Truncated ERG Oncoproteins from TMPRSS2-ERG Fusions Are Resistant to SPOP-Mediated Proteasome Degradation.

SPOP mutations and TMPRSS2-ERG rearrangements occur collectively in up to 65% of human prostate cancers. Although the two events are mutually exclusive, it is unclear whether they are functionally interrelated. Here, we demonstrate that SPOP, functioning as an E3 ubiquitin ligase substrate-binding protein, promotes ubiquitination and proteasome degradation of wild-type ERG by recognizing a degron motif at the N terminus of ERG. Prostate cancer-associated SPOP mutations abrogate the SPOP-mediated degradation function on the ERG oncoprotein. Conversely, the majority of TMPRSS2-ERG fusions encode N-terminal-truncated ERG proteins that are resistant to the SPOP-mediated degradation because of degron impairment. Our findings reveal degradation resistance as a previously uncharacterized mechanism that contributes to elevation of truncated ERG proteins in prostate cancer. They also suggest that overcoming ERG resistance to SPOP-mediated degradation represents a viable strategy for treatment of prostate cancers expressing either mutated SPOP or truncated ERG.

Prostate cancer-associated SPOP mutations lead to genomic instability through disruption of the SPOP-HIPK2 axis.

Speckle-type Poz protein (SPOP), an E3 ubiquitin ligase adaptor, is the most frequently mutated gene in prostate cancer. The SPOP-mutated subtype of prostate cancer shows high genomic instability, but the underlying mechanisms causing this phenotype are still largely unknown. Here, we report that upon DNA damage, SPOP is phosphorylated at Ser119 by the ATM serine/threonine kinase, which potentiates the binding of SPOP to homeodomain-interacting protein kinase 2 (HIPK2), resulting in a nondegradative ubiquitination of HIPK2. This modification subsequently increases the phosphorylation activity of HIPK2 toward HP1γ, and then promotes the dissociation of HP1γ from trimethylated (Lys9) histone H3 (H3K9me3) to initiate DNA damage repair. Moreover, the effect of SPOP on the HIPK2-HP1γ axis is abrogated by prostate cancer-associated SPOP mutations. Our findings provide new insights into the molecular mechanism of SPOP mutations-driven genomic instability in prostate cancer.

CHIP control degradation of mutant ETF:QO through ubiquitylation in late-onset multiple acyl-CoA dehydrogenase deficiency.

Late-onset multiple acyl-CoA dehydrogenase deficiency (MADD) is the most common form of lipid storage myopathy. The disease is mainly caused by mutations in electron-transfer flavoprotein dehydrogenase gene (ETFDH), which leads to decreased levels of ETF:QO in skeletal muscle. However, the specific underlying mechanisms triggering such degradation remain unknown. We constructed expression plasmids containing wild type ETF:QO and mutants ETF:QO-A84T, R175H, A215T, Y333C, and cultured patient-derived fibroblasts containing the following mutations in ETFDH: c.250G>A (p.A84T), c.998A>G (p.Y333C), c.770A>G (p.Y257C), c.1254_1257delAACT (p. L418TfsX10), c.524G>A (p.R175H), c.380T>A (p.L127P), and c.892C>T (p.P298S). We used in vitro expression systems and patient-derived fibroblasts to detect stability of ETF:QO mutants then evaluated their interaction with Hsp70 interacting protein CHIP with active/inactive ubiquitin E3 ligase carboxyl terminus using western blot and immunofluorescence staining. This interaction was confirmed in vitro and in vivo by co-immunoprecipitation and immunofluorescence staining. We confirmed the existence two ubiquitination sites in mutant ETF:QO using mass spectrometry (MS) analysis. We found that mutant ETF:QO proteins were unstable and easily degraded in patient fibroblasts and in vitro expression systems by ubiquitin-proteasome pathway, and identified the specific ubiquitin E3 ligase as CHIP, which forms complex to control mutant ETF:QO degradation through poly-ubiquitination. CHIP-dependent degradation of mutant ETF:QO proteins was confirmed by MS and site-directed mutagenesis of ubiquitination sites. Hsp70 is directly involved in this process as molecular chaperone of CHIP. CHIP plays an important role in ubiquitin-proteasome pathway dependent degradation of mutant ETF:QO by working as a chaperone-assisted E3 ligase, which reveals CHIP's potential role in pathological mechanisms of late-onset MADD.

