Journal of Biological Chemistry最新文献

Peroxisome proliferator-activated receptor-γ (PPARγ) is a nuclear hormone receptor that is a master regulator of adipocyte differentiation and function. ZBTB9 is a widely expressed but poorly studied transcription factor that was predicted to interact with PPARγ based on large-scale protein-protein interaction experiments. In addition, genome-wide association studies (GWAS) revealed associations between ZBTB9 and BMI, T2D risk, and HbA1c levels. Here we show that Zbtb9 deficiency in mature adipocytes decreased PPARγ activity and protein level, and thus acts as a positive regulator of PPARγ signaling. In contrast, Zbtb9 deficiency in 3T3-L1 and human preadipocytes increased PPARγ levels and enhanced adipogenesis. Transcriptomic and transcription factor binding site analyses of Zbtb9 deficient preadipocytes revealed that the E2F pathway, controlled by the E2F family of transcription factors that are classically associated with cell cycle regulation, was among the most upregulated pathways. E2F1 positively regulates adipogenesis by promoting Pparg expression, independent of its cell cycle role, via direct binding to the Pparg promoter early during adipogenesis. RB phosphorylation (pRB), which regulates E2F activity, was also upregulated in Zbtb9 deficient preadipocytes. Critically, an E2F1 inhibitor blocked the effects of Zbtb9 deficiency on adipogenesis. Collectively, these results demonstrate that Zbtb9 inhibits adipogenesis as a negative regulator of Pparg expression via pRB-E2F signaling. Our findings reveal cell-state dependent roles of ZBTB9 in adipocytes, identifying a new molecule that regulates adipocyte biology as both a positive and negative regulator of PPARγ signaling depending on the cellular context, and thus may be important in the pathogenesis of obesity and T2D.

Translational arrest is a phenomenon wherein a temporary pause or slowing of the translation elongation reaction occurs due to the interaction between ribosome and nascent peptide. Recent studies have revealed that translational arrest peptides are involved in intracellular protein homeostasis regulatory functions, such as gene expression regulation at the translational level and regulation of cotranslational protein folding. Herein, we established a method for the large-scale in vitro selection of translational arrest peptides from DNA libraries by combining a modified mRNA display method and deep sequencing. We performed in vitro selection of translational arrest sequences from random-sequence libraries via mRNA display based on the E. coli PURE system or wheat germ extract. Following several rounds of affinity selection, we obtained various candidate sequences that were not similar to known arrest peptides and subsequently confirmed their ribosome stalling activity by peptidyl-tRNA detection and toeprinting assay. Following the site-directed mutagenesis of the selected sequences, these clones were found to contain novel arrest peptide motifs. This method, termed as STALL-seq (Selection of Translational Arrest sequences from Large Library sequencing), could be useful for the large-scale investigation of translational arrest sequences acting on both bacterial and eukaryotic ribosomes and could help discover novel intracellular regulatory mechanisms.

S100B is a multifunctional protein primarily found in the brain, where it plays crucial roles in cell proliferation, differentiation, and survival. It has intra- and extracellular functions and, depending on S100B levels, can exhibit both neurotrophic and neurotoxic activities, both mediated by the receptor for advanced glycation end products (RAGE). Here, we report the discovery and characterization of nanobodies (Nbs) targeting dimeric and tetrameric S100B, which are the two most abundant oligomeric functional forms of the protein, aiming to modulate S100B-mediated RAGE activation. Two Nbs were selected for detailed structural and functional studies, and found to bind tetrameric S100B with high affinity, as determined by biolayer interferometry analysis and SEC-stable binary complex formation. Structural and docking analyses revealed preferential contact sites of Nbs with S100B regions implicated in interactions with RAGE, namely residues at the interfacial cleft on dimeric S100B and the at hydrophobic cleft formed by the association of two homodimeric units in the tetramer. In accordance, assays in SH-SY5Y cells showed that Nbs modulate the RAGE-mediated neurotrophic activity of S100B by hindering its functional interactions with the receptor. Biolayer interferometry competition assays between tetrameric S100B and the RAGE-VC1 domain, confirmed that Nbs selectively block S100B-mediated RAGE engagement, in agreement with cell activation experiments. These findings highlight Nbs as powerful tools for elucidating molecular and cellular mechanisms through the modulation of S100B and RAGE functions, inspiring potential therapeutic applications.

