CUL4A abrogation augments DNA damage response and protection against skin carcinogenesis.

It is intuitively obvious that the ability of a cell to repair DNA damage is saturable, either by limitation of enzymatic activities, the time allotted to achieve their function, or both. However, very little is known regarding the mechanisms that establish such a threshold. Here we demonstrate that the CUL4A ubiquitin ligase restricts the cellular repair capacity by orchestrating the concerted actions of nucleotide excision repair (NER) and the DNA damage-responsive G1/S checkpoint through selective degradation of the DDB2 and XPC DNA damage sensors and the p21/CIP1/WAF1 checkpoint effector. We generated Cul4a conditional knockout mice and observed that skin-specific Cul4a ablation dramatically increased resistance to UV-induced skin carcinogenesis. Our findings reveal that wild-type cells do not operate at their full DNA repair potential, underscore the critical role of CUL4A in establishing the cellular DNA repair threshold, and highlight the potential augmentation of cellular repair proficiency by pharmacological CUL4A inhibition.

Lower Expression of SPRY4 Predicts a Poor Prognosis and Regulates Cell Proliferation in Colorectal Cancer.

BACKGROUND/AIMS: Colorectal cancer (CRC) is the third most common type of cancer worldwide. Sprouty proteins are modulators of mitogeninduced signal transduction processes and therefore can influence the process of cancerogenesis. The encoded protein of Sprouty homolog 4 (SPRY4) is associated with various human cancers. However, its biological role and clinical significance in CRC development and progression are unknown. METHODS: The aim of this study was to evaluate the expression and biological role of SPRY4 in colorectal cancer. qRT-PCR was performed to investigate the expression of SPRY4 in tumor tissues and corresponding non tumor colorectal tissues from 70 patients. The effect of SPRY4 on proliferation was evaluated by MTT and colony formation assays. CRC cells transfected with SPRY4 were injected into nude mice to study the effect of SPRY4 on tumorigenesis in vivo. RESULTS: The lower expression of SPRY4 was remarkably correlated with deep tumor invasion and advanced TNM stage. Multivariate analyses revealed that SPRY4 expression served as an independent predictor for overall survival. Using 5-aza treatment, we also observed that SPRY4 expression can be affected by DNA methylation. Further experiments revealed that overexpressed SPRY4 significantly inhibited CRC cell proliferation both in vitro and in vivo. CONCLUSION: Our study demonstrated that SPRY4 is involved in the development and progression of colorectal cancer by regulating cell proliferation and shows that SPRY4 may be a potential diagnostic and prognostic target in patients with colorectal cancer.

Improved ex vivo expansion of adult hematopoietic stem cells by overcoming CUL4-mediated degradation of HOXB4.

Direct transduction of the homeobox (HOX) protein HOXB4 promotes the proliferation of hematopoietic stem cells (HSCs) without induction of leukemogenesis, but requires frequent administration to overcome its short protein half-life (∼1 hour). We demonstrate here that HOXB4 protein levels are post-translationally regulated by the CUL4 ubiquitin ligase, and define the degradation signal sequence (degron) of HOXB4 required for CUL4-mediated destruction. Additional HOX paralogs share the conserved degron in the homeodomain and are also subject to CUL4-mediated degradation, indicating that CUL4 likely controls the stability of all HOX proteins. Moreover, we engineered a degradation-resistant HOXB4 that conferred a growth advantage over wild-type HOXB4 in myeloid progenitor cells. Direct transduction of recombinant degradation-resistant HOXB4 protein to human adult HSCs significantly enhanced their maintenance in a more primitive state both in vitro and in transplanted NOD/SCID/IL2R-γ(null) mice compared with transduction with wild-type HOXB4 protein. Our studies demonstrate the feasibility of engineering a stable HOXB4 variant to overcome a major technical hurdle in the ex vivo expansion of adult HSCs and early progenitors for human therapeutic use.

Rapid detection of SARS-CoV-2 variants of concern by single nucleotide polymorphism genotyping using TaqMan assays.

