Hook3 is a scaffold for the opposite-polarity microtubule-based motors cytoplasmic dynein-1 and KIF1C.

The unidirectional and opposite-polarity microtubule-based motors, dynein and kinesin, drive long-distance intracellular cargo transport. Cellular observations suggest that opposite-polarity motors may be coupled. We recently identified an interaction between the cytoplasmic dynein-1 activating adaptor Hook3 and the kinesin-3 KIF1C. Here, using in vitro reconstitutions with purified components, we show that KIF1C and dynein/dynactin can exist in a complex scaffolded by Hook3. Full-length Hook3 binds to and activates dynein/dynactin motility. Hook3 also binds to a short region in the "tail" of KIF1C, but unlike dynein/dynactin, this interaction does not activate KIF1C. Hook3 scaffolding allows dynein to transport KIF1C toward the microtubule minus end, and KIF1C to transport dynein toward the microtubule plus end. In cells, KIF1C can recruit Hook3 to the cell periphery, although the cellular role of the complex containing both motors remains unknown. We propose that Hook3's ability to scaffold dynein/dynactin and KIF1C may regulate bidirectional motility, promote motor recycling, or sequester the pool of available dynein/dynactin activating adaptors

Protein aggregation mediates stoichiometry of protein complexes in aneuploid cells

Aneuploidy, a condition characterized by chromosome gains and losses, causes reduced fitness and numerous cellular stresses, including increased protein aggregation. Here, we identify protein complex stoichiometry imbalances as a major cause of protein aggregation in aneuploid cells. Subunits of protein complexes encoded on excess chromosomes aggregate in aneuploid cells, which is suppressed when expression of other subunits is coordinately altered. We further show that excess subunits are either degraded or aggregate and that protein aggregation is nearly as effective as protein degradation at lowering levels of excess proteins. Our study explains why proteotoxic stress is a universal feature of the aneuploid state and reveals protein aggregation as a form of dosage compensation to cope with disproportionate expression of protein complex subunits

Mechanism of cyclin D1-dependent genomic instability and neoplastic transformation

Regulation of cyclin D1-dependent kinase activity is essential for cell cycle progression and DNA replication fidelity. Critically, impaired cyclin D1 phosphorylation and ubiquitin-mediated proteolysis following the G1/S transition drives neoplastic growth, suggesting that posttranslational regulation is required for cell homeostasis. Elucidation of mechanisms facilitating S-phase cyclin D1 accumulation and novel functions of nuclear cyclin D1/CDK4 kinase is critical for understanding the role of cyclin D1 in tumorigenesis. The work presented herein demonstrates that accelerated, Fbx4-dependent cyclin D1 degradation following S-phase DNA damage is essential to maintain genome stability. Furthermore, Fbx4 functions as a bona fide tumor suppressor, as Fbx4-deficient mice develop spontaneous tumors and murine fibroblasts exhibit cyclin D1 stabilization, nuclear accumulation, and associated genomic instability. This work also describes novel regulation of the PRMT5 methyltransferase by nuclear cyclin D1/CDK4, thereby facilitating histone methylation and gene repression during S-phase necessary for neoplastic growth. Finally, current work reveals a synergistic relationship between constitutively nuclear cyclin D1 and impaired DNA damage checkpoint integrity in driving lymphomagenesis in mice. Collectively, these findings define an intricate relationship wherein nuclear cyclin D1/CDK4 activity modulates genetic alterations necessary for perturbed DNA replication, genomic instability, and ultimately neoplasia.

Mechanism of Cyclin D1-Dependent Genomic Instability and Neoplastic Transformation

Regulation of cyclin D1-dependent kinase activity is essential for cell cycle progression and DNA replication fidelity. Critically, impaired cyclin D1 phosphorylation and ubiquitin-mediated proteolysis following the G1/S transition drives neoplastic growth, suggesting that posttranslational regulation is required for cell homeostasis. Elucidation of mechanisms facilitating S-phase cyclin D1 accumulation and novel functions of nuclear cyclin D1/CDK4 kinase is critical for understanding the role of cyclin D1 in tumorigenesis. The work presented herein demonstrates that accelerated, Fbx4-dependent cyclin D1 degradation following S-phase DNA damage is essential to maintain genome stability. Furthermore, Fbx4 functions as a bona fide tumor suppressor, as Fbx4-deficient mice develop spontaneous tumors and murine fibroblasts exhibit cyclin D1 stabilization, nuclear accumulation, and associated genomic instability. This work also describes novel regulation of the PRMT5 methyltransferase by nuclear cyclin D1/CDK4, thereby facilitating histone methylation and gene repression during S-phase necessary for neoplastic growth. Finally, current work reveals a synergistic relationship between constitutively nuclear cyclin D1 and impaired DNA damage checkpoint integrity in driving lymphomagenesis in mice. Collectively, these findings define an intricate relationship wherein nuclear cyclin D1/CDK4 activity modulates genetic alterations necessary for perturbed DNA replication, genomic instability, and ultimately neoplasia

Hook3 is a scaffold for the opposite-polarity microtubule-based motors cytoplasmic dynein-1 and KIF1C.