Integrative analysis of AR-mediated transcriptional regulatory network reveals IRF1 as an inhibitor of prostate cancer progression.

BACKGROUND: Androgen receptor (AR) is crucial for prostate cancer (PCa) initiation and malignant progression. Only half of androgen-responsive genes have been identified as having androgen-responsive elements, suggesting that AR regulates downstream genes through other transcriptional factors. However, whether and how AR regulates the progression via regulating these androgen-responsive genes remains unclear. METHODS: Androgen-responsive and activity-changed (AC) transcriptional factors (TFs) were identified based on the time-course gene-expression array and gene promoter regions analysis. The intersection of androgen-responsive and AC TFs was selected the core TFs, which were used to construct the core transcriptional regulatory network. GO enrichment analysis, cell proliferation assays, glycolysis experiments, and reverse transcription polymerase chain reaction analysis were used to analyze and validate the functions of the network. As one of the core TFs, the function and mechanism of IRF1 have been further explored. RESULTS: We devised a new integrated approach to select core TFs and construct core transcriptional regulatory network in PCa. The 24 core TFs and core transcriptional regulatory network participate in regulating PCa cell proliferation, RNA splicing, and cancer metabolism. Further validations showed that AR signaling could promote glycolysis via inducing glycolytic enzymes in PCa cells. IRF1, a novel target of AR, served as a tumor suppressor by inhibiting PCa proliferation, cell cycle, and glycolysis. CONCLUSIONS: It is the first time to demonstrate the regulating role of the AR-mediated transcriptional regulatory network in a series of important biological processes in PCa cells. IRF1, an AR-regulated TF, acts as tumor suppressor in this core transcriptional regulatory network, which highlights the therapeutic potential of targeting this regulatory network for PCa.

Dysregulation of INF2-mediated mitochondrial fission in SPOP-mutated prostate cancer.

Next-generation sequencing of the exome and genome of prostate cancers has identified numerous genetic alternations. SPOP (Speckle-type POZ Protein) was one of the most frequently mutated genes in primary prostate cancer, suggesting SPOP is a potential driver of prostate cancer development and progression. However, how SPOP mutations contribute to prostate cancer pathogenesis remains poorly understood. SPOP acts as an adaptor protein of the CUL3-RBX1 E3 ubiquitin ligase complex that generally recruits substrates for ubiquitination and subsequent degradation. ER-localized isoform of the formin protein inverted formin 2 (INF2) mediates actin polymerization at ER-mitochondria intersections and facilitates DRP1 recruitment to mitochondria, which is a critical step in mitochondrial fission. Here, we revealed that SPOP recognizes a Ser/Thr (S/T)-rich motif in the C-terminal region of INF2 and triggers atypical polyubiquitination of INF2. These ubiquitination modifications do not lead to INF2 instability, but rather reduces INF2 localization in ER and mitochondrially associated DRP1 puncta formation, therefore abrogates its ability to facilitate mitochondrial fission. INF2 mutant escaping from SPOP-mediated ubiquitination is more potent in prompting mitochondrial fission. Moreover, prostate cancer-associated SPOP mutants increase INF2 localization in ER and promote mitochondrial fission, probably through a dominant-negative effect to inhibit endogenous SPOP. Moreover, INF2 is important for SPOP inactivation-induced prostate cancer cell migration and invasion. These findings reveal novel molecular events underlying the regulation of INF2 function and localization, and provided insights in understanding the relationship between SPOP mutations and dysregulation of mitochondrial dynamics in prostate cancer.

Prostate Cancer-associated SPOP mutations enhance cancer cell survival and docetaxel resistance by upregulating Caprin1-dependent stress granule assembly.

