Aging-associated REGγ proteasome decline predisposes to tauopathy.

The REGγ-20S proteasome is an ubiquitin- and ATP-independent degradation system, targeting selective substrates, possibly helping to regulate aging. The studies we report here demonstrate that aging-associated REGγ decline predisposes to decreasing tau turnover, as in a tauopathy. The REGγ proteasome promotes degradation of human and mouse tau, notably phosphorylated tau and toxic tau oligomers that shuttle between the cytoplasm and nuclei. REGγ-mediated proteasomal degradation of tau was validated in 3- to 12-month-old REGγ KO mice, REGγ KO;PS19 mice, and PS19 mice with forebrain conditional neuron-specific overexpression of REGγ (REGγ OE) and behavioral abnormalities. Coupled with tau accumulation, we found with REGγ-deficiency, neuron loss, dendrite reduction, tau filament accumulation, and microglial activation are much more prominent in the REGγ KO;PS19 than the PS19 model. Moreover, we observed that the degenerative neuronal lesions and aberrant behaviors were alleviated in REGγ OE;PS19 mice. Memory and other behavior analysis substantiate the role of REGγ in prevention of tauopathy-like symptoms. In addition, we investigated the potential mechanism underlying aging-related REGγ decline. This study provides valuable insights into the novel regulatory mechanisms and potential therapeutic targets for tau-related neurodegenerative diseases.

The speckle-type POZ protein (SPOP) inhibits breast cancer malignancy by destabilizing TWIST1.

Epithelial-mesenchymal transition (EMT) inducing transcription factor TWIST1 plays a vital role in cancer metastasis. How the tumor-suppressive E3 ligase, speckle-type POZ protein (SPOP), regulates TWIST1 in breast cancer remains unknown. In this study, we report that SPOP physically interacts with, ubiquitinates, and destabilizes TWIST1. SPOP promotes K63-and K48-linked ubiquitination of TWIST1, predominantly at K73, thereby suppressing cancer cell migration and invasion. Silencing SPOP significantly enhances EMT, which accelerates breast cancer cell migration and invasiveness in vitro and lung metastasis in vivo. Clinically, SPOP is negatively correlated with the levels of TWIST1 in highly invasive breast carcinomas. Reduced SPOP expression, along with elevated TWIST1 levels, is associated with poor prognosis in advanced breast cancer patients, particularly those with metastatic triple-negative breast cancer (TNBC). Taken together, we have disclosed a new mechanism linking SPOP to TWIST1 degradation. Thus SPOP may serve as a prognostic marker and a potential therapeutic target for advanced TNBC patients.

The proteasome activator REGγ promotes diabetic endothelial impairment by inhibiting HMGA2-GLUT1 pathway.

Diabetic vascular endothelial impairment is one of the main causes of death in patients with diabetes lacking adequately defined mechanisms or effective treatments. REGγ, the 11S proteasome activator known to promote the degradation of cellular proteins in a ubiquitin- and ATP-independent manner, emerges as a new regulator in the cardiovascular system. Here, we found that REGγ was upregulated in streptozocin (STZ)-induced diabetic mouse aortic endothelium in vivo and high glucose (HG)-treated vascular endothelial cells (ECs) in vitro. REGγ deficiency ameliorated endothelial impairment in STZ-induced diabetic mice by protecting against a decline in cellular glucose uptake and associated vascular ECs dysfunction by suppressing high mobility group AT-hook 2 (HMGA2) decay. Mechanically, REGγ interacted with and degraded the transcription factor HMGA2 directly, leading to decreased HMGA2 transcriptional activity, subsequently lowered expression of glucose transporter type 1 (GLUT1), and reduced cellular glucose uptake, vascular endothelial dysfunction, and impaired diabetic endothelium. Ablation of endogenous GLUT1 or HMGA2 or overexpressing exogenous HMGA2 in vascular ECs significantly blocked or reestablished the REGγ-dependent action on cellular glucose uptake and vascular endothelial functions of HG stimulation in vitro. Furthermore, exogenously introducing HMGA2 improved diabetic mice endothelial impairment features caused by REGγ in vivo, thereby substantiating a REGγ-HMGA2-GLUT1 pathway in diabetic endothelial impairment. Our findings indicate that modulating REGγ-proteasome activity may be a potential therapeutic approach for diabetic disorders with endothelial impairment.