The ATPase Associated with diverse cellular Activities (AAA+) family of proteases play crucial roles in cellular proteolysis and stress responses. Like other AAA+ proteases, the Lon protease is known to be allosterically regulated by nucleotide and substrate binding. Although it was originally classified as a DNA binding protein, the impact of DNA binding on Lon activity is unclear. In this study, we characterize the regulation of Lon by single-stranded DNA (ssDNA) binding and serendipitously identify general activation strategies for Lon. Upon binding to ssDNA, Lon's ATP hydrolysis rate increases due to improved nucleotide binding, leading to enhanced degradation of protein substrates, including physiologically important targets. We demonstrate that mutations in basic residues that are crucial for Lon's DNA binding not only reduce ssDNA binding but result in charge-specific consequences on Lon activity. Introducing negative charge at these sites induces activation akin to that induced by ssDNA binding, whereas neutralizing the charge reduces Lon's activity. Based on single molecule measurements, we find this change in activity correlated with changes in Lon oligomerization. Our study provides insights into the complex regulation of the Lon protease driven by electrostatic contributions from either DNA binding or mutations.

DNA methylation is mainly catalyzed by three DNA methyltransferase (DNMT) proteins in mammals. Usually DNMT1 is considered the primary DNMT for maintenance DNA methylation, whereas DNMT3A and DNMT3B function in de novo DNA methylation. Interestingly, we found DNMT3A and DNMT3B exerted maintenance and de novo DNA methylation in post-implantation mouse embryos. Together with DNMT1, they maintained DNA methylation at some pluripotent genes and lineage marker genes. Germline-derived DNA methylation at the imprinting control regions (ICRs) is stably maintained in embryos. DNMT1 maintained DNA methylation at most ICRs in post-implantation embryos. Surprisingly, DNA methylation was increased at five ICRs after implantation, and two DNMT3 proteins maintained the newly acquired DNA methylation at two of these five ICRs. Intriguingly, DNMT3A and DNMT3B maintained pre-existing DNA methylation at four other ICRs, similar to what we found in embryonic stem (ES) cells before. These results suggest that DNA methylation is more dynamic than originally thought during embryogenesis including the ICRs of the imprinted regions. DNMT3A and DNMT3B exert both de novo and maintenance DNA methylation functions after implantation. They maintain large portions of newly acquired DNA methylation at variable degrees across the genome in mouse embryos, together with DNMT1. Furthermore, they contribute to maintenance of pre-existing DNA methylation at a subset of ICRs as well as in the CpG islands (CGIs) and certain lineage marker gene. These findings may have some implications for the important roles of DNMT proteins in development and human diseases.

Non-alcoholic fatty liver disease (NAFLD) is a growing health problem worldwide, ranging from non-alcoholic fatty liver (NAFL) to the more severe metabolic non-alcoholic steatohepatitis (NASH). Although many studies have elucidated the pathogenesis of NAFLD, the epigenetic regulatory mechanism from NAFL to NASH remains incompletely understood. The histone H3 lysine 4 methyltransferase, MLL4 (also called KMT2D), is a critical epigenetic transcriptional coactivator that mediates overnutrition-induced steatosis in mice, but its potential role in the progression of NASH remains largely unknown. Here, we show that mice lacking the one allele of the Mll4 gene are resistant to hepatic steatosis, inflammation, and fibrosis in NASH conditions compared to wild-type controls. Transcriptome analysis of the livers of control and Mll4^+/- mice identified pro-inflammatory genes regulated by the nuclear factor kappa B (NF-κB) signaling pathway as major target genes of MLL4. We show that MLL4 binds to p65 and that MLL4 is required for NF-κB transactivation. Myeloid-specific Mll4 knockout mice showed an almost complete block of NASH, while hepatocyte-specific Mll4 knockout mice showed mild inhibition of steatosis. Pro-inflammatory M1 polarization is decreased and anti-inflammatory M2 polarization is increased in liver macrophages from myeloid-specific Mll4 knockout mice. Importantly, we show that histone H3-lysine 4 methylation mediated by the MLL4-complex plays a critical role in promoting the expression of Ccl2 in hepatocytes and M1 marker genes in macrophages. Our results demonstrate that MLL4, through the NF-κB-MLL4 regulatory axis, exacerbates steatohepatitis in the context of an inflammatory response and represents a potential therapeutic target for NASH.