BACKGROUND: The gene encoding the E3 ubiquitin ligase substrate-binding adaptor SPOP is frequently mutated in primary prostate cancer, but how SPOP mutations contribute to prostate cancer pathogenesis remains poorly understood. Stress granules (SG) assembly is an evolutionarily conserved strategy for survival of cells under stress, and often upregulated in human cancers. We investigated the role of SPOP mutations in aberrant activation of the SG in prostate cancer and explored the relevanve of the mechanism in therapy resistance. METHODS: We identified SG nucleating protein Caprin1 as a SPOP interactor by using the yeast two hybrid methods. A series of functional analyses in cell lines, patient samples, and xenograft models were performed to investigate the biological significance and clinical relevance of SPOP regulation of SG signaling in prostate cancer. RESULTS: The cytoplasmic form of wild-type (WT) SPOP recognizes and triggers ubiquitin-dependent degradation of Caprin1. Caprin1 abundance is elevated in SPOP-mutant expressing prostate cancer cell lines and patient specimens. SPOP WT suppresses SG assembly, while the prostate cancer-associated mutants enhance SG assembly in a Caprin1-dependent manner. Knockout of SPOP or expression of prostate cancer-associated SPOP mutants conferred resistance to death caused by SG inducers (e.g. docetaxel, sodium arsenite and H2O2) in prostate cancer cells. CONCLUSIONS: SG assembly is aberrantly elevated in SPOP-mutated prostate cancer. SPOP mutations cause resistance to cellular stress induced by chemtherapeutic drug such as docetaxel in prostate cancer.

A novel androgen-reduced prostate-specific lncRNA, PSLNR, inhibits prostate-cancer progression in part by regulating the p53-dependent pathway.

BACKGROUND: Prostate cancer (PCa) is one of the most common cancers in males in China. Long noncoding RNAs (lncRNAs) reportedly play crucial roles in human cancer progression in many studies. However, the molecular mechanisms underlying PCa progression remain unclear. MATERIALS AND METHODS: We investigated the lncRNA transcriptome using publicly available RNA-sequencing data to identify prostate-specific lncRNAs. Then, the chromatin immunoprecipitation (ChIP) assay identified lncRNA with a direct binding to androgen receptor (AR), hereafter denoted as PSLNR. Quantitative real-time polymerase chain reaction analysis and Western blot analysis were performed to detect the expression of p53 signaling-related genes after overexpression PSLNR. The effects of overexpression of PSLNR on cell proliferation, cell cycle, and cell apoptosis were assessed by using CCK-8 and flow cytometric analysis. We then detected the expression of PSLNR in tissues. RESULT: We reported a novel androgen-reduced prostate-specific lncRNA, PSLNR, that inhibited PCa progression via the p53-dependent pathway. By analyzing the NOCODE data set, we reported that PSLNR was specifically expressed in the prostate, suggesting the potential of PSLNR as a biomarker for PCa treatment. The AR pathway was also confirmed to be an upstream regulation signaling pathway of PSLNR by transcriptionally regulating its expression in androgen-dependent PCa cells. PSLNR also significantly inhibited PCa proliferation by inducing cell apoptosis in a p53-dependent manner. Thus, PSLNR may be a candidate diagnosis and therapeutic target for PCa. CONCLUSIONS: Our study revealed for the first time a novel androgen-reduced prostate-specific lncRNA, PSLNR, which inhibited PCa progression via the p53-dependent pathway, suggesting that PSLNR may be a candidate diagnosis and therapeutic target for PCa.

Front-signal-dependent accumulation of the RHOA inhibitor FAM65B at leading edges polarizes neutrophils.

A hallmark of neutrophil polarization is the back localization of active RHOA and phosphorylated myosin light chain (pMLC, also known as MYL2). However, the mechanism for the polarization is not entirely clear. Here, we show that FAM65B, a newly identified RHOA inhibitor, is important for the polarization. When FAM65B is phosphorylated, it binds to 14-3-3 family proteins and becomes more stable. In neutrophils, chemoattractants stimulate FAM65B phosphorylation largely depending on the signals from the front of the cells that include those mediated by phospholipase Cß (PLCß) and phosphoinositide 3-kinase γ (PI3Kγ), leading to FAM65B accumulation at the leading edge. Concordantly, FAM65B deficiency in neutrophils resulted in an increase in RHOA activity and localization of pMLC to the front of cells, as well as defects in chemotaxis directionality and adhesion to endothelial cells under flow. These data together elucidate a mechanism for RHOA and pMLC polarization in stimulated neutrophils through direct inhibition of RHOA by FAM65B at the leading edge.

Zinc finger protein 191 inhibits hepatocellular carcinoma metastasis through discs large 1-mediated yes-associated protein inactivation.