REGγ drives Lgr5+ stem cells to potentiate radiation induced intestinal regeneration.

Leucine-rich repeat containing G protein-coupled receptor 5 (Lgr5), a marker of intestinal stem cells (ISCs), is considered to play key roles in tissue homoeostasis and regeneration after acute radiation injury. However, the activation of Lgr5 by integrated signaling pathways upon radiation remains poorly understood. Here, we show that irradiation of mice with whole-body depletion or conditional ablation of REGγ in Lgr5+ stem cell impairs proliferation of intestinal crypts, delaying regeneration of intestine epithelial cells. Mechanistically, REGγ enhances transcriptional activation of Lgr5 via the potentiation of both Wnt and Hippo signal pathways. TEAD4 alone or cooperates with TCF4, a transcription factor mediating Wnt signaling, to enhance the expression of Lgr5. Silencing TEAD4 drastically attenuated ß-catenin/TCF4 dependent expression of Lgr5. Together, our study reveals how REGγ controls Lgr5 expression and expansion of Lgr5+ stem cells in the regeneration of intestinal epithelial cells. Thus, REGγ proteasome appears to be a potential therapeutic target for radiation-induced gastrointestinal disorders.

Tomato Ve-resistance locus: resilience in the face of adversity?

MAIN CONCLUSION: The Ve-resistance locus in tomato acts as a resilience gene by affecting both the stress/defense cascade and growth, constituting a signaling intercept with a competitive regulatory mechanism. For decades, the tomato Ve-gene has been recognized as a classical resistance R-gene, inherited as a dominant Mendelian trait and encoding a receptor protein that binds with a fungal effector to provide defense against Verticillium dahliae and V. albo-atrum. However, recent molecular studies suggest that the function and role(s) of the Ve-locus and the two proteins that it encodes are more complex than previously understood. This review summarizes both the background and recent molecular evidence and provides a reinterpretation of the function and role(s) of the Ve1- and Ve2-genes and proteins that better accommodates existing data. It is proposed that these two plasma membrane proteins interact to form a signaling intercept that directly links defense and growth. The induction of Ve1 by infection or wounding promotes growth but also downregulates Ve2 signaling, resulting in a decreased biosynthesis of PR proteins. In this context, the Ve1 R-gene acts as a Resilience gene rather than a Resistance gene, promoting taller more robust tomato plants with reduced symptoms (biotic and abiotic) and Verticillium concentration.

Loss of Smad4 promotes aggressive lung cancer metastasis by de-repression of PAK3 via miRNA regulation.

SMAD4 is mutated in human lung cancer, but the underlying mechanism by which Smad4 loss-of-function (LOF) accelerates lung cancer metastasis is yet to be elucidated. Here, we generate a highly aggressive lung cancer mouse model bearing conditional KrasG12D, p53fl/fl LOF and Smad4fl/fl LOF mutations (SPK), showing a much higher incidence of tumor metastases than the KrasG12D, p53fl/fl (PK) mice. Molecularly, PAK3 is identified as a downstream effector of Smad4, mediating metastatic signal transduction via the PAK3-JNK-Jun pathway. Upregulation of PAK3 by Smad4 LOF in SPK mice is achieved by attenuating Smad4-dependent transcription of miR-495 and miR-543. These microRNAs (miRNAs) directly bind to the PAK3 3'UTR for blockade of PAK3 production, ultimately regulating lung cancer metastasis. An inverse correlation between Smad4 and PAK3 pathway components is observed in human lung cancer. Our study highlights the Smad4-PAK3 regulation as a point of potential therapy in metastatic lung cancer.

Procyanidin B2 Promotes Intestinal Injury Repair and Attenuates Colitis-Associated Tumorigenesis via Suppression of Oxidative Stress in Mice.

