ABSTRACT
BACKGROUND: Lymphatic valves are specialized structures in collecting lymphatic vessels and are crucial for preventing retrograde lymph flow. Mutations in valve-forming genes have been clinically implicated in the pathology of congenital lymphedema. Lymphatic valves form when oscillatory shear stress from lymph flow signals through the PI3K/AKT pathway to promote the transcription of valve-forming genes that trigger the growth and maintenance of lymphatic valves. Conventionally, in many cell types, AKT is phosphorylated at Ser473 by the mTORC2 (mammalian target of rapamycin complex 2). However, mTORC2 has not yet been implicated in lymphatic valve formation. METHODS: In vivo and in vitro techniques were used to investigate the role of Rictor, a critical component of mTORC2, in lymphatic endothelium. RESULTS: Here, we showed that embryonic and postnatal lymphatic deletion of Rictor, a critical component of mTORC2, led to a significant decrease in lymphatic valves and prevented the maturation of collecting lymphatic vessels. RICTOR knockdown in human dermal lymphatic endothelial cells not only reduced the level of activated AKT and the expression of valve-forming genes under no-flow conditions but also abolished the upregulation of AKT activity and valve-forming genes in response to oscillatory shear stress. We further showed that the AKT target, FOXO1 (forkhead box protein O1), a repressor of lymphatic valve formation, had increased nuclear activity in Rictor knockout mesenteric lymphatic endothelial cells in vivo. Deletion of Foxo1 in Rictor knockout mice restored the number of valves to control levels in lymphatic vessels of the ear and mesentery. CONCLUSIONS: Our work identifies a novel role for RICTOR in the mechanotransduction signaling pathway, wherein it activates AKT and prevents the nuclear accumulation of the valve repressor, FOXO1, which ultimately enables the formation and maintenance of lymphatic valves.
Subject(s)
Carrier Proteins , Forkhead Box Protein O1 , Lymphangiogenesis , Lymphatic Vessels , Mechanistic Target of Rapamycin Complex 2 , Mechanotransduction, Cellular , Mice, Knockout , Proto-Oncogene Proteins c-akt , Rapamycin-Insensitive Companion of mTOR Protein , Signal Transduction , Animals , Rapamycin-Insensitive Companion of mTOR Protein/metabolism , Rapamycin-Insensitive Companion of mTOR Protein/genetics , Proto-Oncogene Proteins c-akt/metabolism , Forkhead Box Protein O1/metabolism , Forkhead Box Protein O1/genetics , Lymphatic Vessels/metabolism , Mechanistic Target of Rapamycin Complex 2/metabolism , Mechanistic Target of Rapamycin Complex 2/genetics , Humans , Carrier Proteins/metabolism , Carrier Proteins/genetics , Endothelial Cells/metabolism , Cells, Cultured , TOR Serine-Threonine Kinases/metabolism , Phosphorylation , Forkhead Transcription Factors/metabolism , Forkhead Transcription Factors/genetics , Mice , Multiprotein Complexes/metabolism , Multiprotein Complexes/genetics , Mice, Inbred C57BL , RNA Interference , TransfectionABSTRACT
Protein complexes are fundamental to all cellular processes, so understanding their evolutionary history and assembly processes is important. Gene duplication followed by divergence is considered a primary mechanism for diversifying protein complexes. Nonetheless, to what extent assembly of present-day paralogous complexes has been constrained by their long evolutionary pathways and how cross-complex interference is avoided remain unanswered questions. Subunits of protein complexes are often stabilized upon complex formation, whereas unincorporated subunits are degraded. How such cooperative stability influences protein complex assembly also remains unclear. Here, we demonstrate that subcomplexes determined by cooperative stabilization interactions serve as building blocks for protein complex assembly. We further develop a protein stability-guided method to compare the assembly processes of paralogous complexes in cellulo. Our findings support that oligomeric state and the structural organization of paralogous complexes can be maintained even if their assembly processes are rearranged. Our results indicate that divergent assembly processes by paralogous complexes not only enable the complexes to evolve new functions, but also reinforce their segregation by establishing incompatibility against deleterious hybrid assemblies.
