[High-intensity interval training (HIIT) induces hepatic ketone body production possibly through altering expression of mTORC1, PPARα and FGF21 in mice].

The present study aims to investigate the production of ketone body in the liver of mice after 6 weeks of high-intensity interval training (HIIT) intervention and explore the possible mechanisms. Male C57BL/6J mice (7-week-old) were randomly divided into control and HIIT groups. The control group did not engage in exercise, while the HIIT group underwent a 6-week HIIT (10° slope treadmill exercise). Changes in weight and body composition were recorded, and blood ketone body levels were measured before, immediately after, and 1 h after each HIIT exercise. After 6-week HIIT, the levels of free fatty acids in the liver and serum were detected using reagent kits, and expression levels of regulatory factors and key enzymes of ketone body production in the mouse liver were detected by Western blot and qPCR. The results showed that, the blood ketone body levels in the HIIT group significantly increased immediately after a single HIIT and 1 h after HIIT, compared with that before HIIT. The body weight of the control group gradually increased within 6 weeks, while the HIIT group mice did not show significant weight gain. After 6-week HIIT, compared with the control group, the HIIT group showed decreased body fat ratio, increased lean body weight ratio, and increased free fatty acid levels in liver and serum. Liver carnitine palmitoyl transferase-I (CPT-I), peroxisome proliferator activated receptor α (PPARα), and fibroblast growth factor 21 (FGF21) protein expression levels were up-regulated, whereas mammalian target of rapamycin complex 1 (mTORC1) protein expression level was significantly down-regulated in the HIIT group, compared with those in the control group. These results suggest that HIIT induces hepatic ketone body production through altering mTORC1, PPARα and FGF21 expression in mice.

Important role and underlying mechanism of non­SMC condensin I complex subunit G in tumours (Review).

At present, the incidence of tumours is increasing on a yearly basis, and tumourigenesis is usually associated with chromosomal instability and cell cycle dysregulation. Moreover, abnormalities in the chromosomal structure often lead to DNA damage, further exacerbating gene mutations and chromosomal rearrangements. However, the non­SMC condensin I complex subunit G (NCAPG) of the structural maintenance of chromosomes family is known to exert a key role in tumour development. It has been shown that high expression of NCAPG is closely associated with tumour development and progression. Overexpression of NCAPG variously affects chromosome condensation and segregation during cell mitosis, influences cell cycle regulation, promotes tumour cell proliferation and invasion, and inhibits apoptosis. In addition, NCAPG has been associated with tumour cell stemness, tumour resistance and recurrence. The aim of the present review was to explore the underlying mechanisms of NCAPG during tumour development, with a view towards providing novel targets and strategies for tumour therapy, and through the elucidation of the mechanisms involved, to lay the foundation for future developments in health.

mTORC1 - TFEB pathway was involved in sodium arsenite induced lysosomal alteration, oxidative stress and genetic damage in BEAS-2B cells.

The mechanistic target of rapamycin (RAPA) complex 1 (mTORC1) - transcription factor EB (TFEB) pathway plays a crucial role in response to nutritional status, energy and environmental stress for maintaining cellular homeostasis. But there is few reports on its role in the toxic effects of arsenic exposure and the related mechanisms. Here, we show that the exposure of bronchial epithelial cells (BEAS-2B) to sodium arsenite promoted the activation of mTORC1 (p-mTORC1) and the inactivation of TFEB (p-TFEB), the number and activity of lysosomes decreased, the content of reduced glutathione (GSH) and superoxide dismutase (SOD) decreased, the content of malondialdehyde (MDA) increased, the DNA and chromosome damage elevated. Further, when mTORC1 was inhibited with RAPA, p-mTORC1 and p-TFEB down-regulated, GSH and SOD increased, MDA decreased, the DNA and chromosome damage reduced significantly, as compared with the control group. Our data revealed for the first time that mTORC1 - TFEB pathway was involved in sodium arsenite induced lysosomal alteration, oxidative stress and genetic damage in BEAS-2B cells, and it may be a potential intervention target for the toxic effects of arsenic.

PfCAP-H is essential for assembly of condensin I complex and karyokinesis during asexual proliferation of Plasmodium falciparum.