Aims: Intact intestinal epithelium is essential to maintain normal intestinal physiological function. Irradiation-induced gastrointestinal syndrome or inflammatory bowel disease occurred when epithelial integrity was impaired. This study aims at exploring the mechanism of procyanidin B2 (PB2) administration to promote intestinal injury repair in mice. Results: PB2 treatment reduces reactive oxygen species (ROS) accumulation and protects the intestine damage from irradiation. Mechanistic studies reveal that PB2 could effectively slow down the degradation of nuclear factor-erythroid 2-related factor 2 (Nrf2) and it significantly triggers Nrf2 into the nucleus, which leads to subsequent antioxidant enzyme expression. However, knockdown of Nrf2 attenuates PB2-induced protection in the intestine. More importantly, PB2 also promotes leucine-rich repeat-containing G protein-coupled receptor 5 (Lgr5)-positive intestinal stem cells (Lgr5+ ISCs) driven regeneration via enhancing Wnt/ß-catenin signaling, which depends on, at least in part, activation of the Nrf2 signal. Evidence from an injury model of intestinal organoids is similar with in vivo results. Correspondingly, results from flow cytometric analysis and luciferase reporter assay reveal that PB2 also inhibits the level of ROS and promotes Lgr5 expression in vitro. Finally, PB2 alleviates the severity of experimental colitis and colitis-associated cancer in a long-term inflammatory model via inhibiting nuclear localization of p65. Innovation: This study, for the first time, reveals a role of PB2 for intestinal regeneration and repair after radiation or dextran sulfate sodium-induced injury in mice. Conclusion: Our results indicate that PB2 can repress oxidative stress via Nrf2/ARE signaling and then promote intestinal injury repair.

The Mediator Subunit, Med23 Is Required for Embryonic Survival and Regulation of Canonical WNT Signaling During Cranial Ganglia Development.

Development of the vertebrate head is a complex and dynamic process, which requires integration of all three germ layers and their derivatives. Of special importance are ectoderm-derived cells that form the cranial placodes, which then differentiate into the cranial ganglia and sensory organs. Critical to a fully functioning head, defects in cranial placode and sensory organ development can result in congenital craniofacial anomalies. In a forward genetic screen aimed at identifying novel regulators of craniofacial development, we discovered an embryonically lethal mouse mutant, snouty, which exhibits malformation of the facial prominences, cranial nerves and vasculature. The snouty mutation was mapped to a single nucleotide change in a ubiquitously expressed gene, Med23, which encodes a subunit of the global transcription co-factor complex, Mediator. Phenotypic analyses revealed that the craniofacial anomalies, particularly of the cranial ganglia, were caused by a failure in the proper specification of cranial placode neuronal precursors. Molecular analyses determined that defects in cranial placode neuronal differentiation in Med23 sn/sn mutants were associated with elevated WNT/ß-catenin signaling, which can be partially rescued through combined Lrp6 and Wise loss-of-function. Our work therefore reveals a surprisingly tissue specific role for the ubiquitously expressed mediator complex protein Med23 in placode differentiation during cranial ganglia development. This highlights the importance of coupling general transcription to the regulation of WNT signaling during embryogenesis.

Author Correction: The REGγ inhibitor NIP30 increases sensitivity to chemotherapy in p53-deficient tumor cells.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The REGγ inhibitor NIP30 increases sensitivity to chemotherapy in p53-deficient tumor cells.

A major challenge in chemotherapy is chemotherapy resistance in cells lacking p53. Here we demonstrate that NIP30, an inhibitor of the oncogenic REGγ-proteasome, attenuates cancer cell growth and sensitizes p53-compromised cells to chemotherapeutic agents. NIP30 acts by binding to REGγ via an evolutionarily-conserved serine-rich domain with 4-serine phosphorylation. We find the cyclin-dependent phosphatase CDC25A is a key regulator for NIP30 phosphorylation and modulation of REGγ activity during the cell cycle or after DNA damage. We validate CDC25A-NIP30-REGγ mediated regulation of the REGγ target protein p21 in vivo using p53-/- and p53/REGγ double-deficient mice. Moreover, Phosphor-NIP30 mimetics significantly increase the growth inhibitory effect of chemotherapeutic agents in vitro and in vivo. Given that NIP30 is frequently mutated in the TCGA cancer database, our results provide insight into the regulatory pathway controlling the REGγ-proteasome in carcinogenesis and offer a novel approach to drug-resistant cancer therapy.

