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BioRxiv : the Preprint Server For... Jun 2024Acral melanoma (AM) is an aggressive melanoma variant that arises from palmar, plantar, and nail unit melanocytes. Compared to non-acral cutaneous melanoma (CM), AM is...
Acral melanoma (AM) is an aggressive melanoma variant that arises from palmar, plantar, and nail unit melanocytes. Compared to non-acral cutaneous melanoma (CM), AM is biologically distinct, has an equal incidence across genetic ancestries, typically presents in advanced stage disease, is less responsive to therapy, and has an overall worse prognosis. Independent analysis of published genomic and transcriptomic sequencing identified that receptor tyrosine kinase (RTK) ligands and adapter proteins are frequently amplified, translocated, and/or overexpressed in AM. To target these unique genetic changes, a zebrafish acral melanoma model was exposed to a panel of narrow and broad spectrum multi-RTK inhibitors, revealing that dual FGFR/VEGFR inhibitors decrease acral-analogous melanocyte proliferation and migration. The potent pan-FGFR/VEGFR inhibitor, Lenvatinib, uniformly induces tumor regression in AM patient-derived xenograft (PDX) tumors but only slows tumor growth in CM models. Unlike other multi-RTK inhibitors, Lenvatinib is not directly cytotoxic to dissociated AM PDX tumor cells and instead disrupts tumor architecture and vascular networks. Considering the great difficulty in establishing AM cell culture lines, these findings suggest that AM may be more sensitive to microenvironment perturbations than CM. In conclusion, dual FGFR/VEGFR inhibition may be a viable therapeutic strategy that targets the unique biology of AM.
PubMed: 38948879
DOI: 10.1101/2024.06.15.599116 -
BioRxiv : the Preprint Server For... Jun 2024Faithfull cell division relies on mitotic chromosomes becoming bioriented with each pair of sister kinetochores bound to microtubules oriented toward opposing spindle...
Faithfull cell division relies on mitotic chromosomes becoming bioriented with each pair of sister kinetochores bound to microtubules oriented toward opposing spindle poles. Erroneous kinetochore-microtubule attachments often form during early mitosis, but are destabilized through the phosphorylation of outer kinetochore proteins by centromeric AURORA B kinase (ABK) and centrosomal AURORA A kinase (AAK), thus allowing for re-establishment of attachments until biorientation is achieved. MPS1-mediated phosphorylation of NDC80 has also been shown to directly weaken the kinetochore-microtubule interface in yeast. In human cells, MPS1 has been proposed to transiently accumulate at end-on attached kinetochores and phosphorylate SKA3 to promote microtubule release. Whether MPS1 directly targets NDC80 and/or promotes the activity of AURORA kinases in metazoans remains unclear. Here, we report a novel mechanism involving communication between kinetochores and centrosomes, wherein MPS1 acts upstream of AAK to promote error correction. MPS1 on pole-proximal kinetochores phosphorylates the C-lobe of AAK thereby increasing its activation at centrosomes. This proximity-based activation ensures the establishment of a robust AAK activity gradient that locally destabilizes mal-oriented kinetochores near spindle poles. Accordingly, MPS1 depletion from cells causes severe chromosome misalignment and erroneous kinetochore-microtubule attachments, which can be rescued by tethering either MPS1 or constitutively active AAK mutants to centrosomes. Proximity-based activation of AAK by MPS1 also occurs in human cells to promote AAK-mediated phosphorylation of the NDC80 N-terminal tail. These findings uncover an MPS1-AAK cross-talk that is required for efficient error correction, showcasing the ability of kinetochores to modulate centrosome outputs to ensure proper chromosome segregation.
PubMed: 38948877
DOI: 10.1101/2024.06.11.598300 -
BioRxiv : the Preprint Server For... Jun 2024Nucleoli are large nuclear sub-compartments where vital processes, such as ribosome assembly, take place. Technical obstacles still limit our understanding of the...