Subject(s)
Multiprotein Complexes , Multiprotein Complexes/metabolism , Multiprotein Complexes/chemistry , Multiprotein Complexes/genetics , Protein Stability , Evolution, Molecular , Protein Subunits/metabolism , Protein Subunits/chemistry , Protein Multimerization , Protein Binding , Gene DuplicationABSTRACT
The structural maintenance of chromosome (SMC) complexes-cohesin and condensins-are crucial for chromosome separation and compaction during cell division. During the interphase, mammalian cohesins additionally fold the genome into loops and domains. Here we show that, in Caenorhabditis elegans, a species with holocentric chromosomes, condensin I is the primary, long-range loop extruder. The loss of condensin I and its X-specific variant, condensin IDC, leads to genome-wide decompaction, chromosome mixing and disappearance of X-specific topologically associating domains, while reinforcing fine-scale epigenomic compartments. In addition, condensin I/IDC inactivation led to the upregulation of X-linked genes and unveiled nuclear bodies grouping together binding sites for the X-targeting loading complex of condensin IDC. C. elegans condensin I/IDC thus uniquely organizes holocentric interphase chromosomes, akin to cohesin in mammals, as well as regulates X-chromosome gene expression.
Subject(s)
Adenosine Triphosphatases , Caenorhabditis elegans Proteins , Caenorhabditis elegans , DNA-Binding Proteins , Multiprotein Complexes , X Chromosome , Animals , Caenorhabditis elegans/genetics , Adenosine Triphosphatases/genetics , Adenosine Triphosphatases/metabolism , Multiprotein Complexes/genetics , Multiprotein Complexes/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Caenorhabditis elegans Proteins/genetics , Caenorhabditis elegans Proteins/metabolism , X Chromosome/genetics , Chromosomal Proteins, Non-Histone/metabolism , Chromosomal Proteins, Non-Histone/genetics , Cohesins , Cell Cycle Proteins/metabolism , Cell Cycle Proteins/genetics , Interphase/genetics , Genome, Helminth , Genes, X-Linked , Chromosomes/geneticsABSTRACT
In this issue of Molecular Cell, Yi et al.1 demonstrate that reduced mTORC1 activity induces the CTLH E3 ligase-dependent degradation of HMGCS1, an enzyme in the mevalonate pathway, thus revealing a unique connection between mTORC1 signaling and the degradation of a specific metabolic enzyme via the ubiquitin-proteasome system.
Subject(s)
Mechanistic Target of Rapamycin Complex 1 , Proteasome Endopeptidase Complex , Signal Transduction , Mechanistic Target of Rapamycin Complex 1/metabolism , Mechanistic Target of Rapamycin Complex 1/genetics , Humans , Proteasome Endopeptidase Complex/metabolism , Ubiquitin-Protein Ligases/metabolism , Ubiquitin-Protein Ligases/genetics , Proteolysis , TOR Serine-Threonine Kinases/metabolism , TOR Serine-Threonine Kinases/genetics , Multiprotein Complexes/metabolism , Multiprotein Complexes/genetics , Animals , Mevalonic Acid/metabolism , Ubiquitin/metabolismABSTRACT
In growing E. coli cells, the transcription-translation complexes (TTCs) form characteristic foci; however, the exact molecular composition of these superstructures is not known with certainty. Herein, we report that, during our recently developed "fast" procedures for purification of E. coli RNA polymerase (RP), a fraction of the RP's α/RpoA subunits is displaced from the core RP complexes and copurifies with multiprotein superstructures carrying the nucleic acid-binding protein Hfq and the ribosomal protein S6. We show that the main components of these large multiprotein assemblies are fixed protein copy-number (Hfq6)n≥8 complexes; these complexes have a high level of structural uniformity and are distinctly unlike the previously described (Hfq6)n "head-to-tail" polymers. We describe purification of these novel, structurally uniform (Hfq6)n≥8 complexes to near homogeneity and show that they also contain small nonprotein molecules and accessory S6. We demonstrate that Hfq, S6, and RP have similar solubility profiles and present evidence pointing to a role of the Hfq C-termini in superstructure formation. Taken together, our data offer new insights into the composition of the macromolecular assemblies likely acting as scaffolds for transcription complexes and ribosomes during bacterial cells' active growth.