We evaluated the performance of SARS-CoV-2 TaqMan real-time reverse-transcription PCR (RT-qPCR) assays (ThermoFisher) for detecting 2 nonsynonymous spike protein mutations, E484K and N501Y. Assay accuracy was evaluated by whole genome sequencing (WGS). Residual nasopharyngeal SARS-CoV-2 positive samples (N = 510) from a diverse patient population in New York City submitted for routine SARS-CoV-2 testing during January-April 2020 were used. We detected 91 (18%) N501Y and 101 (20%) E484K variants. Four samples (0.8%) were positive for both variants. The assay had nearly perfect concordance with WGS in the validation subset, detecting B.1.1.7 and B.1.526 variants among others. Sensitivity and specificity ranged from 0.95 to 1.00. Positive and negative predictive values were 0.98-1.00. TaqMan genotyping successfully predicted the presence of B.1.1.7, but had significantly lower sensitivity, 62% (95% CI, 0.53, 0.71), for predicting B.1.526 sub-lineages lacking E484K. This approach is rapid and accurate for detecting SARS-CoV-2 variants and can be rapidly implemented in routine clinical setting.

Increased cell apoptosis in human lung adenocarcinoma and in vivo tumor growth inhibition by RBM10, a tumor suppressor gene.

Tumor suppressor genes are frequently deleted or mutated in lung cancer. The RNA-binding motif protein 10 (RBM10) gene has the ability to suppress tumor activity, but the role of RBM10 during the development of lung cancer has yet to be elucidated. The current study investigated the expression levels of RBM10 in non-tumor and tumor tissues obtained from patients with adenocarcinoma using reverse transcription-polymerase chain reaction and western blot analysis, and identified a reduction in RBM10 expression in lung tumor tissue. To investigate the in vitro and in vivo function of RBM10, A549 human non-small cell lung cancer cells were transfected with the pcDNA-RBM10 vector. Flow cytometry was used to analyze the levels of apoptosis in the transfected cells. Western blot analysis was used to evaluate the expression of B-cell lymphoma 2 (Bcl-2), cleaved caspase-3, caspase-9 and poly (ADP-ribose) polymerase (PARP) proteins in A549 cells and tissues from the A549 xenograft Bagg Albino coat (BALB/c) nude mice model. RBM10 mRNA levels were significantly decreased in adenocarcinoma cells, but not in the non-tumor tissues. The A549 cells and tumor tissues exhibited significant growth inhibition following transfection with the pcDNA-RBM10 vector, which was determined using a cell proliferation assay. Flow cytometry analysis of cells stained with Annexin V/propidium iodide indicated that the overexpression of RBM10 induced apoptosis in A549 cells. The present study demonstrated that the expression levels of Bcl-2 protein were decreased and the expression levels of cleaved caspase-3, caspase-9 and PARP proteins were significantly increased in the A549 cells and cells from ex vivo tumor tissues that were injected with RBM10 vector-containing Salmonella enterica subspecies enterica serovar typhimurium. Notably, the current study identified that the accumulated and stable overexpression of RBM10 in the xenograft BALB/c nude mice model significantly inhibited the tumor growth rate. These results may provide novel insights into the use of RBM10 for lung cancer diagnosis and therapy.

Ectopic targeting of substrates to the ubiquitin pathway.

Explanation of the physiological function of a cellular protein often requires targeted removal of that protein to reveal the associated biochemical and phenotypic alterations. A variety of technologies such as gene targeting and RNAi have been developed to abrogate the biosynthesis of the protein of interest. Recently, targeted protein degradation by harnessing the cellular ubiquitin-proteolytic machinery has emerged as a novel reverse genetic tool for loss-of-function studies. Targeted proteolysis operates at the posttranslational level to directly accelerate the turnover rate of the target protein and opens up new avenues for the dissection of complicated protein functions associated with posttranslational events, which are unattainable by a simple blocking of the biosynthesis of the target protein.

Transient in utero knockout (TIUKO) of C-MYC affects late lung and intestinal development in the mouse.

BACKGROUND: Developmentally important genes often result in early lethality in knockout animals. Thus, the direct role of genes in late gestation organogenesis cannot be assessed directly. In utero delivery of transgenes was shown previously to result in high efficiency transfer to pulmonary and intestinal epithelial stem cells. Thus, this technology can be used to evaluate late gestation development. RESULTS: In utero gene transfer was used to transfer adenovirus with either an antisense c-myc or a C-MYC ubiquitin targeting protein to knockout out c-myc expression in late gestation lung and intestines. Using either antisense or ubiquitin mediated knockout of C-MYC levels in late gestation resulted in similar effects. Decreased complexity was observed in both intestines and lungs. Stunted growth of villi was evident in the intestines. In the lung, hypoplastic lungs with disrupted aveolarization were observed. CONCLUSIONS: These data demonstrated that C-MYC was required for cell expansion and complexity in late gestation lung and intestinal development. In addition they demonstrate that transient in utero knockout of proteins may be used to determine the role of developmentally important genes in the lungs and intestines.