Condensin I is a pentameric complex that regulates the mitotic chromosome assembly in eukaryotes. The kleisin subunit CAP-H of the condensin I complex acts as a linchpin to maintain the structural integrity and loading of this complex on mitotic chromosomes. This complex is present in all eukaryotes and has recently been identified in Plasmodium spp. However, how this complex is assembled and whether the kleisin subunit is critical for this complex in these parasites are yet to be explored. To examine the role of PfCAP-H during cell division within erythrocytes, we generated an inducible PfCAP-H knockout parasite. We find that PfCAP-H is dynamically expressed during mitosis with the peak expression at the metaphase plate. PfCAP-H interacts with PfCAP-G and is a non-SMC member of the condensin I complex. Notably, the absence of PfCAP-H does not alter the expression of PfCAP-G but affects its localization at the mitotic chromosomes. While mitotic spindle assembly is intact in PfCAP-H-deficient parasites, duplicated centrosomes remain clustered over the mass of unsegmented nuclei with failed karyokinesis. This failure leads to the formation of an abnormal nuclear mass, while cytokinesis occurs normally. Altogether, our data suggest that PfCAP-H plays a crucial role in maintaining the structural integrity of the condensin I complex on the mitotic chromosomes and is essential for the asexual development of malarial parasites. IMPORTANCE: Mitosis is a fundamental process for Plasmodium parasites, which plays a vital role in their survival within two distinct hosts-human and Anopheles mosquitoes. Despite its great significance, our comprehension of mitosis and its regulation remains limited. In eukaryotes, mitosis is regulated by one of the pivotal complexes known as condensin complexes. The condensin complexes are responsible for chromosome condensation, ensuring the faithful distribution of genetic material to daughter cells. While condensin complexes have recently been identified in Plasmodium spp., our understanding of how this complex is assembled and its precise functions during the blood stage development of Plasmodium falciparum remains largely unexplored. In this study, we investigate the role of a central protein, PfCAP-H, during the blood stage development of P. falciparum. Our findings reveal that PfCAP-H is essential and plays a pivotal role in upholding the structure of condensin I and facilitating karyokinesis.

Structure-Based Protein Assembly Simulations Including Various Binding Sites and Conformations.

Many biological functions are mediated by large complexes formed by multiple proteins and other cellular macromolecules. Recent progress in experimental structure determination, as well as in integrative modeling and protein structure prediction using deep learning approaches, has resulted in a rapid increase in the number of solved multiprotein assemblies. However, the assembly process of large complexes from their components is much less well-studied. We introduce a rapid computational structure-based (SB) model, GoCa, that allows to follow the assembly process of large multiprotein complexes based on a known native structure. Beyond existing SB Go̅-type models, it distinguishes between intra- and intersubunit interactions, allowing us to include coupled folding and binding. It accounts automatically for the permutation of identical subunits in a complex and allows the definition of multiple minima (native) structures in the case of proteins that undergo global transitions during assembly. The model is successfully tested on several multiprotein complexes. The source code of the GoCa program including a tutorial is publicly available on Github: https://github.com/ZachariasLab/GoCa. We also provide a web source that allows users to quickly generate the necessary input files for a GoCa simulation: https://goca.t38webservices.nat.tum.de.

An evolutionary mechanism to assimilate new nutrient sensors into the mTORC1 pathway.

Animals sense and respond to nutrient availability in their environments, a task coordinated in part by the mTOR complex 1 (mTORC1) pathway. mTORC1 regulates growth in response to nutrients and, in mammals, senses specific amino acids through specialized sensors that bind the GATOR1/2 signaling hub. Given that animals can occupy diverse niches, we hypothesized that the pathway might evolve distinct sensors in different metazoan phyla. Whether such customization occurs, and how the mTORC1 pathway might capture new inputs, is unknown. Here, we identify the Drosophila melanogaster protein Unmet expectations (CG11596) as a species-restricted methionine sensor that directly binds the fly GATOR2 complex in a fashion antagonized by S-adenosylmethionine (SAM). We find that in Dipterans GATOR2 rapidly evolved the capacity to bind Unmet and to thereby repurpose a previously independent methyltransferase as a SAM sensor. Thus, the modular architecture of the mTORC1 pathway allows it to co-opt preexisting enzymes to expand its nutrient sensing capabilities, revealing a mechanism for conferring evolvability on an otherwise conserved system.