Subject(s)
DNA-Directed RNA Polymerases , Escherichia coli Proteins , Escherichia coli , Transcription, Genetic , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/metabolism , Escherichia coli Proteins/genetics , Escherichia coli Proteins/isolation & purification , Escherichia coli/genetics , Escherichia coli/metabolism , DNA-Directed RNA Polymerases/metabolism , DNA-Directed RNA Polymerases/chemistry , DNA-Directed RNA Polymerases/genetics , Host Factor 1 Protein/metabolism , Host Factor 1 Protein/chemistry , Host Factor 1 Protein/genetics , Protein Biosynthesis , Multiprotein Complexes/chemistry , Multiprotein Complexes/genetics , Multiprotein Complexes/isolation & purification , Multiprotein Complexes/metabolismABSTRACT
3C-based methods have significantly advanced our understanding of 3D genome organization. However, it remains a formidable task to precisely capture long-range chromosomal interactions between individual loci, such as those between promoters and distal enhancers. Here, we present Methyltransferase Targeting-based chromosome Architecture Capture (MTAC), a method that maps the contacts between a target site (viewpoint) and the rest of the genome in budding yeast with high resolution and sensitivity. MTAC detects hundreds of intra- and inter-chromosomal interactions within nucleosome-depleted regions (NDRs) that cannot be captured by 4C, Hi-C, or Micro-C. By applying MTAC to various viewpoints, we find that (1) most long-distance chromosomal interactions detected by MTAC reflect tethering by the nuclear pore complexes (NPCs), (2) genes co-regulated by methionine assemble into inter-chromosomal clusters near NPCs upon activation, (3) mediated by condensin, the mating locus forms a highly specific interaction with the recombination enhancer (RE) in a mating-type specific manner, and (4) correlation of MTAC signals among NDRs reveal spatial mixing and segregation of the genome. Overall, these results demonstrate MTAC as a powerful tool to resolve fine-scale long-distance chromosomal interactions and provide insights into the 3D genome organization.
Subject(s)
Chromosomes, Fungal , DNA Methylation , Nucleosomes , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae , Nucleosomes/metabolism , Nucleosomes/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Chromosomes, Fungal/genetics , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Chromosome Mapping/methods , DNA-Binding Proteins/metabolism , DNA-Binding Proteins/genetics , Adenosine Triphosphatases/metabolism , Adenosine Triphosphatases/genetics , Genome, Fungal , Promoter Regions, Genetic/genetics , Multiprotein Complexes/metabolism , Multiprotein Complexes/genetics , Nuclear Pore/metabolism , Nuclear Pore/genetics , Methyltransferases/metabolism , Methyltransferases/geneticsABSTRACT
Mammalian target of rapamycin (mTOR) senses changes in nutrient status and stimulates the autophagic process to recycle amino acids. However, the impact of nutrient stress on protein degradation beyond autophagic turnover is incompletely understood. We report that several metabolic enzymes are proteasomal targets regulated by mTOR activity based on comparative proteome degradation analysis. In particular, 3-hydroxy-3-methylglutaryl (HMG)-coenzyme A (CoA) synthase 1 (HMGCS1), the initial enzyme in the mevalonate pathway, exhibits the most significant half-life adaptation. Degradation of HMGCS1 is regulated by the C-terminal to LisH (CTLH) E3 ligase through the Pro/N-degron motif. HMGCS1 is ubiquitylated on two C-terminal lysines during mTORC1 inhibition, and efficient degradation of HMGCS1 in cells requires a muskelin adaptor. Importantly, modulating HMGCS1 abundance has a dose-dependent impact on cell proliferation, which is restored by adding a mevalonate intermediate. Overall, our unbiased degradomics study provides new insights into mTORC1 function in cellular metabolism: mTORC1 regulates the stability of limiting metabolic enzymes through the ubiquitin system.
