Search

The human AAA-ATPase Bcs1L translocates the fully assembled Rieske iron-sulfur protein (ISP) precursor across the mitochondrial inner membrane, enabling respiratory Complex III assembly. Exactly how the folded substrate is bound to and released from Bcs1L has been unclear, and there has been ongoing debate as to whether subunits of Bcs1L act in sequence or in unison hydrolyzing ATP when moving the protein cargo. Here, we captured Bcs1L conformations by cryo-EM during active ATP hydrolysis in the presence or absence of ISP substrate. In contrast to the threading mechanism widely employed by AAA proteins in substrate translocation, subunits of Bcs1L alternate uniformly between ATP and ADP conformations without detectable intermediates that have different, co-existing nucleotide states, indicating that the subunits act in concert. We further show that the ISP can be trapped by Bcs1 when its subunits are all in the ADP-bound state, which we propose to be released in the apo form., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

In the liver, mitochondria are exposed to different concentrations of nutrients due to their spatial positioning across the periportal and pericentral axis. How the mitochondria sense and integrate these signals to respond and maintain homeostasis is not known. Here, we combine intravital microscopy, spatial proteomics, and functional assessment to investigate mitochondrial heterogeneity in the context of liver zonation. We find that periportal and pericentral mitochondria are morphologically and functionally distinct; beta-oxidation is elevated in periportal regions, while lipid synthesis is predominant in the pericentral mitochondria. In addition, comparative phosphoproteomics reveals spatially distinct patterns of mitochondrial composition and potential regulation via phosphorylation. Acute pharmacological modulation of nutrient sensing through AMPK and mTOR shifts mitochondrial phenotypes in the periportal and pericentral regions, linking nutrient gradients across the lobule and mitochondrial heterogeneity. This study highlights the role of protein phosphorylation in mitochondrial structure, function, and overall homeostasis in hepatic metabolic zonation. These findings have important implications for liver physiology and disease., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Fusion-positive rhabdomyosarcoma (FP-RMS) is an aggressive pediatric sarcoma driven primarily by the PAX3-FOXO1 fusion oncogene, for which therapies targeting PAX3-FOXO1 are lacking. Here, we screen 62,643 compounds using an engineered cell line that monitors PAX3-FOXO1 transcriptional activity identifying a hitherto uncharacterized compound, P3FI-63. RNA-seq, ATAC-seq, and docking analyses implicate histone lysine demethylases (KDMs) as its targets. Enzymatic assays confirm the inhibition of multiple KDMs with the highest selectivity for KDM3B. Structural similarity search of P3FI-63 identifies P3FI-90 with improved solubility and potency. Biophysical binding of P3FI-90 to KDM3B is demonstrated using NMR and SPR. P3FI-90 suppresses the growth of FP-RMS in vitro and in vivo through downregulating PAX3-FOXO1 activity, and combined knockdown of KDM3B and KDM1A phenocopies P3FI-90 effects. Thus, we report KDM inhibitors P3FI-63 and P3FI-90 with the highest specificity for KDM3B. Their potent suppression of PAX3-FOXO1 activity indicates a possible therapeutic approach for FP-RMS and other transcriptionally addicted cancers., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

TOP3B is stabilized by TDRD3. Hypothesizing that TDRD3 recruits a deubiquitinase, we find that TOP3B interacts with USP9X via TDRD3. Inactivation of USP9X destabilizes TOP3B, and depletion of both TDRD3 and USP9X does not promote further TOP3B ubiquitylation. Additionally, we observe that MIB1 mediates the ubiquitylation and proteasomal degradation of TOP3B by directly interacting with TOP3B independently of TDRD3. Combined depletion of USP9X, TDRD3 and MIB1 causes no additional increase in TOP3B levels compared to MIB1 knockdown alone indicating that the TDRD3-USP9X complex works downstream of MIB1. To comprehend why cells degrade TOP3B in the absence of TDRD3, we measured TOP3Bccs. Lack of TDRD3 increases TOP3Bccs in DNA and RNA, and induced R-loops, γH2AX and growth defect. Biochemical experiments confirm that TDRD3 increases the turnover of TOP3B. Our work provides molecular insights into the mechanisms by which TDRD3 protect cells from deleterious TOP3Bccs which are otherwise removed by TRIM41., (© 2023. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Poly(ADP)-ribosylation (PARylation) regulates chromatin structure and recruits DNA repair proteins. Using single-molecule fluorescence microscopy to track topoisomerase I (TOP1) in live cells, we found that sustained PARylation blocked the repair of TOP1 DNA-protein crosslinks (TOP1-DPCs) in a similar fashion as inhibition of the ubiquitin-proteasome system (UPS). PARylation of TOP1-DPC was readily revealed by inhibiting poly(ADP-ribose) glycohydrolase (PARG), indicating the otherwise transient and reversible PARylation of the DPCs. As the UPS is a key repair mechanism for TOP1-DPCs, we investigated the impact of TOP1-DPC PARylation on the proteasome and found that the proteasome is unable to associate with and digest PARylated TOP1-DPCs. In addition, PARylation recruits the deubiquitylating enzyme USP7 to reverse the ubiquitylation of PARylated TOP1-DPCs. Our work identifies PARG as repair factor for TOP1-DPCs by enabling the proteasomal digestion of TOP1-DPCs. It also suggests the potential regulatory role of PARylation for the repair of a broad range of DPCs., (© 2021. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Cutaneous malignant melanoma is an aggressive cancer of melanocytes with a strong propensity to metastasize. We posit that melanoma cells acquire metastatic capability by adopting an embryonic-like phenotype, and that a lineage approach would uncover metastatic melanoma biology. Using a genetically engineered mouse model to generate a rich melanoblast transcriptome dataset, we identify melanoblast-specific genes whose expression contribute to metastatic competence and derive a 43-gene signature that predicts patient survival. We identify a melanoblast gene, KDELR3, whose loss impairs experimental metastasis. In contrast, KDELR1 deficiency enhances metastasis, providing the first example of different disease etiologies within the KDELR-family of retrograde transporters. We show that KDELR3 regulates the metastasis suppressor, KAI1, and report an interaction with the E3 ubiquitin-protein ligase gp78, a regulator of KAI1 degradation. Our work demonstrates that the melanoblast transcriptome can be mined to uncover targetable pathways for melanoma therapy.