UNLABELLED: Interplay between cell polarity module Scribble-Lethal Giant Larvae-Discs Large 1 (DLG1) and Yes-associated protein (YAP) appears critical in tumor metastasis. We identified zinc finger protein 191 (ZNF191) as a metastasis suppressor acting through DLG-YAP crosstalk in hepatocellular carcinoma (HCC). Overexpression of ZNF191 in HCC cells impaired cell motility, while ZNF191 depletion promoted cell migration in vitro and metastasis in vivo through triggering YAP signaling. Chromatin immunoprecipitation-sequencing revealed that ZNF191 specifically bound to the promoter of DLG1, a cell polarity maintainer and a negative regulator of YAP. The binding sequence of ZNF191 at the DLG1 promoter is a seven-repeat of TCAT motif. Double-knockdown experiments inferred that DLG1 was not only the mediator of the function of ZNF191 to suppress migration but also a link between ZNF191 and YAP signaling. Decreased expression of ZNF191 in human metastatic HCC specimens correlated positively with DLG1 levels but inversely with YAP activation. Our findings illustrate a YAP-targeting, antimetastasis function of ZNF191, thereby representing a possible prognostic marker and a potential target for metastasis therapy. CONCLUSION: ZNF191 directly binds to the DLG1 promoter at a typical TCAT repeating motif and activates the expression of DLG1; through up-regulating DLG1, ZNF191 inhibits cell migration and YAP activation in HCC cells and eventually inhibits metastasis. (Hepatology 2016;64:1148-1162).

High TXNDC5 expression predicts poor prognosis in renal cell carcinoma.

Clear cell renal cell carcinoma (ccRCC) is the most common primary kidney cancer in adults, and the identification of biomarkers involved in the pathogenesis and prognosis of ccRCC is crucial for early diagnosis and anticancer treatment. In this study, we demonstrate that thioredoxin domain-containing protein 5 (TXNDC5) expression is markedly upregulated in ccRCC tissues in comparison with adjacent non-cancerous tissues through quantitative RT-PCR, Western blotting, and immunohistochemical analyses. Importantly, TXNDC5 expression is negatively correlated with the overall survival of patients. Knockdown of TXNDC5 by siRNAs inhibits the cell growth, migration, and invasion of ccRCC cells as well as sensitizes ccRCC cells to chemotherapeutic drugs, such as Camptothecin and 5-Fluorouracil. Moreover, we used complementary DNA (cDNA) microarray analyses to explore the underlying molecular mechanisms of TXNDC5 in the pathogenesis of ccRCC. We demonstrate that knockdown of TXNDC5 affects the messenger RNA (mRNA) and protein levels of numerous important genes associated with tumorigenesis. In summary, our findings indicate that TXNDC5 performs an essential function in ccRCC pathogenesis and can serve as a novel prognostic marker of ccRCC.

Genetic variants in miR-196a2 and miR-499 are associated with susceptibility to esophageal squamous cell carcinoma in Chinese Han population.

Esophageal squamous cell carcinoma (ESCC) is the dominant type of esophageal cancer in the East Asian population. MicroRNAs (miRNAs) have been studied to play important roles in tumorigenesis. Single nucleotide polymorphisms (SNPs) in miRNA lead to the aberrant expression and structural alteration of miRNA and are hypothesized to be involved in tumorigenesis and cancer development. We conducted a population-based case-control study to evaluate the association between SNPs in miRNAs and ESCC risk in 1400 ESCC cases and 2185 matched controls. Four SNPs including miR-196a2 rs11614913, miR-146a rs2910164, miR-499 rs3746444, and miR-423 rs6505162 were selected with comprehensive collection strategy and genotyped using the SNaPshot Multiplex System. Odds ratio (OR) and 95 % confidence interval (95 % CI) were used to assess the strength of association. The CC genotype of miR-196a2 rs11614913 was significantly associated with an increased ESCC risk compared with the TT genotype (OR 1.11, 95 % CI 1.01-1.22, P 0.049) and the TT/TC genotypes (OR 1.09, 95 % CI 1.01-1.19, P 0.043). The association was more pronounced in non-drinkers in the recessive model (OR 1.13, 95 % CI 1.01-1.27, P 0.029). A significantly increased risk of ESCC associated with miR-499 rs3746444 polymorphism was evident among patients who never smoking and drinking. This study suggests that miR-196a2 rs11614913 and miR-499 rs3746444 are associated with an increased ESCC risk in a Chinese population.

Destruction of DDIT3/CHOP protein by wild-type SPOP but not prostate cancer-associated mutants.