Subject(s)
Cell Proliferation , Hydroxymethylglutaryl-CoA Synthase , Mechanistic Target of Rapamycin Complex 1 , Proteolysis , Ubiquitin-Protein Ligases , Ubiquitination , Mechanistic Target of Rapamycin Complex 1/metabolism , Mechanistic Target of Rapamycin Complex 1/genetics , Humans , Ubiquitin-Protein Ligases/metabolism , Ubiquitin-Protein Ligases/genetics , HEK293 Cells , Hydroxymethylglutaryl-CoA Synthase/metabolism , Hydroxymethylglutaryl-CoA Synthase/genetics , Proteasome Endopeptidase Complex/metabolism , Proteasome Endopeptidase Complex/genetics , TOR Serine-Threonine Kinases/metabolism , TOR Serine-Threonine Kinases/genetics , Mevalonic Acid/metabolism , Multiprotein Complexes/metabolism , Multiprotein Complexes/genetics , Signal Transduction , Degrons , Adaptor Proteins, Signal TransducingABSTRACT
Structural maintenance of chromosomes (SMC), including cohesin, condensin and the SMC5/6 complex, are protein complexes which maintain the higher structure and dynamic stability of chromatin. Such circular complexes, with similar structures, play pivotal roles in chromatid cohesion, chromosomal condensation, DNA replication and repair, as well as gene transcription. Despite extensive research on the functions of the SMCs, our understanding of the SMC5/6 complex has remained limited compared with the other two complexes. This article has reviewed the architecture and crucial physiological roles of the SMCs, and explored the associated phenotypes resulting from mutations of the SMC components such as Cornelia de Lange syndrome (CdLS) and microcephaly, with an aim to provide insights into their functions in eukaryotic cells and implications for human diseases.
Subject(s)
Cell Cycle Proteins , Chromosomal Proteins, Non-Histone , Humans , Chromosomal Proteins, Non-Histone/genetics , Chromosomal Proteins, Non-Histone/metabolism , Cell Cycle Proteins/genetics , Cohesins , Multiprotein Complexes/genetics , DNA-Binding Proteins/genetics , Adenosine Triphosphatases/genetics , Animals , De Lange Syndrome/genetics , MutationABSTRACT
During mitosis, condensin activity is thought to interfere with interphase chromatin structures. To investigate genome folding principles in the absence of chromatin loop extrusion, we codepleted condensin I and condensin II, which triggered mitotic chromosome compartmentalization in ways similar to that in interphase. However, two distinct euchromatic compartments, indistinguishable in interphase, emerged upon condensin loss with different interaction preferences and dependencies on H3K27ac. Constitutive heterochromatin gradually self-aggregated and cocompartmentalized with facultative heterochromatin, contrasting with their separation during interphase. Notably, some cis-regulatory element contacts became apparent even in the absence of CTCF/cohesin-mediated structures. Heterochromatin protein 1 (HP1) proteins, which are thought to partition constitutive heterochromatin, were absent from mitotic chromosomes, suggesting, surprisingly, that constitutive heterochromatin can self-aggregate without HP1. Indeed, in cells traversing from M to G1 phase in the combined absence of HP1α, HP1ß and HP1γ, constitutive heterochromatin compartments are normally re-established. In sum, condensin-deficient mitotic chromosomes illuminate forces of genome compartmentalization not identified in interphase cells.