Nucleoli are large nuclear sub-compartments where vital processes, such as ribosome assembly, take place. Technical obstacles still limit our understanding of the biological functions of nucleolar proteins in cell homeostasis and cancer pathogenesis. Since most nucleolar proteins are essential, their abrogation cannot be achieved through conventional approaches. Additionally, the biological activities of many nucleolar proteins are connected to their physiological concentration. Thus, artificial overexpression might not fully recapitulate their endogenous functions. Proteolysis-based approaches, such as the Auxin Inducible Degron (AID) system paired with CRISPR/Cas9 knock-in gene-editing, have the potential to overcome these limitations, providing unprecedented characterization of the biological activities of endogenous nucleolar proteins. We applied this system to endogenous nucleolin (NCL), one of the most abundant nucleolar proteins, and characterized the impact of its acute depletion on Triple-Negative Breast Cancer (TNBC) cell behavior. Abrogation of endogenous NCL reduced proliferation and caused defective cytokinesis, resulting in bi-nucleated tetraploid cells. Bioinformatic analysis of patient data, and quantitative proteomics using our experimental NCL-depleted model, indicated that NCL levels are correlated with the abundance of proteins involved in chromosomal segregation. In conjunction with its effects on sister chromatid dynamics, NCL abrogation enhanced the anti-proliferative effects of chemical inhibitors of mitotic modulators such as the Anaphase Promoting Complex. In summary, using the AID system in combination with CRISPR/Cas9 for endogenous gene editing, our findings indicate a novel role for NCL in supporting the completion of the cell division in TNBC models, and that its abrogation could enhance the therapeutic activity of mitotic-progression inhibitors.
PubMed: 38948867
DOI: 10.1101/2024.06.17.599429 -
BioRxiv : the Preprint Server For... Jun 2024Under stress conditions, cells reprogram their molecular machineries to mitigate damage and promote survival. Ubiquitin signaling is globally increased during oxidative...
Under stress conditions, cells reprogram their molecular machineries to mitigate damage and promote survival. Ubiquitin signaling is globally increased during oxidative stress, controlling protein fate and supporting stress defenses at several subcellular compartments. However, the rules driving subcellular ubiquitin localization to promote these concerted response mechanisms remain understudied. Here, we show that K63-linked ubiquitin chains, known to promote proteasome-independent pathways, accumulate primarily in non-cytosolic compartments during oxidative stress induced by sodium arsenite in mammalian cells. Our subcellular ubiquitin proteomic analyses of non-cytosolic compartments expanded 10-fold the pool of proteins known to be ubiquitinated during arsenite stress (2,046) and revealed their involvement in pathways related to immune signaling and translation control. Moreover, subcellular proteome analyses revealed proteins that are recruited to non-cytosolic compartments under stress, including a significant enrichment of helper ubiquitin-binding adaptors of the ATPase VCP that processes ubiquitinated substrates for downstream signaling. We further show that VCP recruitment to non-cytosolic compartments under arsenite stress occurs in a ubiquitin-dependent manner mediated by its adaptor NPLOC4. Additionally, we show that VCP and NPLOC4 activities are critical to sustain low levels of non-cytosolic K63-linked ubiquitin chains, supporting a cyclical model of ubiquitin conjugation and removal that is disrupted by cellular exposure to reactive oxygen species. This work deepens our understanding of the role of localized ubiquitin and VCP signaling in the basic mechanisms of stress response and highlights new pathways and molecular players that are essential to reshape the composition and function of the human subcellular proteome under dynamic environments.
PubMed: 38948861
DOI: 10.1101/2024.06.20.598218 -
BioRxiv : the Preprint Server For... Jun 2024Heterotopic ossifications (HOs) are the pathologic process by which bone inappropriately forms outside of the skeletal system. Despite HOs being a persistent clinical...