Characterization of the exome and genome of prostate cancers by next-generation sequencing has identified numerous genetic alternations. SPOP (speckle-type POZ protein) was identified as one of the most frequently affected genes by somatic point mutations in prostate cancer, suggesting SPOP is potentially a key driver for prostate cancer development and progression. However, how SPOP mutations contribute to prostate cancer remains to be elucidated. SPOP acts as an adaptor protein of the CUL3-RBX1 E3 ubiquitin ligase complex and selectively recruits substrates for their ubiquitination and subsequent degradation. DDIT3 is an endoplasmic reticulum (ER) stress-responsive transcription factor playing an essential role in apoptotic execution pathways triggered by ER stress. Here, we identified DDIT3/CHOP as a bona fide substrate for the SPOP-CUL3-RBX1 E3 ubiquitin ligase complex. SPOP recognizes a Ser/Thr-rich degron in the transactivation domain of DDIT3 and triggers DDIT3 degradation via the ubiquitin-proteasome pathway. Strikingly, prostate cancer-associated mutants of SPOP are defective in promoting DDIT3 degradation. This study reveals novel molecular events underlying the regulation of DDIT3 protein homeostasis and provides insight in understanding the relationship between SPOP mutations and ER stress dysregulation in prostate cancer.

Polycomb group protein PHF1 regulates p53-dependent cell growth arrest and apoptosis.

Polycomb group protein PHF1 is well known as a component of a novel EED-EZH2·Polycomb repressive complex 2 complex and plays important roles in H3K27 methylation and Hox gene silencing. PHF1 is also involved in the response to DNA double-strand breaks in human cells, promotes nonhomologous end-joining processes through interaction with Ku70/Ku80. Here, we identified another function of PHF1 as a potential p53 pathway activator in a pathway screen using luminescence reporter assay. Subsequent studies showed PHF1 directly interacts with p53 proteins both in vivo and in vitro and co-localized in nucleus. PHF1 binds to the C-terminal regulatory domain of p53. Overexpression of PHF1 elevated p53 protein level and prolonged its turnover. Knockdown of PHF1 reduced p53 protein level and its target gene expression both in normal state and DNA damage response. Mechanically, PHF1 protects p53 proteins from MDM2-mediated ubiquitination and degradation. Furthermore, we showed that PHF1 regulates cell growth arrest and etoposide-induced apoptosis in a p53-dependent manner. Finally, PHF1 expression was significantly down-regulated in human breast cancer samples. Taken together, we establish PHF1 as a novel positive regulator of the p53 pathway. These data shed light on the potential roles of PHF1 in tumorigenesis and/or tumor progression.

HDGF-related protein-3 is required for anchorage-independent survival and chemoresistance in hepatocellular carcinomas.

OBJECTIVE: Hepatoma-derived growth factor (HDGF)-related proteins (HRPs) comprise a family of six members and are characterised by a conserved HATH domain. Among the family members, HDGF was the first to be identified as a mitogenic factor and shown to play an important role in hepatocellular carcinoma pathogenesis. The aim of the present study is to examine the relevance of HDGF-related protein-3 (HRP-3), another member of the HRP family in hepatocellular carcinoma (HCC). DESIGN: HRP-3 expression in HCC tissues was measured by quantitative reverse transcriptase PCR, western blot and immunohistochemistry analysis. The biological consequences of overexpression and knockdown of HRP-3 in HCC cell lines were studied in vitro and in vivo. RESULTS: Expression of HRP-3 mRNA and protein was shown to be highly upregulated in HCC tissues. While knockdown of HRP-3 by small interference RNAs failed to affect anchorage-dependent growth of HCC cells, it inhibited anchorage-independent growth of HCC cells in vitro and xenograft tumour growth in vivo. Further, knockdown of HRP-3 was shown to sensitise HCC cells to anoikis. Moreover, HRP-3 specifically activated the extracellular-signal-regulated kinase (ERK) pathway without affecting c-Jun N-terminal kinase (JNK), p38, AKT and signal transducer and activator of transcription 3 (STAT3). Importantly, inhibition of the ERK pathway diminished HRP-3-mediated protection of HCC cells from anoikis. Finally, knockdown of HRP-3 was shown to enhance apoptosis of HCC cells induced by multiple chemotherapeutic drugs. CONCLUSION: These findings indicate that HRP-3 plays an essential role in HCC pathogenesis and suggest that it may serve as a novel prognostic marker and molecular target for development of drugs for treatment of HCC.