Cerebral palsy (CP) is a pediatric onset disorder with poorly understood molecular causes and progression, making early diagnosis difficult. Circular RNAs (circRNAs) are regulatory RNAs that show promise as biomarkers in various diseases but the role of circRNAs in CP is beginning to be understood. This study identified the role of circNFIX in regulating the expression of MEF2C, an important transcription factor for sarcomere development. We found that circNFIX is downregulated in the muscle cells of individuals with CP, and its localization shifts towards the nucleus as visualized using single molecule resolution imaging. The decreased expression of circNFIX, MEF2C, and MEF2C targets persisted throughout myoblasts to myotubes differentiation, and in the skeletal muscle tissue. Bioinformatic and experimental validation confirmed that circNFIX acts as a sponge for miR373-3p, a microRNA that represses MEF2C translation. In normal muscle, circNFIX de-represses MEF2C translation by sponging miR373-3p, allowing for normal sarcomere generation. In CP, reduced circNFIX expression results in loss of miRNA sponging, leading to lower MEF2C expression and downregulation of sarcomere genes, potentially causing shortened and dysfunctional muscle fibers. Knockdown of circNFIX (KD) reduced myogenic capacity of myoblasts to fuse and form myotubes similar to CP cells evident from the lower fusion index in CP and KD as compared to control myotubes. This the first study reporting reduction of MEF2C in CP and single molecule resolution imaging of circNFIX's subcellular distribution and its role in CP, suggesting circNFIX as a potential therapeutic target and biomarker for early CP diagnosis.

Translational control mechanisms modulate microbial latency of eukaryotic pathogens, enabling them to evade immunity and drug treatments. The protozoan parasite Toxoplasma gondii persists in hosts by differentiating from proliferative tachyzoites to latent bradyzoites, which are housed inside tissue cysts. Transcriptional changes facilitating bradyzoite conversion are mediated by a Myb domain transcription factor called BFD1, whose mRNA is present in tachyzoites but not translated into protein until stress is applied to induce differentiation. We addressed the mechanisms by which translational control drives BFD1 synthesis in response to stress-induced parasite differentiation. Using biochemical and molecular approaches, we show that the 5'-leader of BFD1 mRNA is sufficient for preferential translation upon stress. The translational control of BFD1 mRNA is maintained when ribosome assembly near its 5'-cap is impaired by insertion of a 5'-proximal stem-loop and upon knockdown of the Toxoplasma cap-binding protein, eIF4E1. Moreover, we determined that a trans-acting RNA-binding protein called BFD2/ROCY1 is necessary for cap-independent translation of BFD1 through its binding to the 5'-leader. Translation of BFD2 mRNA is also suggested to be preferentially induced under stress, but by a cap-dependent mechanism. These results show that translational control and differentiation in Toxoplasma proceed through cap-independent mechanisms in addition to canonical cap-dependent translation. Our identification of cap-independent translation in protozoa underscores the antiquity of this mode of gene regulation in cellular evolution and its central role in stress-induced life-cycle events.

Enhancing host anti-tumor immunity is paramount for advancing cancer immunotherapy. In this study, we identify CWF19-like cell cycle control factor 1 (CWF19L1) as a novel splicing regulator that enhances T cell-mediated cytotoxicity. CWF19L1 interacts prominently with key splicing factors within the nucleus, including components of the U5 small nuclear ribonucleoprotein (snRNP) and the pre-mRNA processing factor 19 (PRPF19) complex. Deficiency of CWF19L1 disrupts alternative splicing of immune-related genes, resulting in diminished expression of cytotoxic molecules. Furthermore, CWF19L1 plays a critical role in promoting T cell-mediated anti-tumor responses by upregulating the expression of effector cytokines. Our findings unveil previously undocumented functions of CWF19L1 in alternative splicing and its involvement in the regulation of anti-tumor immunity, highlighting its potential as a therapeutic target for novel cancer immunotherapies.

Nuclear dysmorphia, characterized by crumpled or lobulated polymorphic nuclear shapes, has been used as an index for the malignant grades of certain cancers. The expression of vimentin, a type-III intermediate filament protein, is a hallmark of the epithelial-to-mesenchymal transition. However, it remains unclear whether vimentin is involved in cancer cell nuclear dysmorphia. In this study, we found that vimentin intermediate filaments (VIFs) frequently accumulated at the concave of dysmorphic nucleus in breast cancer MDA-MB-231 cells. Depletion of vimentin apparently restored the nuclear shape of the cells, which was devastated by re-expression of vimentin, but not its assembly-defective Y117D mutant. Depletion of plectin, a cytoskeletal linker, partially prevented the perinuclear accumulation of VIFs and concomitantly restored the nuclear shape of the cells. In addition, depletion of vimentin in lung cancer A549 cells largely prevented nuclear dysmorphia during the epithelial-to-mesenchymal transition induced by TGFβ. Moreover, we found that VIF-mediated nuclear dysmorphia led to defects in DNA repair. Together, our results unveil a novel role of VIFs in cancer cell nuclear dysmorphia, which is associated with genome instability.