Heterotopic ossifications (HOs) are the pathologic process by which bone inappropriately forms outside of the skeletal system. Despite HOs being a persistent clinical problem in the general population, there are no definitive strategies for their prevention and treatment due to a limited understanding of the cellular and molecular mechanisms contributing to lesion development. One disease in which the development of heterotopic subcutaneous ossifications (SCOs) leads to morbidity is Albright hereditary osteodystrophy (AHO). AHO is caused by heterozygous inactivation of , the gene that encodes the α-stimulatory subunit (Gα ) of G proteins. Previously, we had shown using our laboratory's AHO mouse model that SCOs develop around hair follicles (HFs). Here we show that SCO formation occurs due to inappropriate expansion and differentiation of HF-resident stem cells into osteoblasts. We also show in AHO patients and mice that ( expression is upregulated in regions of SCO formation and that elimination of in male AHO mice exacerbates SCO development. These studies provide key insights into the cellular and molecular mechanisms contributing to SCO development and have implications for potential therapeutic modalities not only for AHO patients but also for patients suffering from HOs with other etiologies.
PubMed: 38948860
DOI: 10.1101/2024.06.18.599506 -
BioRxiv : the Preprint Server For... Jun 2024The serotonin 2A receptor (5-HT R) and the metabotropic glutamate 2 receptor (mGluR2) form heteromeric G protein-coupled receptor (GPCR) complexes through a direct...
The serotonin 2A receptor (5-HT R) and the metabotropic glutamate 2 receptor (mGluR2) form heteromeric G protein-coupled receptor (GPCR) complexes through a direct physical interaction. Co-translational association of mRNAs encoding subunits of heteromeric ion channels has been reported, but whether complex assembly of GPCRs occurs during translation remains unknown. Our data reveal evidence of co-translational modulation in and mRNAs following siRNA-mediated knockdown. Interestingly, immunoprecipitation of either 5-HT R or mGluR2, using an antibody targeting epitope tags at their N-terminus, results in detection of both transcripts associated with ribonucleoprotein complexes containing RPS24. Additionally, we demonstrate that the mRNA transcripts of and associate autonomously of their respective encoded proteins. Validation of this translation-independent association is extended using mouse frontal cortex samples. Together, these findings provide mechanistic insights into the co-translational assembly of GPCR heteromeric complexes, unraveling regulatory processes governing protein-protein interactions and complex formation.
PubMed: 38948858
DOI: 10.1101/2024.06.17.599432 -
BioRxiv : the Preprint Server For... Jun 2024The intermediate filament (IF) protein vimentin is associated with many diseases with phenotypes of enhanced cellular migration and aggressive invasion through the...
The intermediate filament (IF) protein vimentin is associated with many diseases with phenotypes of enhanced cellular migration and aggressive invasion through the extracellular matrix (ECM) of tissues, but vimentin's role in in-vivo cell migration is still largely unclear. Vimentin is important for proper cellular adhesion and force generation, which are critical to cell migration; yet the vimentin cytoskeleton also hinders the ability of cells to squeeze through small pores in ECM, resisting migration. To identify the role of vimentin in collective cell migration, we generate spheroids of wide-type and vimentin-null mouse embryonic fibroblasts (mEFs) and embed them in a 3D collagen matrix. We find that loss of vimentin significantly impairs the ability of the spheroid to collectively expand through collagen networks and remodel the collagen network. Traction force analysis reveals that vimentin null spheroids exert less contractile force than their wild-type counterparts. In addition, spheroids made of mEFs with only vimentin unit length filaments (ULFs) exhibit similar behavior as vimentin-null spheroids, suggesting filamentous vimentin is required to promote 3D collective cell migration. We find the vimentin-mediated collective cell expansion is dependent on matrix metalloproteinase (MMP) degradation of the collagen matrix. Further, 3D vertex model simulation of spheroid and embedded ECM indicates that wild-type spheroids behave more fluid-like, enabling more active pulling and reconstructing the surrounding collagen network. Altogether, these results signify that VIF plays a critical role in enhancing migratory persistence in 3D matrix environments through MMP transportation and tissue fluidity.
PubMed: 38948855
DOI: 10.1101/2024.06.17.599259 -
The effectiveness of selection in a species affects the direction of amino acid frequency evolution.BioRxiv : the Preprint Server For... Jun 2024Nearly neutral theory predicts that species with higher effective population size ( ) are better able to purge slightly deleterious mutations. We compare evolution in...