Condensin-mediated restriction of retrotransposable elements facilitates brain development in Drosophila melanogaster.

Neural stem and progenitor cell (NSPC) maintenance is essential for ensuring that organisms are born with proper brain volumes and head sizes. Microcephaly is a disorder in which babies are born with significantly smaller head sizes and cortical volumes. Mutations in subunits of the DNA organizing complex condensin have been identified in microcephaly patients. However, the molecular mechanisms by which condensin insufficiency causes microcephaly remain elusive. We previously identified conserved roles for condensins in repression of retrotransposable elements (RTEs). Here, we show that condensin subunit knockdown in NSPCs of the Drosophila larval central brain increases RTE expression and mobility which causes cell death, and significantly decreases adult head sizes and brain volumes. These findings suggest that unrestricted RTE expression and activity may lead to improper brain development in condensin insufficient organisms, and lay the foundation for future exploration of causative roles for RTEs in other microcephaly models.

ER membrane complex (EMC): Structure, functions, and roles in diseases.

The endoplasmic reticulum (ER) is the largest membrane system in eukaryotic cells and is the primary site for the biosynthesis of lipids and carbohydrates, as well as for the folding, assembly, modification, and transport of secreted and integrated membrane proteins. The ER membrane complex (EMC) on the ER membrane is an ER multiprotein complex that affects the quality control of membrane proteins, which is abundant and widely preserved. Its disruption has been found to affect a wide range of processes, including protein and lipid synthesis, organelle communication, endoplasmic reticulum stress, and viral maturation, and may lead to neurodevelopmental disorders and cancer. Therefore, EMC has attracted the attention of many scholars and become a hot field. In this paper, we summarized the main contributions of the research of EMC in the past nearly 15 years, and reviewed the structure and function of EMC as well as its related diseases. We hope this review will promote further progress of research on EMC.

RNAP II antagonizes mitotic chromatin folding and chromosome segregation by condensin.

Condensin shapes mitotic chromosomes by folding chromatin into loops, but whether it does so by DNA-loop extrusion remains speculative. Although loop-extruding cohesin is stalled by transcription, the impact of transcription on condensin, which is enriched at highly expressed genes in many species, remains unclear. Using degrons of Rpb1 or the torpedo nuclease Dhp1XRN2 to either deplete or displace RNAPII on chromatin in fission yeast metaphase cells, we show that RNAPII does not load condensin on DNA. Instead, RNAPII retains condensin in cis and hinders its ability to fold mitotic chromatin and to support chromosome segregation, consistent with the stalling of a loop extruder. Transcription termination by Dhp1 limits such a hindrance. Our results shed light on the integrated functioning of condensin, and we argue that a tight control of transcription underlies mitotic chromosome assembly by loop-extruding condensin.

Targeting DCAF5 suppresses SMARCB1-mutant cancer by stabilizing SWI/SNF.

Whereas oncogenes can potentially be inhibited with small molecules, the loss of tumour suppressors is more common and is problematic because the tumour-suppressor proteins are no longer present to be targeted. Notable examples include SMARCB1-mutant cancers, which are highly lethal malignancies driven by the inactivation of a subunit of SWI/SNF (also known as BAF) chromatin-remodelling complexes. Here, to generate mechanistic insights into the consequences of SMARCB1 mutation and to identify vulnerabilities, we contributed 14 SMARCB1-mutant cell lines to a near genome-wide CRISPR screen as part of the Cancer Dependency Map Project1-3. We report that the little-studied gene DDB1-CUL4-associated factor 5 (DCAF5) is required for the survival of SMARCB1-mutant cancers. We show that DCAF5 has a quality-control function for SWI/SNF complexes and promotes the degradation of incompletely assembled SWI/SNF complexes in the absence of SMARCB1. After depletion of DCAF5, SMARCB1-deficient SWI/SNF complexes reaccumulate, bind to target loci and restore SWI/SNF-mediated gene expression to levels that are sufficient to reverse the cancer state, including in vivo. Consequently, cancer results not from the loss of SMARCB1 function per se, but rather from DCAF5-mediated degradation of SWI/SNF complexes. These data indicate that therapeutic targeting of ubiquitin-mediated quality-control factors may effectively reverse the malignant state of some cancers driven by disruption of tumour suppressor complexes.

DUX4-induced HSATII transcription causes KDM2A/B-PRC1 nuclear foci and impairs DNA damage response.

