An Efficient Endothelial Cell Differentiation Protocol Using Bioactive Lipid O-Cyclic Phytosphingosine-1-Phosphate in Human Embryonic Stem Cells.

Bioactive lipids like sphingosine-1-phosphate (S1P) and lysophosphatidic acid have gained significant attention as signaling molecules with regulatory roles in stem cell proliferation and differentiation. The novel chemically synthesized sphingosine metabolite O-cyclic phytosphingosine-1-phosphate (cP1P) is derived from phytosphingosine-1-phosphate (P1P) and shares structural similarities with S1P. Previously, the role of cP1P in regulating ALK3/BMPR signaling during cardiomyocyte differentiation from human embryonic stem cells (hESCs) was demonstrated. In this study, the applicability of cP1P for endothelial cells (ECs) differentiation from hESCs was investigated an efficient method to obtain a high yield of functional ECs over several passages was standardized. The ECs derived from hESCs showed cellular and molecular characteristics similar to the native ECs. Thus, the results of this study open avenues for further research into cP1P-based stem cell differentiation for regenerative therapies.

USP28 promotes tumorigenesis and cisplatin resistance by deubiquitinating MAST1 protein in cancer cells.

Cisplatin is a chemotherapy drug that causes a plethora of DNA lesions and inhibits DNA transcription and replication, resulting in the induction of apoptosis in cancer cells. However, over time, patients develop resistance to cisplatin due to repeated treatment and thus the treatment efficacy is limited. Therefore, identifying an alternative therapeutic strategy combining cisplatin treatment along with targeting factors that drive cisplatin resistance is needed. CRISPR/Cas9 system-based genome-wide screening for the deubiquitinating enzyme (DUB) subfamily identified USP28 as a potential DUB that governs cisplatin resistance. USP28 regulates the protein level of microtubule-associated serine/threonine kinase 1 (MAST1), a common kinase whose expression is elevated in several cisplatin-resistant cancer cells. The expression level and protein turnover of MAST1 is a major factor driving cisplatin resistance in many cancer types. Here we report that the USP28 interacts and extends the half-life of MAST1 protein by its deubiquitinating activity. The expression pattern of USP28 and MAST1 showed a positive correlation across a panel of tested cancer cell lines and human clinical tissues. Additionally, CRISPR/Cas9-mediated gene knockout of USP28 in A549 and NCI-H1299 cells blocked MAST1-driven cisplatin resistance, resulting in suppressed cell proliferation, colony formation ability, migration and invasion in vitro. Finally, loss of USP28 destabilized MAST1 protein and attenuated tumor growth by sensitizing cells to cisplatin treatment in mouse xenograft model. We envision that targeting the USP28-MAST1 axis along with cisplatin treatment might be an alternative therapeutic strategy to overcome cisplatin resistance in cancer patients.

ßTrCP1 promotes SLC35F2 protein ubiquitination and inhibits cancer progression in HeLa cells.

The solute carrier family 35 F2 (SLC35F2) belongs to membrane-bound carrier proteins that are associated with multiple cancers. The main factor that determines cancer progression is the expression level of SLC35F2. Thus, identifying the E3 ligase that controls SLC35F2 protein abundance in cancer cells is critical. Here, we identified ßTrCP1 interacting with and reducing the SLC35F2 protein level. ßTrCP1 signals SLC35F2 protein ubiquitination and reduces SLC35F2 protein half-life. The mRNA expression pattern between ßTrCP1 and SLC35F2 across a panel of cancer cell lines showed a negative correlation. Additionally, the depletion of ßTrCP1 accumulated SLC35F2 protein and promoted SLC35F2-mediated cell growth, migration, invasion, and colony formation ability in HeLa cells. Overall, we demonstrate that ßTrCP1 acts as a tumor suppressor by controlling SLC35F2 protein abundance in cancer cells. The depletion of ßTrCP1 promotes SLC35F2-mediated carcinogenesis. Thus, we envision that ßTrCP1 may be a potential target for cancer therapeutics.

E3 ubiquitin ligase APC/CCdh1 regulates SLC35F2 protein turnover and inhibits cancer progression in HeLa cells.