The proteasome activator REGγ accelerates cardiac hypertrophy by declining PP2Acα-SOD2 pathway.

Pathological cardiac hypertrophy eventually leads to heart failure without adequate treatment. REGγ is emerging as 11S proteasome activator of 20S proteasome to promote the degradation of cellular proteins in a ubiquitin- and ATP-independent manner. Here, we found that REGγ was significantly upregulated in the transverse aortic constriction (TAC)-induced hypertrophic hearts and angiotensin II (Ang II)-treated cardiomyocytes. REGγ deficiency ameliorated pressure overload-induced cardiac hypertrophy were associated with inhibition of cardiac reactive oxygen species (ROS) accumulation and suppression of protein phosphatase 2A catalytic subunit α (PP2Acα) decay. Mechanistically, REGγ interacted with and targeted PP2Acα for degradation directly, thereby leading to increase of phosphorylation levels and nuclear export of Forkhead box protein O (FoxO) 3a and subsequent of SOD2 decline, ROS accumulation, and cardiac hypertrophy. Introducing exogenous PP2Acα or SOD2 to human cardiomyocytes significantly rescued the REGγ-mediated ROS accumulation of Ang II stimulation in vitro. Furthermore, treatment with superoxide dismutase mimetic, MnTBAP prevented cardiac ROS production and hypertrophy features that REGγ caused in vivo, thereby establishing a REGγ-PP2Acα-FoxO3a-SOD2 pathway in cardiac oxidative stress and hypertrophy, indicates modulating the REGγ-proteasome activity may be a potential therapeutic approach in cardiac hypertrophy-associated disorders.

Proteasome-dependent degradation of Smad7 is critical for lung cancer metastasis.

Lung cancer is one of the cancers with highest morbidity and mortality rates and the metastasis of lung cancer is a leading cause of death. Mechanisms of lung cancer metastasis are yet to be fully understood. Herein, we demonstrate that mice deficient for REGγ, a proteasome activator, exhibited a significant reduction in tumor size, numbers, and metastatic rate with prolonged survival in a conditional Kras/p53 mutant lung cancer model. REGγ enhanced the TGFß-Smad signaling pathway by ubiquitin-ATP-independent degradation of Smad7, an inhibitor of the TGFß pathway. Activated TGFß signaling in REGγ-positive lung cancer cells led to diminished expression of E-cadherin, a biomarker of epithelial-mesenchymal transitions (EMT), and elevated mesenchymal markers compared with REGγ-deficient lung cancer cells. REGγ overexpression was found in lung cancer patients with metastasis, correlating with the reduction of E-Cadherin/Smad7 and a poor prognosis. Overall, our study indicates that REGγ promotes lung cancer metastasis by activating TGF-ß signaling via degradation of Smad7. Thus, REGγ may serve as a novel therapeutic target for lung cancers with poor prognosis.

The proteasome activator REGγ counteracts immunoproteasome expression and autoimmunity.

For quite a long time, the 11S proteasome activator REGɑ and REGß, but not REGγ, are known to control immunoproteasome and promote antigen processing. Here, we demonstrate that REGγ functions as an inhibitor for immunoproteasome and autoimmune disease. Depletion of REGγ promotes MHC class I-restricted presentation to prime CD8+ T cells in vitro and in vivo. Mice deficient for REGγ have elevation of CD8+ T cells and DCs, and develop age-related spontaneous autoimmune symptoms. Mechanistically, REGγ specifically interacts with phosphorylated STAT3 and promotes its degradation in vitro and in cells. Inhibition of STAT3 dramatically attenuates levels of LMP2/LMP7 and antigen presentation in cells lacking REGγ. Importantly, treatment with STAT3 or LMP2/7 inhibitor prevented accumulation of immune complex in REGγ-/- kidney. Moreover, REGγ-/- mice also expedites Pristane-induced lupus. Bioinformatics and immunohistological analyses of clinical samples have correlated lower expression of REGγ with enhanced expression of phosphorylated STAT3, LMP2 and LMP7 in human Lupus Nephritis. Collectively, our results support the concept that REGγ is a new regulator of immunoproteasome to balance autoimmunity.