Subject(s)
Adenosine Triphosphatases , Chromosomal Proteins, Non-Histone , DNA-Binding Proteins , Heterochromatin , Mitosis , Multiprotein Complexes , Adenosine Triphosphatases/genetics , Adenosine Triphosphatases/metabolism , Multiprotein Complexes/genetics , Multiprotein Complexes/metabolism , DNA-Binding Proteins/metabolism , DNA-Binding Proteins/genetics , Mitosis/genetics , Humans , Chromosomal Proteins, Non-Histone/metabolism , Chromosomal Proteins, Non-Histone/genetics , Heterochromatin/metabolism , Heterochromatin/genetics , Interphase/genetics , Chromosomes/genetics , Chromobox Protein Homolog 5 , Chromatin/metabolism , Chromatin/geneticsABSTRACT
Pre-mRNA splicing plays a key role in the regulation of gene expression. Recent discoveries suggest that defects in pre-mRNA splicing, resulting from the dysfunction of certain splicing factors, can impact the expression of genes crucial for genome surveillance mechanisms, including those involved in cellular response to DNA damage. In this study, we analyzed how cells with a non-functional spliceosome-associated Gpl1-Gih35-Wdr83 complex respond to DNA damage. Additionally, we investigated the role of this complex in regulating the splicing of factors involved in DNA damage repair. Our findings reveal that the deletion of any component within the Gpl1-Gih35-Wdr83 complex leads to a significant accumulation of unspliced pre-mRNAs of DNA repair factors. Consequently, mutant cells lacking this complex exhibit increased sensitivity to DNA-damaging agents. These results highlight the importance of the Gpl1-Gih35-Wdr83 complex in regulating the expression of DNA repair factors, thereby protecting the stability of the genome following DNA damage.
Subject(s)
DNA Damage , DNA Repair , RNA Splicing Factors , RNA Splicing , DNA Damage/genetics , DNA Repair/genetics , Gene Expression Regulation, Fungal , RNA Precursors/genetics , RNA Precursors/metabolism , RNA Splicing Factors/metabolism , RNA Splicing Factors/genetics , Schizosaccharomyces/genetics , Schizosaccharomyces/metabolism , Schizosaccharomyces pombe Proteins/genetics , Schizosaccharomyces pombe Proteins/metabolism , Spliceosomes/metabolism , Spliceosomes/genetics , Multiprotein Complexes/genetics , Multiprotein Complexes/metabolismABSTRACT
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.
Subject(s)
Adenosine Triphosphatases , Cell Nucleus Division , DNA-Binding Proteins , Mitosis , Plasmodium falciparum , Humans , Adenosine Triphosphatases/metabolism , Adenosine Triphosphatases/genetics , DNA-Binding Proteins/metabolism , DNA-Binding Proteins/genetics , Erythrocytes/parasitology , Gene Knockout Techniques , Multiprotein Complexes/metabolism , Multiprotein Complexes/genetics , Plasmodium falciparum/genetics , Plasmodium falciparum/metabolism , Plasmodium falciparum/physiology , Plasmodium falciparum/growth & development , Protozoan Proteins/metabolism , Protozoan Proteins/genetics , Cell Nucleus Division/geneticsABSTRACT
Chromosome pairing constitutes an important level of genome organization, yet the mechanisms that regulate pairing in somatic cells and the impact on 3D chromatin organization are still poorly understood. Here, we address these questions in Drosophila, an organism with robust somatic pairing. In Drosophila, pairing preferentially occurs at loci consisting of numerous architectural protein binding sites (APBSs), suggesting a role of architectural proteins (APs) in pairing regulation. Amongst these, the anti-pairing function of the condensin II subunit CAP-H2 is well established. However, the factors that regulate CAP-H2 localization and action at APBSs remain largely unknown. Here, we identify two factors that control CAP-H2 occupancy at APBSs and, therefore, regulate pairing. We show that Z4, interacts with CAP-H2 and is required for its localization at APBSs. We also show that hyperosmotic cellular stress induces fast and reversible unpairing in a Z4/CAP-H2 dependent manner. Moreover, by combining the opposite effects of Z4 depletion and osmostress, we show that pairing correlates with the strength of intrachromosomal 3D interactions, such as active (A) compartment interactions, intragenic gene-loops, and polycomb (Pc)-mediated chromatin loops. Altogether, our results reveal new players in CAP-H2-mediated pairing regulation and the intimate interplay between inter-chromosomal and intra-chromosomal 3D interactions.
