Cyclin D1 has been recognized as an oncogene due to its abnormal upregulation in different types of cancers. Here, we demonstrated that cyclin D1 is SUMOylated, and we identified Itch as a specific E3 ligase recognizing SUMOylated cyclin D1 and mediating SUMO-induced ubiquitination and proteasome degradation of cyclin D1. We generated cyclin D1 mutant mice with mutations in the SUMOylation site, phosphorylation site, or both sites of cyclin D1, and found that double mutant mice developed a Mantle cell lymphoma (MCL)-like phenotype. We showed that arsenic trioxide (ATO) enhances cyclin D1 SUMOylation-mediated degradation through inhibition of cyclin D1 deSUMOylation enzymes, leading to MCL cell apoptosis. Treatment of severe combined immunodeficiency (SCID) mice grafted with MCL cells with ATO resulted in a significant reduction in tumor growth. In this study, we provide novel insights into the mechanisms of MCL tumor development and cyclin D1 regulation and discover a new strategy for MCL treatment.

BACKGROUND: Multiple myeloma (MM) is an incurable hematological malignancy of the plasma cells. The maintenance of protein homeostasis is critical for MM cell survival. Elevated levels of paraproteins in MM cells are cleared by proteasomes or lysosomes, which are independent but inter-connected with each other. Proteasome inhibitors (PIs) work as a backbone agent and successfully improved the outcome of patients; however, the increasing activity of autophagy suppresses the sensitivity to PIs treatment.
METHODS: The transcription levels of CRIP1 were explored in plasma cells obtained from healthy donors, patients with newly diagnosed multiple myeloma (NDMM), and relapsed/refractory multiple myeloma (RRMM) using Gene expression omnibus datasets. Doxycycline-inducible CRIP1-shRNA and CRIP1 overexpressed MM cell lines were constructed to explore the role of CRIP1 in MM pathogenesis. Proliferation, invasion, migration, proteasome activity and autophagy were examined in MM cells with different CRIP1 levels. Co-immunoprecipitation (Co-IP) with Tandem affinity purification/Mass spectrum (TAP/MS) was performed to identify the binding proteins of CRIP1. The mouse xenograft model was used to determine the role of CRIP1 in the proliferation and drug-resistance of MM cells.
RESULTS: High CRIP1 expression was associated with unfavorable clinical outcomes in patients with MM and served as a biomarker for RRMM with shorter overall survival. In vitro and in vivo studies showed that CRIP1 plays a critical role in protein homeostasis via the dual regulation of the activities of proteasome and autophagy in MM cells. A combined analysis of RNA-seq, Co-IP and TAP/MS demonstrated that CRIP1 promotes proteasome inhibitors resistance in MM cells by simultaneously binding to de-ubiquitinase USP7 and proteasome coactivator PA200. CRIP1 promoted proteasome activity and autophagosome maturation by facilitating the dequbiquitination and stabilization of PA200.
CONCLUSIONS: Our findings clarified the pivotal roles of the CRIP1/USP7/PA200 complex in ubiquitin-dependent proteasome degradation and autophagy maturation involved in the pathogenesis of MM.
BACKGROUND: A full list of funding sources can be found in the acknowledgements section.

OBJECTIVE: Vascular endothelial growth factor receptors (VEGFRs) have been demonstrated to play a critical role in ischemic retinal diseases, as VEGFRs mediate hypoxia-induced neovascularization. Not only hypoxia, ischemia also induces the deficiency of glucose, yet its effects on VEGFR signal and neovascularization have seldom been studied. Bioinformatics analysis predicted that VEGFRs may be regulated by O-GlcNAcylation, while glucose deficiency influences the O-GlcNAcylation.
METHODS: In this study, we treated human retinal microvascular endothelial cells with low glucose (LG) alone or in combination with low oxygen (oxygen and glucose deprivation, OGD). Cell viability and apoptosis rate were used to evaluate cell growth characters.
RESULTS: LG (2.8 mmol/L) treatment induced mRNA and protein levels of VEGFR1, 2, 3 even in the presence of the protein synthesis inhibitor, cycloheximide (CHX), suggesting that the increase in VEGFR proteins is partially associated with post-translational modifications. Immunoprecipitation analysis showed that O-GlcNAc level was decreased by LG in both VEGFR1, 2, but a de-O-GlcNAc glycosylase inhibitor restored the O-GlcNAc levels. This inhibitor also abolished the LG-induced increase in VEGFR2 protein, whereas this effect was not disappeared in the presence of the proteasome inhibitor, MG132. Similar results were also observed under OGD condition. VEGFR2 knockdown more significantly retarded the growth of hRMECs and HUVECs than VEGFR1, 3 knockdown under LG and OGD conditions.
CONCLUSIONS: A relatively low glucose suppressed O-GlcNAcylation in VEGFR2, whereby inhibiting its proteasome degradation; up-regulated VEGFR2 promoted the proliferation of vascular endothelial cells under ischemic condition.

