PP2Cδ Controls the Differentiation and Function of Dendritic Cells Through Regulating the NSD2/mTORC2/ACLY Pathway

Dendritic cells (DCs) are recognized as a key orchestrator of immune response and homeostasis, deregulation of which may lead to autoimmunity such as experimental autoimmune encephalomyelitis (EAE). Herein we show that the phosphatase PP2Cδ played a pivotal role in regulating DC activation and function, as PP2Cδ ablation caused aberrant maturation, activation, and Th1/Th17-priming of DCs, and hence induced onset of exacerbated EAE. Mechanistically, PP2Cδ restrained the expression of the essential subunit of mTORC2, Rictor, primarily through de-phosphorylating and proteasomal degradation of the methyltransferase NSD2 via CRL4DCAF2 E3 ligase. Loss of PP2Cδ in DCs accordingly sustained activation of the Rictor/mTORC2 pathway and boosted glycolytic and mitochondrial metabolism. Consequently, ATP-citrate lyse (ACLY) was increasingly activated and catalyzed acetyl-CoA for expression of the genes compatible with hyperactivated DCs under PP2Cδ deletion. Collectively, our findings demonstrate that PP2Cδ has an essential role in controlling DCs activation and function, which is critical for prevention of autoimmunity.

Human gut-microbiome-derived propionate coordinates proteasomal degradation via HECTD2 upregulation to target EHMT2 in colorectal cancer

The human microbiome plays an essential role in the human immune system, food digestion, and protection from harmful bacteria by colonizing the human intestine. Recently, although the human microbiome affects colorectal cancer (CRC) treatment, the mode of action between the microbiome and CRC remains unclear. This study showed that propionate suppressed CRC growth by promoting the proteasomal degradation of euchromatic histone-lysine N-methyltransferase 2 (EHMT2) through HECT domain E3 ubiquitin protein ligase 2 (HECTD2) upregulation. In addition, EHMT2 downregulation reduced the H3K9me2 level on the promoter region of tumor necrosis factor α-induced protein 1 (TNFAIP1) as a novel direct target of EHMT2. Subsequently, TNFAIP1 upregulation induced the apoptosis of CRC cells. Furthermore, using Bacteroides thetaiotaomicron culture medium, we confirmed EHMT2 downregulation via upregulation of HECTD2 and TNFAIP1 upregulation. Finally, we observed the synergistic effect of propionate and an EHMT2 inhibitor (BIX01294) in 3D spheroid culture models. Thus, we suggest the anticancer effects of propionate and EHMT2 as therapeutic targets for colon cancer treatment and may provide the possibility for the synergistic effects of an EHMT2 inhibitor and microbiome in CRC treatment.

TARBP2 Suppresses Ubiquitin-Proteasomal Degradation of HIF-1α in Breast Cancer

TAR (HIV-1) RNA binding protein 2 (TARBP2) is an RNA-binding protein participating in cytoplasmic microRNA processing. Emerging evidence has shown the oncogenic role of TARBP2 in promoting cancer progression, making it an unfavorable prognosis marker for breast cancer. Hypoxia is a hallmark of the tumor microenvironment which induces hypoxia-inducible factor-1α (HIF-1α) for transcriptional regulation. HIF-1α is prone to be rapidly destabilized by the ubiquitination–proteasomal degradation system. In this study, we found that TARBP2 expression is significantly correlated with induced hypoxia signatures in human breast cancer tissues. At a cellular level, HIF-1α protein level was maintained by TARBP2 under either normoxia or hypoxia. Mechanistically, TARBP2 enhanced HIF-1α protein stability through preventing its proteasomal degradation. In addition, downregulation of multiple E3 ligases targeting HIF-1α (VHL, FBXW7, TRAF6) and reduced ubiquitination of HIF-1α were also induced by TARBP2. In support of our clinical findings that TARBP2 is correlated with tumor hypoxia, our IHC staining showed the positive correlation between HIF-1α and TARBP2 in human breast cancer tissues. Taken together, this study indicates the regulatory role of TARBP2 in the ubiquitination–proteasomal degradation of HIF-1α protein in breast cancer.