Subject(s)
Adenosine Triphosphatases , Chromatin , Chromosome Pairing , DNA-Binding Proteins , Drosophila Proteins , Animals , Adenosine Triphosphatases/metabolism , Adenosine Triphosphatases/genetics , Binding Sites , Chromatin/metabolism , Chromosomal Proteins, Non-Histone , DNA-Binding Proteins/metabolism , DNA-Binding Proteins/genetics , Drosophila melanogaster/genetics , Drosophila melanogaster/metabolism , Drosophila Proteins/metabolism , Drosophila Proteins/genetics , Multiprotein Complexes/metabolism , Multiprotein Complexes/genetics , Osmotic Pressure , Protein Binding , Zinc FingersABSTRACT
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.
Subject(s)
Mitochondria , Mitochondrial Proteins , Mutation , Neurodegenerative Diseases , Stress, Physiological , Ubiquitin-Protein Ligases , Apoptosis/drug effects , Ataxia/genetics , Cell Survival/drug effects , Dementia/genetics , Mitochondria/genetics , Mitochondria/metabolism , Mitochondria/pathology , Mitochondrial Proteins/chemistry , Mitochondrial Proteins/metabolism , Multiprotein Complexes/antagonists & inhibitors , Multiprotein Complexes/genetics , Multiprotein Complexes/metabolism , Neurodegenerative Diseases/genetics , Neurodegenerative Diseases/metabolism , Neurodegenerative Diseases/pathology , Protein Stability/drug effects , Protein Transport/drug effects , Proteolysis/drug effects , Stress, Physiological/drug effects , Ubiquitin/metabolism , Ubiquitin-Protein Ligases/antagonists & inhibitors , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolism , Ubiquitination/drug effectsABSTRACT
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.
Subject(s)
Multiprotein Complexes , TOR Serine-Threonine Kinases , Mechanistic Target of Rapamycin Complex 1/genetics , TOR Serine-Threonine Kinases/metabolism , Multiprotein Complexes/genetics , Multiprotein Complexes/metabolism , Signal Transduction , Gene ExpressionABSTRACT
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.
Subject(s)
Gene Expression Regulation, Enzymologic , Multiprotein Complexes , Protein Biosynthesis , Multiprotein Complexes/genetics , Multiprotein Complexes/metabolism , Protein Biosynthesis/genetics , Transcription Factor TFIID/metabolism , Transcription Factors/metabolism , Transcription, Genetic , HumansABSTRACT
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.
Subject(s)
Chromosomes , DNA-Binding Proteins , Animals , DNA-Binding Proteins/physiology , Multiprotein Complexes/genetics , Adenosine Triphosphatases/genetics , MitosisABSTRACT
Eukaryotic chromosomes compact during mitosis into elongated cylinders-and not the spherical globules expected of self-attracting long flexible polymers. This process is mainly driven by condensin-like proteins. Here, we present Brownian-dynamic simulations involving two types of such proteins with different activities. One, which we refer to as looping condensins, anchors long-lived chromatin loops to create bottlebrush structures. The second, referred to as bridging condensins, forms multivalent bridges between distant parts of these loops. We show that binding of bridging condensins leads to the formation of shorter and stiffer mitotic-like cylinders without requiring any additional energy input. These cylinders have several features matching experimental observations. For instance, the axial condensin backbone breaks up into clusters as found by microscopy, and cylinder elasticity qualitatively matches that seen in chromosome pulling experiments. Additionally, simulating global condensin depletion or local faulty condensin loading gives phenotypes seen experimentally and points to a mechanistic basis for the structure of common fragile sites in mitotic chromosomes.