Polycomb repressive complexes regulate developmental gene programs, promote DNA damage repair, and mediate pericentromeric satellite repeat repression. Expression of pericentromeric satellite repeats has been implicated in several cancers and diseases, including facioscapulohumeral dystrophy (FSHD). Here, we show that DUX4-mediated transcription of HSATII regions causes nuclear foci formation of KDM2A/B-PRC1 complexes, resulting in a global loss of PRC1-mediated monoubiquitination of histone H2A. Loss of PRC1-ubiquitin signaling severely impacts DNA damage response. Our data implicate DUX4-activation of HSATII and sequestration of KDM2A/B-PRC1 complexes as a mechanism of regulating epigenetic and DNA repair pathways.

Structural basis for intra- and intermolecular interactions on RAD9 subunit of 9-1-1 checkpoint clamp implies functional 9-1-1 regulation by RHINO.

Eukaryotic DNA clamp is a trimeric protein featuring a toroidal ring structure that binds DNA on the inside of the ring and multiple proteins involved in DNA transactions on the outside. Eukaryotes have two types of DNA clamps: the replication clamp PCNA and the checkpoint clamp RAD9-RAD1-HUS1 (9-1-1). 9-1-1 activates the ATR-CHK1 pathway in DNA damage checkpoint, regulating cell cycle progression. Structure of 9-1-1 consists of two moieties: a hetero-trimeric ring formed by PCNA-like domains of three subunits and an intrinsically disordered C-terminal region of the RAD9 subunit, called RAD9 C-tail. The RAD9 C-tail interacts with the 9-1-1 ring and disrupts the interaction between 9-1-1 and DNA, suggesting a negative regulatory role for this intramolecular interaction. In contrast, RHINO, a 9-1-1 binding protein, interacts with both RAD1 and RAD9 subunits, positively regulating checkpoint activation by 9-1-1. This study presents a biochemical and structural analysis of intra- and inter-molecular interactions on the 9-1-1 ring. Biochemical analysis indicates that RAD9 C-tail binds to the hydrophobic pocket on the PCNA-like domain of RAD9, implying that the pocket is involved in multiple protein-protein interactions. The crystal structure of the 9-1-1 ring in complex with a RHINO peptide reveals that RHINO binds to the hydrophobic pocket of RAD9, shedding light on the RAD9-binding motif. Additionally, the study proposes a structural model of the 9-1-1-RHINO quaternary complex. Together, these findings provide functional insights into the intra- and inter-molecular interactions on the front side of RAD9, elucidating the roles of RAD9 C-tail and RHINO in checkpoint activation.

Selective disruption of mTORC1 and mTORC2 in VTA astrocytes induces depression and anxiety-like behaviors in mice.

Dysfunction of the mechanistic target of rapamycin (mTOR) signaling pathway is implicated in neuropsychiatric disorders including depression and anxiety. Most studies have been focusing on neurons, and the function of mTOR signaling pathway in astrocytes is less investigated. mTOR forms two distinct complexes, mTORC1 and mTORC2, with key scaffolding protein Raptor and Rictor, respectively. The ventral tegmental area (VTA), a vital component of the brain reward system, is enrolled in regulating both depression and anxiety. In the present study, we aimed to examine the regulation effect of VTA astrocytic mTOR signaling pathway on depression and anxiety. We specifically deleted Raptor or Rictor in VTA astrocytes in mice and performed a series of behavioral tests for depression and anxiety. Deletion of Raptor and Rictor both decreased the immobility time in the tail suspension test and the latency to eat in the novelty suppressed feeding test, and increased the horizontal activity and the movement time in locomotor activity. Deletion of Rictor decreased the number of total arm entries in the elevated plus-maze test and the vertical activity in locomotor activity. These data suggest that VTA astrocytic mTORC1 plays a role in regulating depression-related behaviors and mTORC2 is involved in both depression and anxiety-related behaviors. Our results indicate that VTA astrocytic mTOR signaling pathway might be new targets for the treatment of psychiatric disorders.

mTOR Regulates Mineralocorticoid Receptor Transcriptional Activity by ULK1-Dependent and -Independent Mechanisms.

