vitamin C (D) Group treated with vitamin C alone. Mice were treated with water or ibuprofen (100 mg/kg) and vitamin C (150 mg/kg) dissolved in drinking water for 6 weeks. Liver from 6 week treated animals were homogenized and cytosol fractions obtained. The three independent proteolytic proteasome activities, β1 caspase‐like activity, β2 trypsin‐like activity, and β5 chymotrypsin‐like activity were measured in the different cytosolic liver fractions. The proteasome inhibitor bortezomib was utilized to ensure the activity measured was due to the proteasome.Semi‐quantitative western blotting suggests that the 20S proteasome amount was similar in all 4 groups but the immunoproteasome subunit β5i was increased in both male and female ibuprofen treated and ibuprofen with vitamin C groups. The β1 and β2 proteasome activities were significantly decreased in both male and female mice treated with ibuprofen. Although inclusion of vitamin C with the ibuprofen did not fully restore proteasome activity, it resulted in an improvement in the β1 proteasome activity compared to ibuprofen treated mice. The β5 proteasome activity was also significantly decreased in both male and female mice treated with ibuprofen. However, this decrease was significantly more pronounced in female mice than in male mice suggesting gender‐specific effects of ibuprofen on proteasome function.ConclusionThese results suggest that ibuprofen significantly disrupts the UPS and causes proteasome dysfunction in liver tissue. The experimental data also suggests that proteasome dysfunction in livers from ibuprofen treated mice differs in female and male mice.Support or Funding InformationAmerican Heart Association Award #16GRNT31350040This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

OsNAC2 positively affects salt-induced cell death and binds to the OsAP37 and OsCOX11 promoters.

Plant development and adaptation to environmental stresses are intimately associated with programmed cell death (PCD). Although some of the mechanisms regulating PCD [e.g., accumulation of reactive oxygen species (ROS)] are common among responses to different abiotic stresses, the pathways mediating salt-induced PCD remain largely uncharacterized. Here we report that overexpression of OsNAC2, which encodes a plant-specific transcription factor, promotes salt-induced cell death accompanied by the loss of plasma membrane integrity, nuclear DNA fragmentation, and changes to caspase-like activity. In OsNAC2-knockdown lines, cell death was markedly decreased in response to severe salt stress. Additionally, OsNAC2 expression was enhanced in rice seedlings exposed to a high NaCl concentration. Moreover, the results of quantitative real-time PCR, chromatin immunoprecipitation, dual-luciferase, and yeast one-hybrid assays indicated that OsNAC2 targeted genes that encoded an ROS scavenger (OsCOX11) and a caspase-like protease (OsAP37). Furthermore, K+ -efflux channels (OsGORK and OsSKOR) were clearly activated by OsNAC2. Overall, our results suggested that OsNAC2 accelerates NaCl-induced PCD and provide new insights into the mechanisms that affect ROS accumulation, plant caspase-like activity, and K+ efflux.

Indispensable Role of Proteases in Plant Innate Immunity.

Plant defense is achieved mainly through the induction of microbe-associated molecular patterns (MAMP)-triggered immunity (MTI), effector-triggered immunity (ETI), systemic acquired resistance (SAR), induced systemic resistance (ISR), and RNA silencing. Plant immunity is a highly complex phenomenon with its own unique features that have emerged as a result of the arms race between plants and pathogens. However, the regulation of these processes is the same for all living organisms, including plants, and is controlled by proteases. Different families of plant proteases are involved in every type of immunity: some of the proteases that are covered in this review participate in MTI, affecting stomatal closure and callose deposition. A large number of proteases act in the apoplast, contributing to ETI by managing extracellular defense. A vast majority of the endogenous proteases discussed in this review are associated with the programmed cell death (PCD) of the infected cells and exhibit caspase-like activities. The synthesis of signal molecules, such as salicylic acid, jasmonic acid, and ethylene, and their signaling pathways, are regulated by endogenous proteases that affect the induction of pathogenesis-related genes and SAR or ISR establishment. A number of proteases are associated with herbivore defense. In this review, we summarize the data concerning identified plant endogenous proteases, their effect on plant-pathogen interactions, their subcellular localization, and their functional properties, if available, and we attribute a role in the different types and stages of innate immunity for each of the proteases covered.