A protein knockdown strategy to study the function of beta-catenin in tumorigenesis.

BACKGROUND: The Wnt signaling pathway plays critical roles in cell proliferation and cell fate determination at many stages of development. A critical downstream target of Wnt signaling is the cytosolic beta-catenin, which is stabilized upon Wnt activation and promotes transcription of a variety of target genes including c-myc and cyclin D. Aberrant Wnt signaling, which results from mutations of either beta-catenin or adenomatous polyposis coli (APC), renders beta-catenin resistant to degradation, and has been associated with multiple types of human cancers. RESULTS: A protein knockdown strategy was designed to reduce the cytosolic beta-catenin levels through accelerating its turnover rate. By engineering a chimeric protein with the beta-catenin binding domain of E-cadherin fused to betaTrCP ubiquitin-protein ligase, the stable beta-catenin mutant was recruited to the cellular SCF (Skp1, Cullin 1, and F-box-containing substrate receptor) ubiquitination machinery for ubiquitination and degradation. The DLD1 colon cancer cells express wild type beta-catenin at abnormally high levels due to loss of APC. Remarkably, conditional expression of betaTrCP-E-cadherin under the control of a tetracycline-repressive promoter in DLD1 cells selectively knocked down the cytosolic, but not membrane-associated subpopulation of beta-catenin. As a result, DLD1 cells were impaired in their growth and clonogenic ability in vitro, and lost their tumorigenic potential in nude mice. CONCLUSION: We have designed a novel approach to induce degradation of stabilized/mutated beta-catenin. Our results suggest that a high concentration of cytoplasmic beta-catenin is critical for the growth of colorectal tumor cells. The protein knockdown strategy can be utilized not only as a novel method to dissect the role of oncoproteins in tumorigenesis, but also as a unique tool to delineate the function of a subpopulation of proteins localized to a specific subcellular compartment.

DDB1 is essential for genomic stability in developing epidermis.

The mammalian epidermis is maintained by proliferation and differentiation of epidermal progenitor cells in a stereotyped developmental program. Here we report that tissue-specific deletion of the UV-damaged DNA-binding protein 1 (DDB1) in mouse epidermis led to dramatic accumulation of c-Jun and p21Cip1, arrest of cell cycle at G(2)/M, selective apoptosis of proliferating cells, and as a result, a nearly complete loss of the epidermis and hair follicles. Deletion of the p53 tumor suppressor gene partially rescued the epithelial progenitor cells from death and allowed for the accumulation of aneuploid cells in the epidermis. Our results suggest that DDB1 plays an important role in development by controlling levels of cell cycle regulators and thereby maintaining genomic stability.

A kinase-independent function of c-Abl in promoting proteolytic destruction of damaged DNA binding proteins.

Damaged DNA binding proteins (DDBs) play a critical role in the initial recognition of UV-damaged DNA and mediate recruitment of nucleotide excision repair factors. Previous studies identified DDB2 as a target of the CUL-4A ubiquitin ligase. However, the biochemical mechanism governing DDB proteolysis and its underlying physiological function in the removal of UV-induced DNA damage are largely unknown. Here, we report that the c-Abl nonreceptor tyrosine kinase negatively regulates the repair of UV-induced photolesions on genomic DNA. Biochemical studies revealed that c-Abl promotes CUL-4A-mediated DDB ubiquitination and degradation in a manner that does not require its tyrosine kinase activity both under normal growth conditions and following UV irradiation. Moreover, c-Abl activates DDB degradation in part by alleviating the inhibitory effect of CAND1/TIP120A on CUL-4A. These results revealed a kinase-independent function of c-Abl in a ubiquitin-proteolytic pathway that regulates the damage recognition step of nucleotide excision repair.

Deletion of DDB1 in mouse brain and lens leads to p53-dependent elimination of proliferating cells.