FBXL4 mutation-caused mitochondrial DNA depletion syndrome is driven by BNIP3/BNIP3L-dependent excessive mitophagy.

Encephalomyopathic mitochondrial DNA (mtDNA) depletion syndrome 13 (MTDPS13) is an autosomal recessive disorder arising from biallelic F-box and leucine-rich repeat (LRR) protein 4 (FBXL4) gene mutations. Recent advances have shown that excessive BCL2 interacting protein 3 (BNIP3)/ BCL2 interacting protein 3 like (BNIP3L)-dependent mitophagy underlies the molecular pathogenesis of MTDPS13. Here, we provide an overview of these groundbreaking findings and discuss potential therapeutic strategies for this fatal disease.

Excessive BNIP3- and BNIP3L-dependent mitophagy underlies the pathogenesis of FBXL4-mutated mitochondrial DNA depletion syndrome.

Mitophagy, the process of removing damaged mitochondria to promote cell survival, plays a crucial role in cellular functionality. However, excessive, or uncontrolled mitophagy can lead to reduced mitochondrial content that burdens the remaining organelles, triggering mitophagy-mediated cell death. FBXL4 mutations, which affect the substrate-binding adaptor of the CUL1 (cullin 1)-RING ubiquitin ligase complex (CRL1), have been linked to mitochondrial DNA depletion syndrome type 13 (MTDPS13) characterized by reduced mtDNA content and impaired energy production in affected organs. However, the mechanism behind FBXL4 mutation-driven MTDPS13 remain poorly understood. In a recent study, we demonstrate that the CRL1-FBXL4 complex promotes the degradation of BNIP3 and BNIP3L, two key mitophagy cargo receptors. Deficiency of FBXL4 results in a strong accumulation of BNIP3 and BNIP3L proteins and triggers high levels of BNIP3- and BNIP3L-dependent mitophagy. Patient-derived FBXL4 mutations do not affect its interaction with BNIP3 and BNIP3L but impair the assembly of an active CRL1-FBXL4 complex. Furthermore, excessive mitophagy is observed in knockin mice carrying a patient-derived FBXL4 mutation, and in cortical neurons generated from human patient induced pluripotent stem cells (hiPSCs). These findings support the model that the CRL1-FBXL4 complex tightly restricts basal mitophagy, and its dysregulation leads to severe symptoms of MTDPS13.

DDHD2 promotes lipid droplet catabolism by acting as a TAG lipase and a cargo receptor for lipophagy.

Mutations in the DDHD2 (DDHD domain containing 2) gene cause autosomal recessive spastic paraplegia type 54 (SPG54), a rare neurodegenerative disorder characterized by the early childhood onset of progressive spastic paraplegia. DDHD2 is reported as the principal brain triacylglycerol (TAG) lipase whose dysfunction causes massive lipid droplet (LD) accumulation in the brains of SPG54 patients. However, the precise functions of DDHD2 in regulating LD catabolism are not yet fully understood. In a recent study, we demonstrate that DDHD2 interacts with multiple members of the Atg8-family proteins (MAP1LC3/LC3s, GABARAPs), which play crucial roles in lipophagy. DDHD2 possesses two LC3-interacting region (LIR) motifs that contribute to its LD-eliminating activity. Moreover, DDHD2 enhances the colocalization between LC3B and LDs to promote lipophagy. LD·ATTEC, a compound that tethers LC3 to LDs to enhance their macroautophagic/autophagic clearance, effectively counteracts DDHD2 deficiency-induced LD accumulation. These findings provide insights into the dual functions of DDHD2 as a TAG lipase and cargo receptor for lipophagy in neuronal LD catabolism, and also suggest a potential therapeutic approach for treating SPG54 patients.

DDHD2, whose mutations cause spastic paraplegia type 54, enhances lipophagy via engaging ATG8 family proteins.