Drugging the lncRNA MALAT1 via LNA gapmeR ASO inhibits gene expression of proteasome subunits and triggers anti-multiple myeloma activity

The biological role and therapeutic potential of long non-coding RNAs (lncRNAs) in multiple myeloma (MM) are still to be investigated. Here, we studied the functional significance and the druggability of the oncogenic lncRNA MALAT1 in MM. Targeting MALAT1 by novel LNA-gapmeR antisense oligonucleotide antagonized MM cell proliferation and triggered apoptosis both in vitro and in vivo in a murine xenograft model of human MM. Of note, antagonism of MALAT1 downmodulated the two major transcriptional activators of proteasome subunit genes, namely NRF1 and NRF2, and resulted in reduced trypsin, chymotrypsin and caspase-like proteasome activities and in accumulation of polyubiquitinated proteins. NRF1 and NRF2 decrease upon MALAT1 targeting was due to transcriptional activation of their negative regulator KEAP1, and resulted in reduced expression of anti-oxidant genes and increased ROS levels. In turn, NRF1 promoted MALAT1 expression thus establishing a positive feedback loop. Our findings demonstrate a crucial role of MALAT1 in the regulation of the proteasome machinery, and provide proof-of-concept that its targeting is a novel powerful option for the treatment of MM.

Quercetin suppresses the chymotrypsin-like activity of proteasome via inhibition of MEK1/ERK1/2 signaling pathway in hepatocellular carcinoma HepG2 cells.

The proteasomal system is a promising target for cancer treatment. Quercetin (Que), a flavonoid compound with antitumor ability, displays the inhibitory effect on proteasome activity. However, the underlying molecular mechanisms are ill defined. The present study found that Que treatment significantly reduced the chymotrypsin-like protease activity of proteasome whereas the trypsin- and caspase-like protease activities remained unchanged in HepG2 cancer cells, along with activation of p38 MAPK and JNK and reduction of ERK1/2 phosphorylation. Que-reduced proteasome activity could not be reverted by inhibition of p38 MAPK and JNK signaling pathway. In addition, MEK1 overexpression or knockdown upregulated or downregulated the chymotrypsin-like protease activity of proteasome, respectively. Both Que and MEK1/ERK1/2 inhibitor attenuated the expression levels of proteasome β subunits. These results indicate that Que-induced suppression of MEK1/ERK1/2 signaling and subsequent reduction of proteasome β subunits is responsible for its inhibitory impacts on proteasome activity.

RELATIONS OF PROTEASOMAL SYSTEM WITH TUMOR STAGE IN LUMINAL BREAST CANCER

The aim of this study was to investigate characteristics of the proteasomal system in luminal breast cancer. There were included 124 patients with primary luminal breast cancer in stage Т13N0-2M0 who had not received neoadjuvant treatment. The process of lymphogenous metastasis was associated with a significant change in caspase-like activity (CL) and subunit composition of proteasomes. CL activity of proteasomes was increased in luminal A breast cancer with extensive lymphogenic metastasis (N2), while it was decreased in the luminal B subtype of cancer. It was accompanied by an increase in the composition of the proteasomal total pool subunits, regulatory and immune subunits. Decrease in CL activity of proteasomes can be poor prognostic sign which is associated with the lymphogenous invasion of the tumor process in luminal B breast cancer.

Insufficient Apaf-1 expression in early stages of neural differentiation of human embryonic stem cells might protect them from apoptosis

Recent evidence suggests that mitochondrial apoptosis regulators and executioners may regulate differentiation, without being involved in cell death. However, the involved factors and their roles in differentiation and apoptosis are still not fully determined. In the present study, we compared mitochondrial pathway of cell death during early neural differentiation from human embryonic stem cells (hESCs). Our results demonstrated that ROS generation, cytosolic cytochrome c release, caspases activation and rise in p53 protein level occurred upon either neural or apoptosis induction in hESCs. However, unlike apoptosis, no remarkable increase in apoptotic protease activating factor-1 (Apaf-1) level at early stages of differentiation was observed. Also the caspase-like activity of caspase-9 and caspase-3/7 were seen less than apoptosis. The results suggest that low levels of Apaf-1 as an adaptor protein might be considered as a possible regulatory barrier by which differentiating cells control cell death upon rise in ROS production and cytochrome c release from mitochondria. Better understanding of mechanisms via which mitochondria-mediated apoptotic pathway promote neural differentiation can result in development of novel therapeutic approaches.