The mineralocorticoid receptor (MR) is a transcription factor for genes mediating diverse, cell-specific functions, including trophic effects as well as promoting fluid/electrolyte homeostasis. It was reported that in intercalated cells, phosphorylation of the MR at serine 843 (S843) by Unc-51-like kinase (ULK1) inhibits MR activation and that phosphorylation of ULK1 by mechanistic target of rapamycin (mTOR) inactivates ULK1, and thereby prevents MR inactivation. We extended these findings with studies in M1 mouse cortical collecting duct cells stably expressing the rat MR and a reporter gene. Pharmacological inhibition of ULK1 dose-dependently increased ligand-induced MR transactivation, while ULK1 activation had no effect. Pharmacological inhibition of mTOR and CRISPR/gRNA gene knockdown of rapamycin-sensitive adapter protein of mTOR (Raptor) or rapamycin-insensitive companion of mTOR (Rictor) decreased phosphorylated ULK1 and ligand-induced activation of the MR reporter gene, as well as transcription of endogenous MR-target genes. As predicted, ULK1 inhibition had no effect on aldosterone-mediated transcription in M1 cells with the mutated MR-S843A (alanine cannot be phosphorylated). In contrast, mTOR inhibition dose-dependently decreased transcription in the MR-S843A cells, though not as completely as in cells with the wild-type MR-S843. mTOR, Raptor, and Rictor coprecipitated with the MR and addition of aldosterone increased their phosphorylated, active state. These results suggest that mTOR significantly regulates MR activity in at least 2 ways: by suppressing MR inactivation by ULK1, and by a yet ill-defined mechanism that involves direct association with MR. They also provide new insights into the diverse functions of ULK1 and mTOR, 2 key enzymes that monitor the cell's energy status.

New insights into GATOR2-dependent interactions and its conformational changes in amino acid sensing.

Eukaryotic cells coordinate growth under different environmental conditions via mechanistic target of rapamycin complex 1 (mTORC1). In the amino-acid-sensing signalling pathway, the GATOR2 complex, containing five evolutionarily conserved subunits (WDR59, Mios, WDR24, Seh1L and Sec13), is required to regulate mTORC1 activity by interacting with upstream CASTOR1 (arginine sensor) and Sestrin2 (leucine sensor and downstream GATOR1 complex). GATOR2 complex utilizes ß-propellers to engage with CASTOR1, Sestrin2 and GATOR1, removal of these ß-propellers results in substantial loss of mTORC1 capacity. However, structural information regarding the interface between amino acid sensors and GATOR2 remains elusive. With the recent progress of the AI-based tool AlphaFold2 (AF2) for protein structure prediction, structural models were predicted for Sentrin2-WDR24-Seh1L and CASTOR1-Mios ß-propeller. Furthermore, the effectiveness of relevant residues within the interface was examined using biochemical experiments combined with molecular dynamics (MD) simulations. Notably, fluorescence resonance energy transfer (FRET) analysis detected the structural transition of GATOR2 in response to amino acid signals, and the deletion of Mios ß-propeller severely impeded that change at distinct arginine levels. These findings provide structural perspectives on the association between GATOR2 and amino acid sensors and can facilitate future research on structure determination and function.

Stress response silencing by an E3 ligase mutated in neurodegeneration.

Stress response pathways detect and alleviate adverse conditions to safeguard cell and tissue homeostasis, yet their prolonged activation induces apoptosis and disrupts organismal health1-3. How stress responses are turned off at the right time and place remains poorly understood. Here we report a ubiquitin-dependent mechanism that silences the cellular response to mitochondrial protein import stress. Crucial to this process is the silencing factor of the integrated stress response (SIFI), a large E3 ligase complex mutated in ataxia and in early-onset dementia that degrades both unimported mitochondrial precursors and stress response components. By recognizing bifunctional substrate motifs that equally encode protein localization and stability, the SIFI complex turns off a general stress response after a specific stress event has been resolved. Pharmacological stress response silencing sustains cell survival even if stress resolution failed, which underscores the importance of signal termination and provides a roadmap for treating neurodegenerative diseases caused by mitochondrial import defects.

Post-translational regulation of the mTORC1 pathway: A switch that regulates metabolism-related gene expression.