BACKGROUND: The solute carrier family 35 F2 (SLC35F2), belongs to membrane-bound carrier proteins that control various physiological functions and are activated in several cancers. However, the molecular mechanism regulating SLC35F2 protein turnover and its implication in cancer progression remains unexplored. Therefore, screening for E3 ligases that promote SLC35F2 protein degradation is essential during cancer progression. METHODS: The immunoprecipitation and Duolink proximity ligation assays (PLA) were used to determine the interaction between APC/CCdh1 and SLC35F2 proteins. A CRISPR/Cas9-mediated knockdown and rescue experiment were used to validate the functional significance of APC/CCdh1 on SLC35F2 protein stabilization. The ubiquitination function of APC/CCdh1 on SLC35F2 protein was validated using in vitro ubiquitination assay and half-life analysis. The role of APC/CCdh1 regulating SLC35F2-mediated tumorigenesis was confirmed by in vitro oncogenic experiments in HeLa cells. RESULTS: Based on the E3 ligase screen and in vitro biochemical experiments, we identified that APC/CCdh1 interacts with and reduces SLC35F2 protein level. APC/CCdh1 promotes SLC35F2 ubiquitination and decreases the half-life of SLC35F2 protein. On the other hand, the CRISPR/Cas9-mediated depletion of APC/CCdh1 increased SLC35F2 protein levels. The mRNA expression analysis revealed a negative correlation between APC/CCdh1 and SLC35F2 across a panel of cancer cell lines tested. Additionally, we demonstrated that depletion in APC/CCdh1 promotes SLC35F2-mediated cell proliferation, colony formation, migration, and invasion in HeLa cells. CONCLUSION: Our study highlights that APC/CCdh1 is a critical regulator of SLC35F2 protein turnover and depletion of APC/CCdh1 promotes SLC35F2-mediated tumorigenesis. Thus, we envision that APC/CCdh1-SLC35F2 axis might be a therapeutic target in cancer.

USP19 Negatively Regulates p53 and Promotes Cervical Cancer Progression.

p53 is a tumor suppressor gene activated in response to cellular stressors that inhibits cell cycle progression and induces pro-apoptotic signaling. The protein level of p53 is well balanced by the action of several E3 ligases and deubiquitinating enzymes (DUBs). Several DUBs have been reported to negatively regulate and promote p53 degradation in tumors. In this study, we identified USP19 as a negative regulator of p53 protein level. We demonstrate a direct interaction between USP19 and p53 by pull down assay. The overexpression of USP19 promoted ubiquitination of p53 and reduced its protein half-life. We also demonstrate that CRISPR/Cas9-mediated knockout of USP19 in cervical cancer cells elevates p53 protein levels, resulting in reduced colony formation, cell migration, and cell invasion. Overall, our results indicate that USP19 negatively regulates p53 protein levels in cervical cancer progression.

CRISPR/Cas9-based genome-wide screening of the deubiquitinase subfamily identifies USP3 as a protein stabilizer of REST blocking neuronal differentiation and promotes neuroblastoma tumorigenesis.