Nitric oxide suppresses aluminum-induced programmed cell death in peanut (Arachis hypoganea L.) root tips by improving mitochondrial physiological properties

Aluminum (Al) stress alters nitric oxide (NO) and induces programmed cell death (PCD) in plants. Recent study has shown that NO inhibits Al-induced PCD. However, the mechanism of NO inhibiting Al-induced PCD has not been revealed yet. Here, we investigated the behavior of mitochondria during Al-induced PCD suppressed by NO in peanut. Seedlings of peanut was grown hydroponically in a controllable growth room. The mitochondrial physiological parameters were determined spectrophotometrically. The expression of AhANT and AhHsp70 was determined by quantitative RT-PCR. Al-induced cell death rapidly in peanut root tips is mitochondria-dependent PCD. There was a significantly negative relationship between PCD and mitochondrial NO/H2O2 level. Compared with Al treatment alone, the addition of NO donor sodium nitroprusside (SNP) increased the ratio of NO/H2O2, down-regulated AhANT expression and inhibited the opening of mitochondrial permeability transition pore (MPTP), up-regulated AhHsp70 expression and increased mitochondrial inner membrane potential (ΔΨm), reduced cytochrome c (Cyt c) release from mitochondria and caspase 3-like protease activity, while the effect of NO specific scavenger cPTIO supplement was opposite. NO suppresses Al-induce PCD in peanut root tips by improving mitochondrial physiological properties.

Spermidine sprays alleviate the water deficit-induced oxidative stress in finger millet (Eleusine coracana L. Gaertn.) plants.

Severe drought stress (water deficit) in finger millet (Eleusine coracana L. Gaertn.) plants significantly reduced total leaf chlorophyll and relative water content in shoots and roots, whereas electrolyte leakage, concentrations of proline and hydrogen peroxide, as well as caspase-like activity were significantly increased. The role of spermidine in plant defence to water-stress was investigated after subjected to various drought treatments. Three weeks of daily spermidine sprays (0.2mM) at early flowering stage significantly changed shoot and root growth, in both fresh and dry weights terms. At 75% of water deficit stress, leaves accumulated twice as much proline as unstressed plants, and roots accumulated thrice. Plants treated with spermidine under water stress showed lower electrolyte leakage, hydrogen peroxide and caspase-like activity than unstressed and untreated control.

Measurement of the Caspase-1-Like Activity of Vacuolar Processing Enzyme in Plants.

Caspase-like activities are essential to regulate programed cell death in plants. Although no caspase orthologous enzymes with aspartic acid specificity have been identified in plants, vacuolar processing enzyme (VPE) exhibits a caspase-1-like activity. In this chapter, we introduce two methods for the measurement of the caspase-1-like/VPE activity. These methods are based on the cleavage of caspase-1 specific synthetic substrates and on monitoring the active forms of VPE using a biotinylated-inhibitor blot analysis. Both methods are also adaptable to other plant caspase-like activities.

Nitric oxide prevents H2O2-induced apoptosis in SK-N-MC human neuroblastoma cells.

Nitric oxide (NO) is a cellular signaling molecule in many physiological and pathological processes including neuroprotector. Here we examined the antiapoptotic effect of NO in SK-N-MC cells. H2O2 treatment (10-200 μM) induced cell death in a dose-dependent manner and pretreatment of cells with 100 μM S-nitroso-N-acetylpenicillamine (SNAP), an NO donor, attenuated the occurrence of H2O2-induced cell death. DAPI staining showed H2O2-induced nuclear fragmentation and NO treatment suppressed it. NO inhibited the proteolytic activation of caspase-3 and mitochondrial cytochrome c release. Treatment of soluble guanylyl cyclase inhibitor ODQ decreased the protective effect of SNAP on H2O2-treated cells and increased caspase 3-like enzyme activity and activation, cytochrome c release, PARP cleavage, and DNA fragmentation, indicating that cGMP is a key mediator in NO-mediated antiapoptosis. The cGMP analog 8-Br-cGMP blocked H2O2-induced apoptotic cell death; reduction of caspase-3 enzyme, cytochrome c release, and caspase-8 and -9. These preventive effects of SNAP and 8-Br-cGMP were suppressed by PKG inhibitor KT5823. Levels of PKGI, PKGII, and p-VASP proteins were increased by SNAP and 8-Br-cGMP and suppressed by KT5823 treatment. These results indicate that PKG is a downstream signal mediator in the suppression of apoptosis by NO and cGMP. Akt activation was inhibited the PI3K inhibitors LY294002 and Wortmannin, resulting in the inhibition of cell viability and increase of cytochrome c release. SNAP induced phosphorylation of Akt and Bad and then increased the interactions between 14-3-3β and p-Bad. These data suggest that the NO suppresses H2O2-induced SK-N-MC cell apoptosis by suppressing apoptosis signal mediating the interaction between 14‐3‐3β and Bad phosphorylation via PKG/PI3K/Akt.