Subject(s)
Adenosine Triphosphatases , Chromosomes , DNA-Binding Proteins , Multiprotein Complexes , Adenosine Triphosphatases/genetics , Adenosine Triphosphatases/metabolism , Chromatin/genetics , Chromosomes/genetics , Chromosomes/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Mitosis , Multiprotein Complexes/genetics , Multiprotein Complexes/metabolismABSTRACT
Condensin is a structural maintenance of chromosomes (SMC) complex family member thought to build mitotic chromosomes by DNA loop extrusion. However, condensin variants unable to extrude loops, yet proficient in chromosome formation, were recently described. Here, we explore how condensin might alternatively build chromosomes. Using bulk biochemical and single-molecule experiments with purified fission yeast condensin, we observe that individual condensins sequentially and topologically entrap two double-stranded DNAs (dsDNAs). Condensin loading transitions through a state requiring DNA bending, as proposed for the related cohesin complex. While cohesin then favors the capture of a second single-stranded DNA (ssDNA), second dsDNA capture emerges as a defining feature of condensin. We provide complementary in vivo evidence for DNA-DNA capture in the form of condensin-dependent chromatin contacts within, as well as between, chromosomes. Our results support a "diffusion capture" model in which condensin acts in mitotic chromosome formation by sequential dsDNA-dsDNA capture.
Subject(s)
DNA-Binding Proteins , Schizosaccharomyces , DNA-Binding Proteins/genetics , DNA-Binding Proteins/chemistry , Multiprotein Complexes/genetics , Multiprotein Complexes/chemistry , DNA/genetics , Chromosomes , Cell Cycle Proteins/genetics , Schizosaccharomyces/genetics , MitosisABSTRACT
mTORC1 (mechanistic target of rapamycin complex 1) is a metabolic sensor that promotes growth when nutrients are abundant. Ubiquitous inhibition of mTORC1 extends lifespan in multiple organisms but also disrupts several anabolic processes resulting in stunted growth, slowed development, reduced fertility, and disrupted metabolism. However, it is unclear if these pleiotropic effects of mTORC1 inhibition can be uncoupled from longevity. Here, we utilize the auxin-inducible degradation (AID) system to restrict mTORC1 inhibition to C. elegans neurons. We find that neuron-specific degradation of RAGA-1, an upstream activator of mTORC1, or LET-363, the ortholog of mammalian mTOR, is sufficient to extend lifespan in C. elegans. Unlike raga-1 loss of function genetic mutations or somatic AID of RAGA-1, neuronal AID of RAGA-1 robustly extends lifespan without impairing body size, developmental rate, brood size, or neuronal function. Moreover, while degradation of RAGA-1 in all somatic tissues alters the expression of thousands of genes, demonstrating the widespread effects of mTORC1 inhibition, degradation of RAGA-1 in neurons only results in around 200 differentially expressed genes with a specific enrichment in metabolism and stress response. Notably, our work demonstrates that targeting mTORC1 specifically in the nervous system in C. elegans uncouples longevity from growth and reproductive impairments, and that many canonical effects of low mTORC1 activity are not required to promote healthy aging. These data challenge previously held ideas about the mechanisms of mTORC1 lifespan extension and underscore the potential of promoting longevity by neuron-specific mTORC1 modulation.
Subject(s)
Caenorhabditis elegans Proteins , Caenorhabditis elegans , Animals , Mechanistic Target of Rapamycin Complex 1/genetics , Mechanistic Target of Rapamycin Complex 1/metabolism , Caenorhabditis elegans/metabolism , Longevity/genetics , Multiprotein Complexes/genetics , Reproduction/genetics , Caenorhabditis elegans Proteins/genetics , Caenorhabditis elegans Proteins/metabolism , Mammals/metabolismABSTRACT
Three distinct structural maintenance of chromosomes (SMC) complexes facilitate chromosome folding and segregation in eukaryotes, presumably by DNA loop extrusion. How SMCs interact with DNA to extrude loops is not well understood. Among the SMC complexes, Smc5/6 has dedicated roles in DNA repair and preventing a buildup of aberrant DNA junctions. In the present study, we describe the reconstitution of ATP-dependent DNA loading by yeast Smc5/6 rings. Loading strictly requires the Nse5/6 subcomplex which opens the kleisin neck gate. We show that plasmid molecules are topologically entrapped in the kleisin and two SMC subcompartments, but not in the full SMC compartment. This is explained by the SMC compartment holding a looped DNA segment and by kleisin locking it in place when passing between the two flanks of the loop for neck-gate closure. Related segment capture events may provide the power stroke in subsequent DNA extrusion steps, possibly also in other SMC complexes, thus providing a unifying principle for DNA loading and extrusion.