DDB1, a component of the Cul4 ubiquitin ligase complex, promotes protein ubiquitination in diverse cellular functions, including nuclear excision repair, regulation of the cell cycle, and DNA replication. To investigate its physiological significance, we generated mice with null and floxed alleles of the DDB1 gene. Here we report that null mutation of DDB1 caused early embryonic lethality, while conditional inactivation of the gene in brain and lens led to neuronal and lens degeneration, brain hemorrhages, and neonatal death. These defects stemmed from a selective elimination of nearly all proliferating neuronal progenitor cells and lens epithelial cells by apoptosis. The cell death was preceded by aberrant accumulation of cell cycle regulators and increased genomic instability and could be partially rescued by removal of the tumor suppressor protein p53. Our results indicate that DDB1 plays an essential role in maintaining viability and genomic integrity of dividing cells.

Characterization of a human regulatory subunit of protein phosphatase 3 gene (PPP3RL) expressed specifically in testis.

Protein phosphatase 3 (PPP3, formerly PP2B, Calcineurin), a serine/threonine protein phosphatase, is a heterodimer composed of one catalytic subunit (PPP3C, Calcineurin A) and one regulatory subunit (PPP3R, Calcineurin B). PPP3R, an EF-hand Ca2+ binding protein, contains four high-affinity EF-hand calcium-binding sites, indicating that PPP3 plays critical roles in many calcium-mediated signal transduction pathways. PPP3R has two isoforms, PPP3R1 (also known as PP2Bbeta1) and PPP3R2 (also known as PP2BB2). While PPP3R1 is ubiquitously expressed in different tissues, PPP3R2 is exclusively expressed in testis. PPP3R2 has only been identified in rat and mouse. Here we report a human homologue of PPP3R2, which is designated PPP3RL (PPP3R like protein). PPP3RL gene was predicated to encode 171 amino acid residues with four EF-hand calcium-binding domains and this putative protein shares 82.9% and 80.5% identity with PPP3R2 of rat and mouse, respectively. Our results show that PPP3RL gene localizes to human chromosome 9q22 and transcripts of PPP3RL gene are specifically expressed in the testis, moreover, this tissue-specific expression is due to demethylation of its promoter region in testis.

Exploring the functional complexity of cellular proteins by protein knockout.

Comprehensive dissection of protein functions entails more complicated manipulations than simply eliminating the protein of interest. Established knockdown technologies, such as RNA interference, antisense oligodeoxynucleotides, or ribozymes, are limited for specific applications such as modulating protein levels or specific targeting of a posttranslationally modified subpopulation. Here we show that the engineered Skp1, Cullin 1, and F-box-containing betaTrCP substrate receptor ubiquitin-proteolytic system, designated protein knockout, could achieve not only total elimination but also rapid and systematic reduction of a given cellular protein. Stable expression of a single engineered betaTrCP demonstrated simultaneous and sustained degradation of the entire retinoblastoma family proteins. Furthermore, the engineered betaTrCP was capable of selecting hypo- but not hyperphosphorylated forms of retinoblastoma for degradation. The engineered betaTrCP has been extensively modified to increase its specificity in substrate selection. This optimized protein-knockout system offers a powerful and versatile proteomic tool to dissect diverse functional properties of cellular proteins in somatic cells.

CUL-4A stimulates ubiquitylation and degradation of the HOXA9 homeodomain protein.

The HOXA9 homeodomain protein is a key regulator of hematopoiesis and embryonic development. HOXA9 is expressed in primitive hematopoietic cells, and its prompt downregulation is associated with myelocytic maturation. Although transcriptional inactivation of HOXA9 during hematopoietic differentiation has been established, little is known about the biochemical mechanisms underlying the subsequent removal of HOXA9 protein. Here we report that the CUL-4A ubiquitylation machinery controls the stability of HOXA9 by promoting its ubiquitylation and proteasome-dependent degradation. The homeodomain of HOXA9 is responsible for CUL-4A-mediated degradation. Interfering CUL-4A biosynthesis by ectopic expression or by RNA-mediated interference resulted in alterations of the steady-state levels of HOXA9, mirrored by impairment of the ability of 32D myeloid progenitor cells to undergo proper terminal differentiation into granulocytes. These results revealed a novel regulatory mechanism of hematopoiesis by ubiquitin-dependent proteolysis.