BACKGROUND: The repressor element-1 silencing transcription factor (REST), a master transcriptional repressor, is essential for maintenance, self-renewal, and differentiation in neuroblastoma. An elevated expression of REST is associated with impaired neuronal differentiation, which results in aggressive neuroblastoma formation. E3 ligases are known to regulate REST protein abundance through the 26 S proteasomal degradation pathway in neuroblastoma. However, deubiquitinating enzymes (DUBs), which counteract the function of E3 ligase-mediated REST protein degradation and their impact on neuroblastoma tumorigenesis have remained unexplored. METHODS: We employed a CRISPR/Cas9 system to perform a genome-wide knockout of ubiquitin-specific proteases (USPs) and used western blot analysis to screen for DUBs that regulate REST protein abundance. The interaction between USP3 and REST was confirmed by immunoprecipitation and Duolink in situ proximity assays. The deubiquitinating effect of USP3 on REST protein degradation, half-life, and neuronal differentiation was validated by immunoprecipitation, in vitro deubiquitination, protein-turnover, and immunostaining assays. The correlation between USP3 and REST expression was assessed using patient neuroblastoma datasets. The USP3 gene knockout in neuroblastoma cells was performed using CRISPR/Cas9, and the clinical relevance of USP3 regulating REST-mediated neuroblastoma tumorigenesis was confirmed by in vitro and in vivo oncogenic experiments. RESULTS: We identified a deubiquitinase USP3 that interacts with, stabilizes, and increases the half-life of REST protein by counteracting its ubiquitination in neuroblastoma. An in silico analysis showed a correlation between USP3 and REST in multiple neuroblastoma cell lines and identified USP3 as a prognostic marker for overall survival in neuroblastoma patients. Silencing of USP3 led to a decreased self-renewal capacity and promoted retinoic acid-induced differentiation in neuroblastoma. A loss of USP3 led to attenuation of REST-mediated neuroblastoma tumorigenesis in a mouse xenograft model. CONCLUSION: The findings of this study indicate that USP3 is a critical factor that blocks neuronal differentiation, which can lead to neuroblastoma. We envision that targeting USP3 in neuroblastoma tumors might provide an effective therapeutic differentiation strategy for improved survival rates of neuroblastoma patients.

Deubiquitinase USP19 enhances phenylalanine hydroxylase protein stability and its enzymatic activity.

Phenylalanine hydroxylase (PAH) is the key enzyme in phenylalanine metabolism, deficiency of which is associated with the most common metabolic phenotype of phenylketonuria (PKU) and hyperphenylalaninemia (HPA). A bulk of PKU disease-associated missense mutations in the PAH gene have been studied, and the consequence of each PAH variant vary immensely. Prior research established that PKU-associated variants possess defects in protein folding with reduced cellular stability leading to rapid degradation. However, recent evidence revealed that PAH tetramers exist as a mixture of resting state and activated state whose transition depends upon the phenylalanine concentration and certain PAH variants that fail to modulate the structural equilibrium are associated with PKU disease. Collectively, these findings framed our understanding of the complex genotype-phenotype correlation in PKU. In the current study, we substantiate a link between PAH protein stability and its degradation by the ubiquitin-mediated proteasomal degradation system. Here, we provide an evidence that PAH protein undergoes ubiquitination and proteasomal degradation, which can be reversed by deubiquitinating enzymes (DUBs). We identified USP19 as a novel DUB that regulates PAH protein stability. We found that ectopic expression of USP19 increased PAH protein level, whereas depletion of USP19 promoted PAH protein degradation. Our study indicates that USP19 interacts with PAH and prevents polyubiquitination of PAH subsequently extending the half-life of PAH protein. Finally, the increase in the level of PAH protein by the deubiquitinating activity of USP19 resulted in enhanced metabolic function of PAH. In summary, our study identifies the role of USP19 in regulating PAH protein stability and promotes its metabolic activity. Graphical highlights 1. E3 ligase Cdh1 promotes PAH protein degradation leading to insufficient cellular amount of PAH causing PKU. 2. A balance between E3 ligase and DUB is important to regulate the proteostasis of PAH. 3. USP19 deubiquitinates and stabilizes PAH further protecting it from rapid degradation. 4. USP19 increases the enzymatic activity of PAH, thus maintaining normal Phe levels.

Human pluripotent stem cell-derived macrophages and macrophage-derived exosomes: therapeutic potential in pulmonary fibrosis.

Pulmonary fibrosis (PF) is a fatal chronic disease characterized by accumulation of extracellular matrix and thickening of the alveolar wall, ultimately leading to respiratory failure. PF is thought to be initiated by the dysfunction and aberrant activation of a variety of cell types in the lung. In particular, several studies have demonstrated that macrophages play a pivotal role in the development and progression of PF through secretion of inflammatory cytokines, growth factors, and chemokines, suggesting that they could be an alternative therapeutic source as well as therapeutic target for PF. In this review, we describe the characteristics, functions, and origins of subsets of macrophages involved in PF and summarize current data on the generation and therapeutic application of macrophages derived from pluripotent stem cells for the treatment of fibrotic diseases. Additionally, we discuss the use of macrophage-derived exosomes to repair fibrotic lung tissue.