The mechanistic target of rapamycin complex 1 (mTORC1) is a kinase complex that plays a crucial role in coordinating cell growth in response to various signals, including amino acids, growth factors, oxygen, and ATP. Activation of mTORC1 promotes cell growth and anabolism, while its suppression leads to catabolism and inhibition of cell growth, enabling cells to withstand nutrient scarcity and stress. Dysregulation of mTORC1 activity is associated with numerous diseases, such as cancer, metabolic disorders, and neurodegenerative conditions. This review focuses on how post-translational modifications, particularly phosphorylation and ubiquitination, modulate mTORC1 signaling pathway and their consequential implications for pathogenesis. Understanding the impact of phosphorylation and ubiquitination on the mTORC1 signaling pathway provides valuable insights into the regulation of cellular growth and potential therapeutic targets for related diseases.

Regulation of condensin II by self-suppression and release mechanisms.

In vertebrates, two distinct condensin complexes, condensin I and condensin II, cooperate to drive mitotic chromosome assembly. It remains largely unknown how the two complexes differentially contribute to this process at a mechanistic level. We have previously dissected the role of individual subunits of condensin II by introducing recombinant complexes into Xenopus egg extracts. Here we extend these efforts by introducing a modified functional assay using extracts depleted of topoisomerase IIα (topo IIα), which allows us to further elucidate the functional similarities and differences between condensin I and condensin II. The intrinsically disordered C-terminal region of the CAP-D3 subunit (the D3 C-tail) is a major target of Cdk1 phosphorylation, and phosphorylation-deficient mutations in this region impair condensin II functions. We also identify a unique helical structure in CAP-D3 (the D3 HEAT docker) that is predicted to directly interact with CAP-G2. Deletion of the D3 HEAT docker, along with the D3 C-tail, enhances the ability of condensin II to assemble mitotic chromosomes. Taken together, we propose a self-suppression mechanism unique to condensin II that is released by mitotic phosphorylation. Evolutionary implications of our findings are also discussed.

Co-translational Assembly Pathways of Nuclear Multiprotein Complexes Involved in the Regulation of Gene Transcription.

Most factors that regulate gene transcription in eukaryotic cells are multimeric, often large, protein complexes. The understanding of the biogenesis pathways of such large and heterogeneous protein assemblies, as well as the dimerization partner choice among transcription factors, is crucial to interpret and control gene expression programs and consequent cell fate decisions. Co-translational assembly (Co-TA) is thought to play key roles in the biogenesis of protein complexes by directing complex formation during protein synthesis. In this review we discuss the principles of Co-TA with a special focus for the assembly of transcription regulatory complexes. We outline the expected molecular advantages of establishing co-translational interactions, pointing at the available, or missing, evidence for each of them. We hypothesize different molecular mechanisms based on Co-TA to explain the allocation "dilemma" of paralog proteins and subunits shared by different transcription complexes. By taking as a paradigm the different assembly pathways employed by three related transcription regulatory complexes (TFIID, SAGA and ATAC), we discuss alternative Co-TA strategies for nuclear multiprotein complexes and the widespread - yet specific - use of Co-TA for the formation of nuclear complexes involved in gene transcription. Ultimately, we outlined a series of open questions which demand well-defined lines of research to investigate the principles of gene regulation that rely on the coordinated assembly of protein complexes.

Coordination between human DNA polymerase ß and apurinic/apyrimidinic endonuclease 1 in the course of DNA repair.

Coordination of enzymatic activities in the course of base excision repair (BER) is essential to ensure complete repair of damaged bases. Two major mechanisms underlying the coordination of BER are known today: the "passing the baton" model and a model of preassembled stable multiprotein repair complexes called "repairosomes." In this work, we aimed to elucidate the coordination between human apurinic/apyrimidinic (AP) endonuclease APE1 and DNA polymerase Polß in BER through studying an impact of APE1 on Polß-catalyzed nucleotide incorporation into different model substrates that mimic different single-strand break (SSB) intermediates arising along the BER pathway. It was found that APE1's impact on separate stages of Polß's catalysis depends on the nature of a DNA substrate. In this complex, APE1 removed 3' blocking groups and corrected Polß-catalyzed DNA synthesis in a coordinated manner. Our findings support the hypothesis that Polß not only can displace APE1 from damaged DNA within the "passing the baton" model but also performs the gap-filling reaction in the ternary complex with APE1 according to the "repairosome" model. Taken together, our results provide new insights into coordination between APE1 and Polß during the BER process.