Hereditary spastic paraplegia (HSP) is a group of inherited neurodegenerative disorders characterized by progressive lower limb spasticity and weakness. One subtype of HSP, known as SPG54, is caused by biallelic mutations in the DDHD2 gene. The primary pathological feature observed in patients with SPG54 is the massive accumulation of lipid droplets (LDs) in the brain. However, the precise mechanisms and roles of DDHD2 in regulating lipid homeostasis are not yet fully understood. Through Affinity Purification-Mass Spectroscopy (AP-MS) analysis, we identify that DDHD2 interacts with multiple members of the ATG8 family proteins (LC3, GABARAPs), which play crucial roles in lipophagy. Mutational analysis reveals the presence of two authentic LIR motifs in DDHD2 protein that are essential for its binding to LC3/GABARAPs. We show that DDHD2 deficiency leads to LD accumulation, while enhanced DDHD2 expression reduces LD formation. The LC3/GABARAP-binding capacity of DDHD2 and the canonical autophagy pathway both contribute to its LD-eliminating activity. Moreover, DDHD2 enhances the colocalization between LC3B and LDs to promote lipophagy. LD·ATTEC, a small molecule that tethers LC3 to LDs to enhance their autophagic clearance, effectively counteracts DDHD2 deficiency-induced LD accumulation. These findings provide valuable insights into the regulatory roles of DDHD2 in LD catabolism and offer a potential therapeutic approach for treating SPG54 patients.

mTORC1/S6K1 signaling promotes sustained oncogenic translation through modulating CRL3IBTK-mediated ubiquitination of eIF4A1 in cancer cells.

Enhanced protein synthesis is a crucial molecular mechanism that allows cancer cells to survive, proliferate, metastasize, and develop resistance to anti-cancer treatments, and often arises as a consequence of increased signaling flux channeled to mRNA-bearing eukaryotic initiation factor 4F (eIF4F). However, the post-translational regulation of eIF4A1, an ATP-dependent RNA helicase and subunit of the eIF4F complex, is still poorly understood. Here, we demonstrate that IBTK, a substrate-binding adaptor of the Cullin 3-RING ubiquitin ligase (CRL3) complex, interacts with eIF4A1. The non-degradative ubiquitination of eIF4A1 catalyzed by the CRL3IBTK complex promotes cap-dependent translational initiation, nascent protein synthesis, oncogene expression, and cervical tumor cell growth both in vivo and in vitro. Moreover, we show that mTORC1 and S6K1, two key regulators of protein synthesis, directly phosphorylate IBTK to augment eIF4A1 ubiquitination and sustained oncogenic translation. This link between the CRL3IBTK complex and the mTORC1/S6K1 signaling pathway, which is frequently dysregulated in cancer, represents a promising target for anti-cancer therapies.

Zinc finger transcription factor 191, directly binding to ß-catenin promoter, promotes cell proliferation of hepatocellular carcinoma.

UNLABELLED: Activation of ß-catenin, the central effector of the canonical wingless-type (Wnt) pathway, has been implicated in hepatocellular carcinoma (HCC). However, the transcription regulation mechanism of the ß-catenin gene in HCC remains unknown. Here we report that human zinc finger protein 191 (ZNF191) is a potential regulator of ß-catenin transcription. ZNF191, a Krüppel-like protein, specifically interacts with the TCAT motif, which constitutes the HUMTH01 microsatellite in the tyrosine hydroxylase (TH) gene ex vivo. We demonstrate that ZNF191 is significantly overexpressed in human HCC specimens and is associated with growth of human HCC cells. Global profiling of gene expression in ZNF191 knockdown human hepatic L02 cells revealed that the important Wnt signal pathway genes ß-catenin and cyclin D1 messenger RNAs (mRNAs) are significantly down-regulated. In agreement with transcription level, ß-catenin and cyclin D1 proteins are also down-regulated in transient and stable ZNF191 knockdown L02 and hepatoma Hep3B cell lines. Moreover, significant correlation between ZNF191 and ß-catenin mRNA expression was detected in human HCCs. Promoter luciferase assay indicated that ZNF191 can increase transcription activity of the full-length ß-catenin (CTNNB1) promoter, and nucleotide (nt)-1407/-907 of the CTNNB1 promoter exhibited the maximum transcriptional activity. Electrophoretic mobility shift assay showed that purified ZNF191 protein can directly bind to the CTNNB1 promoter, and the binding region is located at nt-1254/-1224. Finally, we demonstrate that the key binding sequence of ZNF191 in vivo is ATTAATT. CONCLUSION: ZNF191 can directly bind to the CTNNB1 promoter and activate the expression of ß-catenin and its downstream target genes such as cyclin D1 in hepatoma cell lines. This study uncovers a new molecular mechanism of transcription regulation of the ß-catenin gene in HCC.