Alpha-synuclein aggregation, Ubiquitin proteasome system impairment, and L-Dopa response in zinc-induced Parkinsonism: resemblance to sporadic Parkinson's disease.

Alpha-synuclein (α-synuclein) aggregation and impairment of the Ubiquitin proteasome system (UPS) are implicated in Parkinson's disease (PD) pathogenesis. While zinc (Zn) induces dopaminergic neurodegeneration resulting in PD phenotype, its effect on protein aggregation and UPS has not yet been deciphered. The current study investigated the role of α-synuclein aggregation and UPS in Zn-induced Parkinsonism. Additionally, levodopa (L-Dopa) response was assessed in Zn-induced Parkinsonian model to establish its closeness with idiopathic PD. Male Wistar rats were treated with zinc sulfate (Zn; 20mg/kg; i.p.) twice weekly for 12 weeks along with respective controls. In few subsets, animals were subsequently treated with L-Dopa for 21 consecutive days following Zn exposure. A significant increase in total and free Zn content was observed in the substantia nigra of the brain of exposed groups. Zn treatment caused neurobehavioral anomalies, striatal dopamine decline, and dopaminergic neuronal cell loss accompanied with a marked increase in α-synuclein expression/aggregation and Ubiquitin-conjugated protein levels in the exposed groups. Zn exposure substantially reduced UPS-associated trypsin-like, chymotrypsin-like, and caspase-like activities along with the expression of SUG1 and β-5 subunits of UPS in the nigrostriatal tissues of exposed groups. L-Dopa treatment rescued from Zn-induced neurobehavioral deficits and restored dopamine levels towards normalcy; however, Zn-induced dopaminergic neuronal loss, reduction in tyrosine hydroxylase expression, and increase in oxidative stress were unaffected. The results suggest that Zn caused UPS impairment, resulting in α-synuclein aggregation subsequently leading to dopaminergic neurodegeneration, and that Zn-induced Parkinsonism exhibited positive L-Dopa response similar to sporadic PD.

Milder degenerative effects of Carfilzomib vs. Bortezomib in the Drosophila model: a link to clinical adverse events

Proteasome inhibitors, e.g. Bortezomib (BTZ) and Carfilzomib (CFZ), have demonstrated clinical efficacy against haematological cancers. Interestingly, several adverse effects are less common, compared to BTZ, in patients treated with CFZ. As the molecular details of these observations remain not well understood we assayed the pathophysiological effects of CFZ vs. BTZ in the Drosophila experimental model. Mass Spectrometry analyses showed that neither CFZ nor BTZ are hydrolysed in flies’ tissues, while at doses inducing similar inhibition of the rate limiting for protein breakdown chymotrypsin-like (CT-L) proteasomal activity, CFZ treatment resulted in less intense increase of oxidative stress or activation of antioxidant and proteostatic modules. Also, despite comparable cardiotoxicity likely due to disrupted mitochondrial function, CFZ did not affect developmental processes, showed minimal neuromuscular defects and reduced to a lesser extent flies’ healthspan. Studies in flies, human cancer cell lines and blood cells isolated from Multiple Myeloma patients treated with CFZ or BTZ revealed, that the increased BTZ toxicity likely relates to partial co-inhibition of the caspase-like (C-L) proteasomal activity Supportively, co-treating flies with CFZ and a C-L selective proteasome inhibitor exacerbated CFZ-mediated toxicity. Our findings provide a reasonable explanation for the differential adverse effects of CFZ and BTZ in the clinic.