Metabolomics Reveals Dysregulated Sphingolipid and Amino Acid Metabolism Associated with Chronic Obstructive Pulmonary Disease.

Purpose: Chronic obstructive pulmonary disease (COPD) is a heterogeneous disease presenting as multiple phenotypes, such as declining lung function, emphysema, or persistent airflow limitation caused by several risk factors, including cigarette smoking and air pollution. The inherent complexity of COPD phenotypes propounds difficulties for accurate diagnosis and prognosis. Although metabolomic profiles on COPD have been reported, the role of metabolism in COPD-related phenotypes is yet to be determined. In this study, we investigated the association between plasma sphingolipids and amino acids, and between COPD and COPD-related phenotypes in a Korean cohort. Patients and Methods: Blood samples were collected from 120 patients with COPD and 80 control participants who underwent spirometry and quantitative computed tomography. The plasma metabolic profiling was carried out using LC-MS/MS analysis. Results: Among the evaluated plasma sphingolipids, an increase in the metabolism of two specific sphingomyelins, SM (d18:1/24:0) and SM (d18:1/24:1) were significantly associated with COPD. There was no significant correlation between any of the SMs and the emphysema index, FVC and FEV1 in the COPD cohort. Meanwhile, Cer (d18:1/18:0) and Cer (d18:1/24:1) were significantly associated with reduced FEV1. Furthermore, the levels of several amino acids were altered in the COPD group compared to that in the non-COPD group; glutamate and alpha AAA were substantial associated with emphysema in COPD. Kynurenine was the only amino acid significantly associated with reduced FEV1 in COPD. In contrast, there was no correlation between FVC and the elevated metabolites. Conclusion: Our results provide dysregulated plasma metabolites impacting COPD phenotypes, although more studies are needed to explore the underlying mechanism related to COPD pathogenesis.

Deubiquitinase USP19 extends the residual enzymatic activity of phenylalanine hydroxylase variants.

Phenylalanine hydroxylase (PAH) is a key enzyme in mammals that maintains the phenylalanine (Phe) concentration at an appropriate physiological level. Some genetic mutations in the PAH gene lead to destabilization of the PAH enzyme, leading to phenylketonuria (PKU). Destabilized PAH variants can have a certain amount of residual enzymatic activity that is sufficient for metabolism of Phe. However, accelerated degradation of those variants can lead to insufficient amounts of cellular PAH protein. The optimal protein level of PAH in cells is regulated by a balancing act between E3 ligases and deubiquitinating enzymes (DUBs). In this work, we analyzed the protein expression and stability of two PKU-linked PAH protein variants, R241C and R243Q, prevalent in the Asian population. We found that the tested PAH variants were highly ubiquitinated and thus targeted for rapid protein degradation. We demonstrated that USP19, a DUB that interacts with both PAH variants, plays a regulatory role by extending their half-lives. The deubiquitinating activity of USP19 prevents protein degradation and increases the abundance of both PAH protein variants. Thus, our study reveals a novel mechanism by which deubiquitinating activity of USP19 extends the residual enzymatic activity of PAH variants.

CRISPR/Cas9-based genome-wide screening for deubiquitinase subfamily identifies USP1 regulating MAST1-driven cisplatin-resistance in cancer cells.