The use of the SPT-based seismic soil liquefaction triggering evaluation methodology in engineering hazard assessments.

Probabilistic and deterministic seismic soil liquefaction triggering methodologies are proposed in Cetin et al. [1]. This manuscript: i) presents the protocols, which need to be followed for the correct use of this methodology for forward engineering (design) assessments, ii) guides the engineers through the procedure, and iii) discusses the "tricks" alongside the protocol. An illustrative soil profile shaken by a scenario earthquake is presented, through which consistent estimations of representative SPT blow-counts along with fines content are discussed. Additionally, the estimation of CSR input parameters are illustrated. Last but not least the uncertainty estimations of these input parameters are presented along with the probability and factory of safety for the assessment of liquefaction triggering. •A simplified methodology and its use to assess liquefaction triggering hazard of a soil site under an earthquake scenario event.•The consistent and unbiased mean estimates of input parameters of SPT blow-counts( N 1,60 ), fines content ( F C ), vertical effective ( σ ' v ) and total ( σ v ) stresses, maximum ground acceleration ( a m a x ), stress reduction (or non-linear shear mass participation) factor ( r d ) and moment magnitude ( M w ) along with their uncertainties are discussed.•Outlined methodology enables engineers to estimate the probability of- and factor of safety against- seismic soil liquefaction triggering for design problems.

Dataset on SPT-based seismic soil liquefaction.

This data article provides a summary of seismic soil liquefaction triggering and non-triggering case histories, which were compiled, screened for data completeness and quality, and then processed for the development of triggering relationships proposed in "SPT-based probabilistic and deterministic assessment of seismic soil liquefaction triggering hazard" [1]. The database is composed of 113 liquefaction, 95 non-liquefaction, and 2 marginal liquefaction case histories, from seismic events with moment magnitude Mw values varying in the range of 5.9 to 8.3. A spreadsheet summary of these case histories are included along with a separate spreadsheet, by which maximum likelihood assessment was performed. These data transparently enable researchers to access case history input parameters and processing details, and to compare the case history processing protocols with the ones of different researchers (e.g.: "The influence of SPT procedures in soil liquefaction resistance evaluations." [2], "SPT-based liquefaction triggering procedures." [3]).

A Graft Mimic Strategy for Verticillium Resistance in Tomato.

Grafting vegetables for disease resistance has increased greatly in popularity over the past 10 years. Verticillium wilt of tomato is commonly controlled through grafting of commercial varieties on resistant rootstocks expressing the Ve1 R-gene. To mimic the grafted plant, proteomic analyses in tomato were used to identify a suitable root-specific promoter (TMVi), which was used to express the Ve1-allele in susceptible Craigella (Cs) tomato plants. The results indicate that when infected with Verticillim dahliae, race 1, the transformed plants are comparable to resistant cultivars (Cr) or grafted plants.

REGγ Controls Hippo Signaling and Reciprocal NF-κB-YAP Regulation to Promote Colon Cancer.