Acute myeloid leukemia (AML) is a quickly progressive and devastated hematological malignancy with large rate of relapse and the appearance of chemotherapy resistance. Therefore, the identification of new therapeutic targets is urgent. ZFP91 is a hidden oncogene. Nevertheless, how ZFP91 takes part in regulating AML is less clear. Our research aims at investigating the molecular mechanisms and uncovering the effects of ZFP91 on AML. This research demonstrates that ZFP91 boosts AML cell proliferation and stops AML cell apoptosis. Mechanistically, experimental results showed the interaction between ZFP91 and RIP1 and inhibitory effect of ZFP91 on the K48-linked ubiquitination of endogenous RIP1, which is an important molecule in AML. Taken together, our results provide the evidence that targeted inhibition of ZFP91 could be a hopeful measure to treat AML.

BACKGROUND: Neuroinflammation is thought to be a cause of Alzheimer\'s disease (AD), which is partly caused by inadequate mitophagy. As a receptor of mitophagy, we aimed to reveal the regulatory roles of optineurin (OPTN) on neuroinflammation in the pathogenesis of AD.
METHODS: BV2 cells and APP/PS1 transgenic (Tg) mice were used as in vitro and in vivo experimental models to determine the regulatory roles of OPTN in neuroinflammation of AD. Sophisticated molecular technologies including quantitative (q) RT-PCR, western blot, enzyme linked immunosorbent assay (ELISA), co-immunoprecipitation (Co-IP) and immunofluorescence (IF) were employed to reveal the inherent mechanisms.
RESULTS: As a consequence, key roles of OPTN in regulating neuroinflammation were identified by depressing the activity of absent in melanoma 2 (AIM2) inflammasomes and receptor interacting serine/threonine kinase 1 (RIPK1)-mediated NF-κB inflammatory mechanisms. In detail, we found that expression of OPTN was downregulated, which resulted in activation of AIM2 inflammasomes due to a deficiency in mitophagy in APP/PS1 Tg mice. By ectopic expression, OPTN blocks the effects of Aβ oligomer (Aβo) on activating AIM2 inflammasomes by inhibiting mRNA expression of AIM2 and apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain (ASC), leading to a reduction in the active form of caspase-1 and interleukin (IL)-1β in microglial cells. Moreover, RIPK1 was also found to be negatively regulated by OPTN via ubiquitin protease hydrolysis, resulting in the synthesis of IL-1β by activating the transcriptional activity of NF-κB in BV2 cells. As an E3 ligase, the UBAN domain of OPTN binds to the death domain (DD) of RIPK1 to facilitate its ubiquitination. Based on these observations, ectopically expressed OPTN in APP/PS1 Tg mice deactivated microglial cells and astrocytes via the AIM2 inflammasome and RIPK-dependent NF-κB pathways, leading to reduce neuroinflammation.
CONCLUSIONS: These results suggest that OPTN can alleviate neuroinflammation through AIM2 and RIPK1 pathways, suggesting that OPTN deficiency may be a potential factor leading to the occurrence of AD.

5,10-methylenetetrahydrofolate reductase (MTHFR) deficiency is a rare hereditary disease characterized by defects in folate and homocysteine metabolism. Individuals with inherited MTHFR gene mutations have a higher tendency to develop neurodegeneration disease as Alzheimer\' disease and atherosclerosis. MTHFR is a rate-limiting enzyme catalyzing folate production, various SNPs/mutations in the MTHFR gene have been correlated to MTHFR deficiency. However, the molecular mechanisms underpinning the pathogenic effects of these SNPs/mutations have not been clearly understood. In the present study, we reported a severe MTHFR deficiency patient with late-onset motor dysfunction and sequenced MTHFR gene exons of the family. The patient carries an MD-associating SNP (rs748289202) in one MTHFR allele and the rs545086633 SNP with unknown disease relevance in the other. The rs545086633 SNP (p.Leu439Pro) results in an L439P substitution in MTHFR protein, and drastically decreases mutant protein expression by promoting proteasomal degradation. L439 in MTHFR is highly conserved in vertebrates. Our study demonstrated that p.Leu439Pro in MTHFR is the first mutation causing significant intracellular defects of MTHFR, and rs545086633 should be examined for the in-depth diagnosis and treatment of MD.

NOD-like receptors (NLRs) are traditionally recognized as major inflammasome components. The role of NLRs in germ cell differentiation and reproduction is not known. Here, we identified the gonad-specific Nlrp14 as a pivotal regulator in primordial germ cell-like cell (PGCLC) differentiation in vitro. Physiologically, knock out of Nlrp14 resulted in reproductive failure in both female and male mice. In adult male mice, Nlrp14 knockout (KO) inhibited differentiation of spermatogonial stem cells (SSCs) and meiosis, resulting in trapped SSCs in early stages, severe oligozoospermia, and sperm abnormality. Mechanistically, NLRP14 promoted spermatogenesis by recruiting a chaperone cofactor, BAG2, to bind with HSPA2 and form the NLRP14-HSPA2-BAG2 complex, which strongly inhibited ChIP-mediated HSPA2 polyubiquitination and promoted its nuclear translocation. Finally, loss of HSPA2 protection and BAG2 recruitment by NLRP14 was confirmed in a human nonsense germline variant associated with male sterility. Together, our data highlight a unique proteasome-mediated, noncanonical function of NLRP14 in PGCLC differentiation and spermatogenesis, providing mechanistic insights of gonad-specific NLRs in mammalian germline development.