The spindle assembly checkpoint (SAC) prevents premature chromosome segregation by inactivating the anaphase promoting complex/cyclosome (APC/C) until all chromosomes are properly attached to mitotic spindles. Here we identify a role for Cullin-RING ubiquitin ligase complex 4 (CRL4), known for modulating DNA replication, as a crucial mitotic regulator that triggers the termination of the SAC and enables chromosome segregation. CRL4 is recruited to chromatin by the replication origin binding protein RepID/DCAF14/PHIP. During mitosis, CRL4 dissociates from RepID and replaces it with RB Binding Protein 7 (RBBP7), which ubiquitinates the SAC mediator BUB3 to enable mitotic exit. During interphase, BUB3 is protected from CRL4-mediated degradation by associating with promyelocytic leukemia (PML) nuclear bodies, ensuring its availability upon mitotic onset. Deficiencies in RepID, CRL4 or RBBP7 delay mitotic exit, increase genomic instability and enhance sensitivity to paclitaxel, a microtubule stabilizer and anti-tumor drug.

Mitochondrial topoisomerase IB (TOP1MT) is a nuclear-encoded topoisomerase, exclusively localized to mitochondria, which resolves topological stress generated during mtDNA replication and transcription. Here, we report that TOP1MT is overexpressed in cancer tissues and demonstrate that TOP1MT deficiency attenuates tumor growth in human and mouse models of colon and liver cancer. Due to their mitochondrial dysfunction, TOP1MT-KO cells become addicted to glycolysis, which limits synthetic building blocks and energy supply required for the proliferation of cancer cells in a nutrient-deprived tumor microenvironment. Mechanistically, we show that TOP1MT associates with mitoribosomal subunits, ensuring optimal mitochondrial translation and assembly of oxidative phosphorylation complexes that are critical for sustaining tumor growth. The TOP1MT genomic signature profile, based on Top1mt-KO liver cancers, is correlated with enhanced survival of hepatocellular carcinoma patients. Our results highlight the importance of TOP1MT for tumor development, providing a potential rationale to develop TOP1MT-targeted drugs as anticancer therapies.

TP53 mutants (mutp53) are involved in the pathogenesis of most human cancers. Specific mutp53 proteins gain oncogenic functions (GOFs) distinct from the tumor suppressor activity of the wild-type protein. Tumor-associated macrophages (TAMs), a hallmark of solid tumors, are typically correlated with poor prognosis. Here, we report a non-cell-autonomous mechanism, whereby human mutp53 cancer cells reprogram macrophages to a tumor supportive and anti-inflammatory state. The colon cancer cells harboring GOF mutp53 selectively shed miR-1246-enriched exosomes. Uptake of these exosomes by neighboring macrophages triggers their miR-1246-dependent reprogramming into a cancer-promoting state. Mutp53-reprogammed TAMs favor anti-inflammatory immunosuppression with increased activity of TGF-β. These findings, associated with poor survival in colon cancer patients, strongly support a microenvironmental GOF role for mutp53 in actively engaging the immune system to promote cancer progression and metastasis.

Δ133p53α, a p53 isoform that can inhibit full-length p53, is downregulated at replicative senescence in a manner independent of mRNA regulation and proteasome-mediated degradation. Here we demonstrate that, unlike full-length p53, Δ133p53α is degraded by autophagy during replicative senescence. Pharmacological inhibition of autophagy restores Δ133p53α expression levels in replicatively senescent fibroblasts, without affecting full-length p53. The siRNA-mediated knockdown of pro-autophagic proteins (ATG5, ATG7 and Beclin-1) also restores Δ133p53α expression. The chaperone-associated E3 ubiquitin ligase STUB1, which is known to regulate autophagy, interacts with Δ133p53α and is downregulated at replicative senescence. The siRNA knockdown of STUB1 in proliferating, early-passage fibroblasts induces the autophagic degradation of Δ133p53α and thereby induces senescence. Upon replicative senescence or STUB1 knockdown, Δ133p53α is recruited to autophagosomes, consistent with its autophagic degradation. This study reveals that STUB1 is an endogenous regulator of Δ133p53α degradation and senescence, and identifies a p53 isoform-specific protein turnover mechanism that orchestrates p53-mediated senescence.