The unidirectional and opposite-polarity microtubule-based motors, dynein and kinesin, drive long-distance intracellular cargo transport. Cellular observations suggest that opposite-polarity motors may be coupled. We recently identified an interaction between the cytoplasmic dynein-1 activating adaptor Hook3 and the kinesin-3 KIF1C. Here, using in vitro reconstitutions with purified components, we show that KIF1C and dynein/dynactin can exist in a complex scaffolded by Hook3. Full-length Hook3 binds to and activates dynein/dynactin motility. Hook3 also binds to a short region in the "tail" of KIF1C, but unlike dynein/dynactin, this interaction does not activate KIF1C. Hook3 scaffolding allows dynein to transport KIF1C toward the microtubule minus end, and KIF1C to transport dynein toward the microtubule plus end. In cells, KIF1C can recruit Hook3 to the cell periphery, although the cellular role of the complex containing both motors remains unknown. We propose that Hook3's ability to scaffold dynein/dynactin and KIF1C may regulate bidirectional motility, promote motor recycling, or sequester the pool of available dynein/dynactin activating adaptors

A Systematic Analysis of Factors Localized to Damaged Chromatin Reveals PARP-Dependent Recruitment of Transcription Factors

Localization to sites of DNA damage is a hallmark of DNA damage response (DDR) proteins. To identify DDR factors, we screened epitope-tagged proteins for localization to sites of chromatin damaged by UV laser microirradiation and found >120 proteins that localize to damaged chromatin. These include the BAF tumor suppressor complex and the amyotrophic lateral sclerosis (ALS) candidate protein TAF15. TAF15 contains multiple domains that bind damaged chromatin in a poly-(ADP-ribose) polymerase (PARP)-dependent manner, suggesting a possible role as glue that tethers multiple PAR chains together. Many positives were transcription factors; > 70% of randomly tested transcription factors localized to sites of DNA damage, and of these, ∼90% were PARP dependent for localization. Mutational analyses showed that localization to damaged chromatin is DNA-binding-domain dependent. By examining Hoechst staining patterns at damage sites, we see evidence of chromatin decompaction that is PARP dependent. We propose that PARP-regulated chromatin remodeling at sites of damage allows transient accessibility of DNA-binding proteins

Ferritinophagy via NCOA4 is required for erythropoiesis and is regulated by iron dependent HERC2-mediated proteolysis

NCOA4 is a selective cargo receptor for the autophagic turnover of ferritin, a process critical for regulation of intracellular iron bioavailability. However, how ferritinophagy flux is controlled and the roles of NCOA4 in iron-dependent processes are poorly understood. Through analysis of the NCOA4-FTH1 interaction, we demonstrate that direct association via a key surface arginine in FTH1 and a C-terminal element in NCOA4 is required for delivery of ferritin to the lysosome via autophagosomes. Moreover, NCOA4 abundance is under dual control via autophagy and the ubiquitin proteasome system. Ubiquitin-dependent NCOA4 turnover is promoted by excess iron and involves an iron-dependent interaction between NCOA4 and the HERC2 ubiquitin ligase. In zebrafish and cultured cells, NCOA4 plays an essential role in erythroid differentiation. This work reveals the molecular nature of the NCOA4-ferritin complex and explains how intracellular iron levels modulate NCOA4-mediated ferritinophagy in cells and in an iron-dependent physiological setting. DOI: http://dx.doi.org/10.7554/eLife.10308.00

Excessive Cell Growth Causes Cytoplasm Dilution And Contributes to Senescence

Cell size varies greatly between cell types, yet within a specific cell type and growth condition, cell size is narrowly distributed. Why maintenance of a cell-type specific cell size is important remains poorly understood. Here we show that growing budding yeast and primary mammalian cells beyond a certain size impairs gene induction, cell-cycle progression, and cell signaling. These defects are due to the inability of large cells to scale nucleic acid and protein biosynthesis in accordance with cell volume increase, which effectively leads to cytoplasm dilution. We further show that loss of scaling beyond a certain critical size is due to DNA becoming limiting. Based on the observation that senescent cells are large and exhibit many of the phenotypes of large cells, we propose that the range of DNA:cytoplasm ratio that supports optimal cell function is limited and that ratios outside these bounds contribute to aging. Optimal cell function requires maintenance of a narrow range of DNA:cytoplasm ratios and when cell size exceeds this ratio cytoplasmic dilution contributes to senescenceNational Institutes of Health (Grant HD085866)National Institutes of Health (Grant 1U54CA217377

A multi-scale map of cell structure fusing protein images and interactions

info:eu-repo/semantics/publishe

Architecture of the human interactome defines protein communities and disease networks

The physiology of a cell can be viewed as the product of thousands of proteins acting in concert to shape the cellular response. Coordination is achieved in part through networks of protein-protein interactions that assemble functionally related proteins into complexes, organelles, and signal transduction pathways. Understanding the architecture of the human proteome has the potential to inform cellular, structural, and evolutionary mechanisms and is critical to elucidation of how genome variation contributes to disease1–3. Here, we present BioPlex 2.0 (Biophysical Interactions of ORFEOME-derived complexes), which employs robust affinity purification-mass spectrometry (AP-MS) methodology4 to elucidate protein interaction networks and co-complexes nucleated by more than 25% of protein coding genes from the human genome, and constitutes the largest such network to date. With >56,000 candidate interactions, BioPlex 2.0 contains >29,000 previously unknown co-associations and provides functional insights into hundreds of poorly characterized proteins while enhancing network-based analyses of domain associations, subcellular localization, and co-complex formation. Unsupervised Markov clustering (MCL)5 of interacting proteins identified more than 1300 protein communities representing diverse cellular activities. Genes essential for cell fitness6,7 are enriched within 53 communities representing central cellular functions. Moreover, we identified 442 communities associated with more than 2000 disease annotations, placing numerous candidate disease genes into a cellular framework. BioPlex 2.0 exceeds previous experimentally derived interaction networks in depth and breadth, and will be a valuable resource for exploring the biology of incompletely characterized proteins and for elucidating larger-scale patterns of proteome organization