Background: Cisplatin is one of the frontline anticancer agents. However, development of cisplatin-resistance limits the therapeutic efficacy of cisplatin-based treatment. The expression of microtubule-associated serine/threonine kinase 1 (MAST1) is a primary factor driving cisplatin-resistance in cancers by rewiring the MEK pathway. However, the mechanisms responsible for MAST1 regulation in conferring drug resistance is unknown. Methods: We implemented a CRISPR/Cas9-based, genome-wide, dual screening system to identify deubiquitinating enzymes (DUBs) that govern cisplatin resistance and regulate MAST1 protein level. We analyzed K48- and K63-linked polyubiquitination of MAST1 protein and mapped the interacting domain between USP1 and MAST1 by immunoprecipitation assay. The deubiquitinating effect of USP1 on MAST1 protein was validated using rescue experiments, in vitro deubiquitination assay, immunoprecipitation assays, and half-life analysis. Furthermore, USP1-knockout A549 lung cancer cells were generated to validate the deubiquitinating activity of USP1 on MAST1 abundance. The USP1-MAST1 correlation was evaluated using bioinformatics tool and in different human clinical tissues. The potential role of USP1 in regulating MAST1-mediated cisplatin resistance was confirmed using a series of in vitro and in vivo experiments. Finally, the clinical relevance of the USP1-MAST1 axis was validated by application of small-molecule inhibitors in a lung cancer xenograft model in NSG mice. Results: The CRISPR/Cas9-based dual screening system identified USP1 as a novel deubiquitinase that interacts, stabilizes, and extends the half-life of MAST1 by preventing its K48-linked polyubiquitination. The expression analysis across human clinical tissues revealed a positive correlation between USP1 and MAST1. USP1 promotes MAST1-mediated MEK1 activation as an underlying mechanism that contributes to cisplatin-resistance in cancers. Loss of USP1 led to attenuation of MAST1-mediated cisplatin-resistance both in vitro and in vivo. The combined pharmacological inhibition of USP1 and MAST1 using small-molecule inhibitors further abrogated MAST1 level and synergistically enhanced cisplatin efficacy in a mouse xenograft model. Conclusions: Overall, our study highlights the role of USP1 in the development of cisplatin resistance and uncovers the regulatory mechanism of MAST1-mediated cisplatin resistance in cancers. Co-treatment with USP1 and MAST1 inhibitors abrogated tumor growth and synergistically enhanced cisplatin efficacy, suggesting a novel alternative combinatorial therapeutic strategy that could further improve MAST1-based therapy in patients with cisplatin-resistant tumors.

Dual role of deubiquitinating enzyme USP19 regulates mitotic progression and tumorigenesis by stabilizing survivin.

Survivin is a component of the chromosomal passenger complex, which includes Aurora B, INCENP, and Borealin, and is required for chromosome segregation and cytokinesis. We performed a genome-wide screen of deubiquitinating enzymes for survivin. For the first time, we report that USP19 has a dual role in the modulation of mitosis and tumorigenesis by regulating survivin expression. Our results found that USP19 stabilizes and interacts with survivin in HCT116 cells. USP19 deubiquitinates survivin protein and extends its half-life. We also found that USP19 functions as a mitotic regulator by controlling the downstream signaling of survivin protein. Targeted genome knockout verified that USP19 depletion leads to several mitotic defects, including cytokinesis failure. In addition, USP19 depletion results in significant enrichment of apoptosis and reduces the growth of tumors in the mouse xenograft. We envision that simultaneous targeting of USP19 and survivin in oncologic drug development would increase therapeutic value and minimize redundancy.

Genome-wide screening for deubiquitinase subfamily identifies ubiquitin-specific protease 49 as a novel regulator of odontogenesis.

Proteins expressed by the paired box gene 9 (PAX9) and Msh Homeobox 1 (MSX1) are intimately involved in tooth development (odontogenesis). The regulation of PAX9 and MSX1 protein turnover by deubiquitinating enzymes (DUBs) plausibly maintain the required levels of PAX9 and MSX1 during odontogenesis. Herein, we used a loss-of-function CRISPR-Cas9-mediated DUB KO library kit to screen for DUBs that regulate PAX9 and MSX1 protein levels. We identify and demonstrate that USP49 interacts with and deubiquitinates PAX9 and MSX1, thereby extending their protein half-lives. On the other hand, the loss of USP49 reduces the levels of PAX9 and MSX1 proteins, which causes transient retardation of odontogenic differentiation in human dental pulp stem cells and delays the differentiation of human pluripotent stem cells into the neural crest cell lineage. USP49 depletion produced several morphological defects during tooth development, such as reduced dentin growth with shrunken enamel space, and abnormal enamel formation including irregular mineralization. In sum, our results suggest that deubiquitination of PAX9 and MSX1 by USP49 stabilizes their protein levels to facilitate successful odontogenesis.