Purpose: Colorectal cancer is one of the most commonly diagnosed cancers closely associated with inflammation and hyperactive growth. We previously demonstrated a regulatory circuit between the proteasome activator REGγ and NF-kappaB (NF-κB) during colon inflammation, known to be important in the development of colitis-associated cancer as well as sporadic colorectal cancer. How the inflammatory microenvironment affects the Hippo pathway during colorectal cancer development is largely unknown.Experimental Design: Here, we used REGγ-deficient colon cancer cell lines, REGγ knockout mice, and human colorectal cancer samples to identify the novel molecular mechanism by which REGγ functions as an oncoprotein in the development of colorectal cancer.Results: REGγ can directly interact with Lats1 and promote its degradation, which facilitates Yes-associated protein (YAP) activation in colon cancer cells. REGγ deficiency significantly attenuated colon cancer growth, associated with decreased YAP activity. Suppression of tumor growth due to REGγ depletion was overcome by constitutively active YAP. Surprisingly, reciprocal activation of the YAP and NF-κB pathways was observed in human colon cancer cells. REGγ overexpression was found in over 60% of 172 colorectal cancer specimens, highly correlating with the elevation of YAP and p65. Postoperative follow-up revealed a significantly lower survival rate in patients with concomitantly high expression of REGγ, YAP, and p-p65.Conclusions: REGγ could be a master regulator during colorectal cancer development to promote YAP signaling and reinforce cross-talks between inflammation and growth pathways, and REGγ might be a new marker for prognosis of colorectal cancer patients. Clin Cancer Res; 24(8); 2015-25. ©2018 AACR.

The Case for Insurance Reimbursement of Couple Therapy.

A case is made for why it may now be in the best interest of insurance companies to reimburse for marital therapy to treat marital distress. Relevant literature is reviewed with a considerable focus on the reasons that insurance companies would benefit from reimbursing marital therapy - the high costs of marital distress, the growing link between marital distress and a host of related physical and mental health problems, as well as the availability of empirically supported treatments for marital distress. This is followed by a focus on the major reasons insurance companies cite for not reimbursing marital therapy, along with a discussion of advances in several growing bodies of research to address these concerns. Main arguments include the direct medical offset costs of couple and family therapy (including for high utilizers of health insurance), and the fact that insurance companies already find it cost effective to reimburse for prevention of other health and psychological problems. This is followed by implications for practitioners and researchers.

The REGγ-proteasome forms a regulatory circuit with IκBɛ and NFκB in experimental colitis.

Increasing incidence of inflammatory bowel disorders demands a better understanding of the molecular mechanisms underlying its multifactorial aetiology. Here we demonstrate that mice deficient for REGγ, a proteasome activator, show significantly attenuated intestinal inflammation and colitis-associated cancer in dextran sodium sulfate model. Bone marrow transplantation experiments suggest that REGγ's function in non-haematopoietic cells primarily contributes to the phenotype. Elevated expression of REGγ exacerbates local inflammation and promotes a reciprocal regulatory loop with NFκB involving ubiquitin-independent degradation of IκBɛ. Additional deletion of IκBɛ restored colitis phenotypes and inflammatory gene expression in REGγ-deficient mice. In sum, this study identifies REGγ-mediated control of IκBɛ as a molecular mechanism that contributes to NFκB activation and promotes bowel inflammation and associated tumour formation in response to chronic injury.

Upregulation of GSK3ß Contributes to Brain Disorders in Elderly REGγ-knockout Mice.

GSK3ß regulates some functions of the brain, but the mechanisms involved in the maintenance of GSK3ß protein stability remain ambiguous. REGγ, an important proteasome activator for ubiquitin-independent protein degradation, has been shown to degrade certain intact proteins and is involved in the regulation of important biological processes. Here we demonstrate that REGγ promotes the degradation of GSK3ß protein in vitro and in vivo. With increased GSK3ß activity, REGγ knockout (REGγ-/-) mice exhibit late-onset sensorimotor gating and cognitive deficiencies including decreased working memory, hyperlocomotion, increased stereotype, defective prepulse inhibition (PPI), and disability in nest building, at the age of 8 months or older. Inhibition of GSK3ß rescued the compromised PPI phenotypes and working memory deficiency in the knockout mice. Also, we found an age-dependent decrease in the trypsin-like proteasomal activity in REGγ-/- mice brains, which may be reflective of a lack of degradation of GSK3ß. Collectively, our findings reveal a novel regulatory pathway in which the REGγ-proteasome controls the steady-state level of GSK3ß protein. Dysfunction in this non-canonical proteasome degradation pathway may contribute to the sensorimotor gating deficiency and cognitive disorders in aging mice.