Abstract 20950: An Intrinsic Mechanism for a Gain in Proteasome Activity Preserving Cardiac Function

Cardiac remodeling and heart failure are characterized by misaligned adaptations in protein synthesis and degradation, harboring the therapeutic potential for interventional harmonization. Partial inhibition of protein degradation via the ubiquitin-proteasome system was reported to reduce acute hypertrophic remodeling as well as recover systolic function. The studies are suggestive of an inadequate gain in proteasome activity, contributing to cardiac remodeling and heart failure. Aim was to identify a common mechanism promoting proteasome activity during cardiac remodeling and heart failure and to test whether it contributes to the pathogenesis. Incorporation of the inducible proteasome subunit Lmp2 has been described to increase proteasome activity. In fact, it is increasingly expressed and incorporated in proteasome complexes during isoproterenol-induced hypertrophic remodeling, which is associated with a gain in proteasome activity. We found that Lmp2 is also increasingly expressed upon transverse aortic constriction and in a murine model of familial hypertrophic cardiomyopathy. Knock-out (KO) of Lmp2 in mice increased the caspase-like proteasome activity by 19% (n=5, p<0.05), but had no apparent influence on cardiac morphology or function. As predicted, lack of Lmp2 confined the regulatory capacity of proteasomes to gain activity during hypertrophic remodeling induced by isoproterenol. Unexpectedly, the restriction in additional activity did not reduce cardiac remodeling as previously described for proteasome inhibition. In contrast, lack of Lmp2 exacerbated hypertrophic remodeling and caused loss of systolic function (fractional shortening: -32% vs. wildtype, n≥8, p<0.01). Cardiomyocyte-specific expression of Lmp2 via gene transfer into adult Lmp2 KO mice completely rescued the isoproterenol-induced deterioration of cardiac remodeling and function. Interestingly, the deprived gain in proteasome activity in Lmp2 KO hearts rather affected a specific subset than the general pool of ubiquitinated proteins. In conclusion, a gain in proteasome activity during acute cardiac remodeling is not entirely maladaptive. At least in part, it is dependent on Lmp2, controlling remodeling and preserving cardiac function.

Anticandidal activity of hetero-dinuclear copper(II) Mn(II) Schiff base and its potential action of the mechanism.

Invasive fungal infections are one of the major challenges especially for immunosuppressed patients since they are drug resistant and pathogen to patients. Therefore, developing new, efficient and nonresistant antifungal agents have been a primary focus of international research. In the current study, a novel Schiff base [hetero-dinuclear copper(II) Mn(II) complex] (SB) derivative was investigated for its anticandidal activity against Candida albicans and possible mechanisms inducing cell death. The results revealed that SB treatment induces apoptotic and necrotic pathways in C. albicans ATCC10231 strain. Intracellular reactive oxygen species production determined by 2',7'-dichlorofluorescein diacetate staining was triggered by SB and amphotericin B administrations in a dose-dependent manner. Gene expression analysis demonstrated that SB exposure resulted in regulation of critical development and stress related gene expressions. SB treatment directly upregulated expression of stress related genes, DDR48 and RIM101, while suppressed important cell signaling and antibiotic resistance acquiring related genes such as HSP90, ERG11 and EFG1. Furthermore, CaMCA1 mRNA levels were found to be significantly high in SB-treated yeast cells, indicating possible caspase-like mechanism activation. Scanning electron microscopy analysis confirmed that SB treatment led to severe cell wall integrity disruption and wrinkling. The study will encourage development of SB-based anticandidal regimens but further studies are highly warranted to understand limitations and the extended use in the routine.