Genome-Wide CRISPR/Cas9-Based Screening for Deubiquitinase Subfamily Identifies Ubiquitin-Specific Protease 11 as a Novel Regulator of Osteogenic Differentiation.

The osteoblast differentiation capacity of mesenchymal stem cells must be tightly regulated, as inadequate bone mineralization can lead to osteoporosis, and excess bone formation can cause the heterotopic ossification of soft tissues. The balanced protein level of Msh homeobox 1 (MSX1) is critical during normal osteogenesis. To understand the factors that prevent MSX1 protein degradation, the identification of deubiquitinating enzymes (DUBs) for MSX1 is essential. In this study, we performed loss-of-function-based screening for DUBs regulating MSX1 protein levels using the CRISPR/Cas9 system. We identified ubiquitin-specific protease 11 (USP11) as a protein regulator of MSX1 and further demonstrated that USP11 interacts and prevents MSX1 protein degradation by its deubiquitinating activity. Overexpression of USP11 enhanced the expression of several osteogenic transcriptional factors in human mesenchymal stem cells (hMSCs). Additionally, differentiation studies revealed reduced calcification and alkaline phosphatase activity in USP11-depleted cells, while overexpression of USP11 enhanced the differentiation potential of hMSCs. These results indicate the novel role of USP11 during osteogenic differentiation and suggest USP11 as a potential target for bone regeneration.

CYLD destabilizes NoxO1 protein by promoting ubiquitination and regulates prostate cancer progression.

The NADPH oxidase (Nox) family of enzymes is solely dedicated in the generation of reactive oxygen species (ROS). ROS generated by Nox are involved in multiple signaling cascades and a myriad of pathophysiological conditions including cancer. As such, ROS seem to have both detrimental and beneficial roles in a number of cellular functions, including cell signaling, growth, apoptosis and proliferation. Regulatory mechanisms are required to control the activity of Nox enzymes in order to maintain ROS balance within the cell. Here, we performed genome-wide screening for deubiquitinating enzymes (DUBs) regulating Nox organizer 1 (NoxO1) protein expression using a CRISPR/Cas9-mediated DUB-knockout library. We identified cylindromatosis (CYLD) as a binding partner regulating NoxO1 protein expression. We demonstrated that the overexpression of CYLD promotes ubiquitination of NoxO1 protein and reduces the NoxO1 protein half-life. The destabilization of NoxO1 protein by CYLD suppressed excessive ROS generation. Additionally, CRISPR/Cas9-mediated knockout of CYLD in PC-3 cells promoted cell proliferation, migration, colony formation and invasion in vitro. In xenografted mice, injection of CYLD-depleted cells consistently led to tumor development with increased weight and volume. Taken together, these results indicate that CYLD acts as a destabilizer of NoxO1 protein and could be a potential tumor suppressor target for cancer therapeutics.

USP32 confers cancer cell resistance to YM155 via promoting ER-associated degradation of solute carrier protein SLC35F2.

Background: The most commonly preferred chemotherapeutic agents to treat cancers are small-molecule drugs. However, the differential sensitivity of various cancer cells to small molecules and untargeted delivery narrow the range of potential therapeutic applications. The mechanisms responsible for drug resistance in a variety of cancer cells are also largely unknown. Several deubiquitinating enzymes (DUBs) are the main determinants of drug resistance in cancer cells. Methods: We used CRISPR-Cas9 to perform genome-scale knockout of the entire set of genes encoding ubiquitin-specific proteases (USPs) and systematically screened for DUBs resistant to the clinically evaluated anticancer compound YM155. A series of in vitro and in vivo experiments were conducted to reveal the relationship between USP32 and SLC35F2 on YM155-mediated DNA damage in cancer cells. Results: CRISPR-based dual-screening method identified USP32 as a novel DUB that governs resistance for uptake of YM155 by destabilizing protein levels of SLC35F2, a solute-carrier protein essential for the uptake of YM155. The expression of USP32 and SLC35F2 was negatively correlated across a panel of tested cancer cell lines. YM155-resistant cancer cells in particular exhibited elevated expression of USP32 and low expression of SLC35F2. Conclusion: Collectively, our DUB-screening strategy revealed a resistance mechanism governed by USP32 associated with YM155 resistance in breast cancers, one that presents an attractive molecular target for anti-cancer therapies. Targeted genome knockout verified that USP32 is the main determinant of SLC35F2 protein stability in vitro and in vivo, suggesting a novel way to treat tumors resistant to small-molecule drugs.