Transforming growth factor β (TGFβ) signaling plays an important role in regulating tumor malignancy, including in non-small cell lung cancer (NSCLC). The major biological responses of TGFβ signaling are determined by the effector proteins SMAD2 and SMAD3. However, the regulators of TGFβ-SMAD signaling are not completely revealed yet. Here, we showed that the scaffolding protein PDLIM5 (PDZ and LIM domain protein 5, ENH) critically promotes TGFβ signaling by maintaining SMAD3 stability in NSCLC. First, PDLIM5 was highly expressed in NSCLC compared with that in adjacent normal tissues, and high PDLIM5 expression was associated with poor outcome. Knockdown of PDLIM5 in NSCLC cells decreased migration and invasion in vitro and lung metastasis in vivo In addition, TGFβ signaling and TGFβ-induced epithelial-mesenchymal transition was repressed by PDLIM5 knockdown. Mechanistically, PDLIM5 knockdown resulted in a reduction of SMAD3 protein levels. Overexpression of SMAD3 reversed the TGFβ-signaling-repressing and anti-migration effects induced by PDLIM5 knockdown. Notably, PDLIM5 interacted with SMAD3 but not SMAD2 and competitively suppressed the interaction between SMAD3 and its E3 ubiquitin ligase STUB1. Therefore, PDLIM5 protected SMAD3 from STUB1-mediated proteasome degradation. STUB1 knockdown restored SMAD3 protein levels, cell migration, and invasion in PDLIM5-knockdown cells. Collectively, our findings indicate that PDLIM5 is a novel regulator of basal SMAD3 stability, with implications for controlling TGFβ signaling and NSCLC progression.

Death-associated protein kinase 1 (DAPK) is a calcium/calmodulin kinase that plays a vital role as a suppressor gene in various cancers. Yet its role and target gene independent of p53 is still unknown in hepatocellular carcinoma (HCC). In this study, we discovered that DAPK suppressed HCC cell migration and invasion instead of proliferation or colony formation. Using a proteomics approach, we identified DEAD-box helicase 20 (DDX20) as an important downstream target of DAPK in HCC cells and critical for DAPK-mediated inhibition of HCC cell migration and invasion. Using integrin inhibitor RGD and GTPase activity assays, we discovered that DDX20 suppressed HCC cell migration and invasion through the CDC42-integrin pathway, which was previously reported as an important downstream pathway of DAPK in cancer. Further research using cycloheximide found that DAPK attenuates the proteasomal degradation of DDX20 protein, which is dependent on the kinase activity of DAPK. Our results shed light on new functions and regulation for both DAPK and DDX20 in carcinogenesis and identifies new potential therapeutic targets for HCC.

Regulated intramembrane proteolysis (RIP) is a key mechanism for activating transmembrane proteins such as epithelial cell adhesion molecule (EpCAM) for cellular signaling and degradation. EpCAM is highly expressed in carcinomas and progenitor and embryonic stem cells and is involved in the regulation of cell adhesion, proliferation, and differentiation. Strictly sequential cleavage of EpCAM through RIP involves initial shedding of the extracellular domain by α-secretase (ADAM) and β-secretase (BACE) sheddases, generating a membrane-tethered C-terminal fragment EpCTF. Subsequently, the rate-limiting γ-secretase complex catalyzes intramembrane cleavage of EpCTF, generating an extracellular EpCAM-Aβ-like fragment and an intracellular EpICD fragment involved in nuclear signaling. Here, we have combined biochemical approaches with live-cell imaging of fluorescent protein tags to investigate the kinetics of γ-secretase-mediated intramembrane cleavage of EpCTF. We demonstrate that γ-secretase-mediated proteolysis of exogenously and endogenously expressed EpCTF is a slow process with a 50% protein turnover in cells ranging from 45 min to 5.5 h. The slow cleavage was dictated by γ-secretase activity and not by EpCTF species, as indicated by cross-species swapping experiments. Furthermore, both human and murine EpICDs generated from EpCTF by γ-secretase were degraded efficiently (94-99%) by the proteasome. Hence, proteolytic cleavage of EpCTF is a comparably slow process, and EpICD generation does not appear to be suited for rapidly transducing extracellular cues into nuclear signaling, but appears to provide steady signals that can be further controlled through efficient proteasomal degradation. Our approach provides an unbiased bioassay to investigate proteolytic processing of EpCTF in single living cells.