Immunoproteasome subunit \xdf5i/LMP7-deficiency in atherosclerosis

Management of protein homeostasis by the ubiquitin-proteasome system is critical for atherosclerosis development. Recent studies showed controversial results on the role of immunoproteasome (IP) subunit β5i/LMP7 in maintenance of protein homeostasis under cytokine induced oxidative stress. The present study aimed to investigate the effect of β5i/LMP7-deficiency on the initiation and progression of atherosclerosis as a chronic inflammatory, immune cell driven disease. LDLR−/−LMP7−/− and LDLR−/− mice were fed a Western-type diet for either 6 or 24 weeks to induce early and advanced stage atherosclerosis, respectively. Lesion burden was similar between genotypes in both stages. Macrophage content and abundance of polyubiquitin conjugates in aortic root plaques were unaltered by β5i/LMP7-deficiency. In vitro experiments using bone marrow-derived macrophages (BMDM) showed that β5i/LMP7-deficiency did not influence macrophage polarization or accumulation of polyubiquitinated proteins and cell survival upon hydrogen peroxide and interferon-γ treatment. Analyses of proteasome core particle composition by Western blot revealed incorporation of standard proteasome subunits in β5i/LMP7-deficient BMDM and spleen. Chymotrypsin-, trypsin- and caspase-like activities assessed by using short fluorogenic peptides in BMDM whole cell lysates were similar in both genotypes. Taken together, deficiency of IP subunit β5i/LMP7 does not disturb protein homeostasis and does not aggravate atherogenesis in LDLR−/− mice.

Excessive UDPG resulting from the mutation of UAP1 causes programmed cell death by triggering reactive oxygen species accumulation and caspase-like activity in rice.

Lesion mimic mutants are valuable to unravel the mechanisms governing the programmed cell death (PCD) process. Uridine 5'-diphosphoglucose-glucose (UDPG) functions as a signaling molecule activating multiple pathways in animals, but little is known about its function in plants. Two novel allelic mutants of spl29 with typical PCD characters and reduced pollen viability were obtained by ethane methyl sulfonate mutagenesis in rice cv Kitaake. The enzymatic analyses showed that UDP-N-acetylglucosamine pyrophosphorylase 1 (UAP1) irreversibly catalyzed the decomposition of UDPG. Its activity was severely destroyed and caused excessive UDPG accumulation, with the lesion occurrence associated with the enhanced caspase-like activities in spl29-2. At the transcriptional level, several key genes involved in endoplasmic reticulum stress and the unfolded protein response were abnormally expressed. Moreover, exogenous UDPG could aggravate lesion initiation and development in spl29-2. Importantly, exogenous UDPG and its derivative UDP-N-acetylglucosamine could induce reactive oxygen species (ROS) accumulation and lesion mimics in Kitaake seedlings. These results suggest that the excessive accumulation of UDPG, caused by the mutation of UAP1, was a key biochemical event resulting in the lesion mimics in spl29-2. Thus, our findings revealed that UDPG might be an important component involved in ROS accumulation, PCD execution and lesion mimicking in rice, which also provided new clues for investigating the connection between sugar metabolism and PCD process.

Uncoupling Protein 2 Inhibition Exacerbates Glucose Fluctuation-Mediated Neuronal Effects.

Though glucose fluctuations have been considered as an adverse factor for the development of several diabetes-related complications, their impact in the central nervous system is still not fully elucidated. This study was conducted to evaluate the responses of neuronal cells to different glycemic exposures alongside to elucidate the role of uncoupling protein 2 (UCP2) in regulating such responses. To achieve our goals, primary cortical neurons were submitted to constant high (HG)/low (LG) or glucose level variations (GVs), and the pharmacological inhibition of UCP2 activity was performed using genipin. Results obtained show that GV decreased neuronal cells' viability, mitochondrial membrane potential, and manganese superoxide dismutase activity and increased reactive oxygen species (ROS) production. GV also caused an increase in the glutathione/glutathione disulfide ratio and in the protein expression levels of nuclear factor E2-related factor 2 (NRF2), UCP2, NADH-ubiquinone oxidoreductase chain 1 (ND1), and mitochondrially encoded cytochrome c oxidase I (MTCO1), both mitochondrial DNA encoded subunits of the electron transport chain. Contrariwise, genipin abrogated all those compensations and increased the levels of caspase 3-like activity, potentiated mitochondrial ROS levels, and the loss of neuronal synaptic integrity, decreased the protein expression levels of NRF1, and increased the protein expression levels of UCP5. Further, in the control and LG conditions, genipin increased mitochondrial ROS and the protein expression levels of UCP4, postsynaptic density protein 95 (PSD95), ND1, and MTCO1. Overall, these observations suggest that UCP2 is in the core of neuronal cell protection and/or adaptation against GV-mediated effects and that other isoforms of neuronal UCPs can be upregulated to compensate the inhibition of UCP2 activity.