EDEM1 Regulates Amyloid Precursor Protein (APP) Metabolism and Amyloid-β Production

Endoplasmic reticulum (ER) degradation-enhancing α-mannosidase-like protein 1 (EDEM1) is a quality control factor directly involved in the endoplasmic reticulum-associated degradation (ERAD) process. It recognizes terminally misfolded proteins and directs them to retrotranslocation which is followed by proteasomal degradation in the cytosol. The amyloid-β precursor protein (APP) is synthesized and N-glycosylated in the ER and transported to the Golgi for maturation before being delivered to the cell surface. The amyloidogenic cleavage pathway of APP leads to production of amyloid-β (Aβ), deposited in the brains of Alzheimer’s disease (AD) patients. Here, using biochemical methods applied to human embryonic kidney, HEK293, and SH-SY5Y neuroblastoma cells, we show that EDEM1 is an important regulatory factor involved in APP metabolism. We find that APP cellular levels are significantly reduced after EDEM1 overproduction and are increased in cells with downregulated EDEM1. We also report on EDEM1-dependent transport of APP from the ER to the cytosol that leads to proteasomal degradation of APP. EDEM1 directly interacts with APP. Furthermore, overproduction of EDEM1 results in decreased Aβ40 and Aβ42 secretion. These findings indicate that EDEM1 is a novel regulator of APP metabolism through ERAD.

The Kinase MAP4K1 Inhibits Cytosolic RNA-Induced Antiviral Signaling by Promoting Proteasomal Degradation of TBK1/IKKε

TANK-binding kinase 1 (TBK1)/IκB kinase-ε (IKKε) mediates robust production of type I interferons (IFN-I) and proinflammatory cytokines to restrict the spread of invading viruses. However, excessive or prolonged production of IFN-I is harmful to the host by causing autoimmune disorders.

Herpesvirus-mediated stabilization of ICP0 expression neutralizes restriction by TRIM23

Herpes simplex virus (HSV) infection relies on immediate early proteins that initiate viral replication. Among them, ICP0 is known, for many years, to facilitate the onset of viral gene expression and reactivation from latency. However, how ICP0 itself is regulated remains elusive. Through genetic analyses, we identify that the viral γ134.5 protein, an HSV virulence factor, interacts with and prevents ICP0 from proteasomal degradation. Furthermore, we show that the host E3 ligase TRIM23, recently shown to restrict the replication of HSV-1 (and certain other viruses) by inducing autophagy, triggers the proteasomal degradation of ICP0 via K11- and K48-linked ubiquitination. Functional analyses reveal that the γ134.5 protein binds to and inactivates TRIM23 through blockade of K27-linked TRIM23 autoubiquitination. Deletion of γ134.5 or ICP0 in a recombinant HSV-1 impairs viral replication, whereas ablation of TRIM23 markedly rescues viral growth. Herein, we show that TRIM23, apart from its role in autophagy-mediated HSV-1 restriction, down-regulates ICP0, whereas viral γ134.5 functions to disable TRIM23. Together, these results demonstrate that posttranslational regulation of ICP0 by virus and host factors determines the outcome of HSV-1 infection.

Hepatitis C virus core protein inhibits hepatitis B virus replication by downregulating HBx levels via Siah-1-mediated proteasomal degradation during coinfection

Most clinical and experimental studies have suggested that hepatitis C virus (HCV) is dominant over hepatitis B virus (HBV) during coinfection, although the mechanism remains unclear. Here, we found that HCV core protein inhibits HBV replication by downregulating HBx levels during coinfection in human hepatoma cells. For this effect, HCV core protein increased reactive oxygen species levels in the mitochondria and activated the ataxia telangiectasia mutated-checkpoint kinase two pathway in the nucleus, resulting in an upregulation of p53 levels. Accordingly, HCV core protein induced p53-dependent activation of seven in absentia homolog one expression, an E3 ligase of HBx, resulting in the ubiquitination and proteasomal degradation of HBx. The effect of the HCV core protein on HBx levels was accurately reproduced in both a 1.2-mer HBV replicon and in vitro HBV infection systems, providing evidence for the inhibition of HBV replication by HCV core protein. The present study may provide insights into the mechanism of HCV dominance in HBV- and HCV-coinfected patients.