USP48 Governs Cell Cycle Progression by Regulating the Protein Level of Aurora B.

Deubiquitinating enzymes play key roles in the precise modulation of Aurora B-an essential cell cycle regulator. The expression of Aurora B increases before the onset of mitosis and decreases during mitotic exit; an imbalance in these levels has a severe impact on the fate of the cell cycle. Dysregulation of Aurora B can lead to aberrant chromosomal segregation and accumulation of errors during mitosis, eventually resulting in cytokinesis failure. Thus, it is essential to identify the precise regulatory mechanisms that modulate Aurora B levels during the cell division cycle. Using a deubiquitinase knockout strategy, we identified USP48 as an important candidate that can regulate Aurora B protein levels during the normal cell cycle. Here, we report that USP48 interacts with and stabilizes the Aurora B protein. Furthermore, we showed that the deubiquitinating activity of USP48 helps to maintain the steady-state levels of Aurora B protein by regulating its half-life. Finally, USP48 knockout resulted in delayed progression of cell cycle due to accumulation of mitotic defects and ultimately cytokinesis failure, suggesting the role of USP48 in cell cycle regulation.

Bioactive Lipid O-cyclic phytosphingosine-1-phosphate Promotes Differentiation of Human Embryonic Stem Cells into Cardiomyocytes via ALK3/BMPR Signaling.

Adult human cardiomyocytes have an extremely limited proliferative capacity, which poses a great barrier to regenerative medicine and research. Human embryonic stem cells (hESCs) have been proposed as an alternative source to generate large numbers of clinical grade cardiomyocytes (CMs) that can have potential therapeutic applications to treat cardiac diseases. Previous studies have shown that bioactive lipids are involved in diverse cellular responses including cardiogenesis. In this study, we explored the novel function of the chemically synthesized bioactive lipid O-cyclic phytosphingosine-1-phosphate (cP1P) as an inducer of cardiac differentiation. Here, we identified cP1P as a novel factor that significantly enhances the differentiation potential of hESCs into cardiomyocytes. Treatment with cP1P augments the beating colony number and contracting area of CMs. Furthermore, we elucidated the molecular mechanism of cP1P regulating SMAD1/5/8 signaling via the ALK3/BMP receptor cascade during cardiac differentiation. Our result provides a new insight for cP1P usage to improve the quality of CM differentiation for regenerative therapies.

Ubiquitin Specific Protease 29 Functions as an Oncogene Promoting Tumorigenesis in Colorectal Carcinoma.

Colorectal carcinoma is the third foremost cause of cancer-related deaths and accounts for 5.8% of all deaths globally. The molecular mechanisms of colon cancer progression and metastasis control are not well studied. Ubiquitin-specific protease 29 (USP29), a deubiquitinating enzyme, is involved in the occurrence and development of wide variety of cancers. However, its clinical significance and biological roles in colorectal carcinoma (CRC) remain unexplored. In this research, we observed that the rate of USP29 overexpression was higher in colon cancer patient tissues relative to its corresponding normal tissues. CRISPR-Cas9-mediated depletion of USP29 triggered DNA double strand breaks and delayed cell-cycle progression in HCT116 cells. We also demonstrated that USP29 depletion hampers the colony formation and increases apoptosis of HCT116 cells. USP29 knockdown significantly decreased CRC cell proliferation in vitro. Depletion of USP29 in HCT116 cells substantially reduced the tumor volume of mouse xenografts. In conclusion, our study shows that elevated expression of USP29 promotes malignancy in CRC, suggesting that USP29 could be a promising target for colon cancer therapy.