Human herpesvirus 6A U4 inhibits proteasomal degradation of amyloid precursor protein

Human herpesvirus 6 (HHV-6) belongs to the betaherpesvirus subfamily and is divided into two distinct species, HHV-6A and HHV-6B. HHV-6 can infect nerve cells and is associated with a variety of nervous system diseases. Recently, the association of HHV-6A infection with Alzheimer's disease (AD) has been suggested. The main pathological phenomena of AD are the accumulation of β-amyloid (Aβ), neurofibrillary tangles, and neuroinflammation, however, the specific molecular mechanism of pathogenesis of AD is not fully clear. In this study, we focused on the effect of HHV-6A U4 gene function on Aβ expression. Co-expression of HHV-6A U4 with APP resulted in inhibition of ubiquitin-mediated proteasomal degradation of amyloid precursor protein (APP). Consequently, accumulation of β-amyloid peptide (Aβ), insoluble neurofibrillary tangles, and loss of neural cells may occur. Immunoprecipitation coupled to mass spectrometry (IP-MS) showed that HHV-6A U4 protein interacts with E3 ubiquitin ligase composed of DDB1 and Cullin 4B which is also responsible for APP degradation. We hypothesize that HHV-6A U4 protein competes with APP for binding to E3 ubiquitin ligase, resulting in inhibition of APP ubiquitin modification and clearance. Finally, this is leading to the increase of APP expression and Aβ deposition, which is the hallmark of AD. These findings provide novel evidence for the etiological hypothesis of AD that can contribute to the further analysis of HHV-6A role in AD. IMPORTANCE The association of HHV-6A infection with Alzheimer’s disease has attracted increasing attention, although its role and molecular mechanism remain to be established. Our results here indicate that HHV-6A U4 inhibits APP (amyloid precursor protein) degradation. U4 protein interacts with CRLs (Cullin-RING E3 ubiquitin-protein ligases) which is also responsible for APP degradation. We propose a model that U4 competitively binds to CRLs with APP, resulting in APP accumulation and Aβ generation. Our findings provide new insights into the etiological hypothesis of HHV-6A in AD that can help further analyses.

Unspliced XBP1 Counteracts β-catenin to Inhibit Vascular Calcification

Background: Vascular calcification is a prevalent complication in chronic kidney disease and contributes to increased cardiovascular morbidity and mortality. XBP1 (X-box binding protein 1), existing as the unspliced (XBP1u) and spliced (XBP1s) forms, is a key component of the endoplasmic reticulum stress involved in vascular diseases. However, whether XBP1u participates in the development of vascular calcification remains unclear. Methods: We aim to investigate the role of XBP1u in vascular calcification.XBP1u protein levels were reduced in high phosphate (Pi)-induced calcified vascular smooth muscle cells (VSMCs), calcified aortas from mice with adenine diet-induced chronic renal failure (CRF) and calcified radial arteries from CRF patients. Results: Inhibition of XBP1u rather than XBP1s upregulated in the expression of the osteogenic markers runt-related transcription factor 2 (Runx2) and msh homeobox2 (Msx2), and exacerbated high Pi-induced VSMC calcification, as verified by calcium deposition and Alizarin red S staining. In contrast, XBP1u overexpression in high Pi-induced VSMCs significantly inhibited osteogenic differentiation and calcification. Consistently, SMC-specific XBP1 deficiency in mice markedly aggravated the adenine diet- and 5/6 nephrectomy-induced vascular calcification compared with that in the control littermates. Further interactome analysis revealed that XBP1u bound directly to β-catenin, a key regulator of vascular calcification, via aa 205-230 in its C-terminal degradation domain. XBP1u interacted with β-catenin to promote its ubiquitin-proteasomal degradation and thus inhibited β-catenin/T-cell factor (TCF)-mediated Runx2 and Msx2 transcription. Knockdown of β-catenin abolished the effect of XBP1u deficiency on VSMC calcification, suggesting a β-catenin-mediated mechanism. Moreover, the degradation of β-catenin promoted by XBP1u was independent of glycogen synthase kinase 3β (GSK-3β)-involved destruction complex. Conclusions: Our study identified XBP1u as a novel endogenous inhibitor of vascular calcification by counteracting β-catenin and promoting its ubiquitin-proteasomal degradation, which represents a new regulatory pathway of β-catenin and a promising target for vascular calcification treatment.