UNLABELLED
Nearly neutral theory predicts that species with higher effective population size ( ) are better able to purge slightly deleterious mutations. We compare evolution in high- vs. low- vertebrates to reveal which amino acid frequencies are subject to subtle selective preferences. We take three complementary approaches, two measuring flux and one measuring outcomes. First, we fit non-stationary substitution models of amino acid flux using maximum likelihood, comparing the high- clade of rodents and lagomorphs to its low- sister clade of primates and colugos. Second, we compare evolutionary outcomes across a wider range of vertebrates, via correlations between amino acid frequencies and . Third, we dissect the details of flux in human, chimpanzee, mouse, and rat, as scored by parsimony - this also enables comparison to a historical paper. All three methods agree on which amino acids are preferred under more effective selection. Preferred amino acids tend to be smaller, less costly to synthesize, and to promote intrinsic structural disorder. Parsimony-induced bias in the historical study produces an apparent reduction in structural disorder, perhaps driven by slightly deleterious substitutions. Within highly exchangeable pairs of amino acids, arginine is strongly preferred over lysine, and valine over isoleucine, consistent with more effective selection preferring a marginally larger free energy of folding. These two preferences match differences between thermophiles and mesophilic relatives. These results reveal the biophysical consequences of mutation-selection-drift balance, and demonstrate the utility of nearly neutral theory for understanding protein evolution.
SIGNIFICANCE STATEMENT
According to the nearly neutral theory of molecular evolution, selection is less able to distinguish between similar alleles in species with lower population size. We identify which amino acids are subject to such weak preferences - these tend to be smaller, to be less costly to make, to promote structural disorder of proteins, and to be enriched in thermophiles relative to mesophiles. The latter agrees with theories of marginal protein stability under mutation-selection-drift balance.
PubMed: 38948853
DOI: 10.1101/2023.02.01.526552 -
BioRxiv : the Preprint Server For... Jun 2024The XPD/Rad3-like helicase, YoaA, and DNA polymerase III subunit, χ, are involved in DNA damage tolerance and repair. YoaA and χ promote tolerance to the DNA...
The XPD/Rad3-like helicase, YoaA, and DNA polymerase III subunit, χ, are involved in DNA damage tolerance and repair. YoaA and χ promote tolerance to the DNA chain-terminator, 3□-azidothymidine (AZT), and together form the functional helicase complex, YoaA-χ. How YoaA-χ contributes to DNA damage tolerance is not well understood. single-stranded DNA binding protein (SSB) accumulates at stalled replication forks, and the SSB-χ interaction is required to promote AZT tolerance via an unknown mechanism. YoaA-χ and SSB interactions were investigated to better understand this DNA damage tolerance mechanism, and we discovered YoaA-χ and SSB have a functional interaction. SSB confers a substrate-specific effect on the helicase activity of YoaA-χ, barely affecting YoaA-χ on an overhang DNA substrate but inhibiting YoaA-χ on forked DNA. A paralog helicase, DinG, unwinds SSB-bound DNA in a similar manner to YoaA-χ on the substrates tested. Through use of ensemble experiments, we believe SSB binds behind YoaA-χ relative to the DNA ds/ss junction and show via single-molecule assays that SSB translocates along ssDNA with YoaA-χ. This is, to our knowledge, the first demonstration of a mechanoenzyme pulling SSB along ssDNA.
PubMed: 38948847
DOI: 10.1101/2024.06.21.600097 -
BioRxiv : the Preprint Server For... Jun 2024The marine annelid is a model organism used in many research areas including evolution and development, neurobiology, ecology and regeneration. Here we present the...
The marine annelid is a model organism used in many research areas including evolution and development, neurobiology, ecology and regeneration. Here we present the genomes of and of the closely related and , to facilitate comparative genomic approaches and help explore biology. We used long-read sequencing technology and chromosomal-conformation capture along with extensive transcriptomic resources to obtain and annotate a draft genome assembly of ∼1.47 Gbp for , of which more than half represent repeat elements. We predict around 29,000 protein-coding genes, with relatively large intron sizes, over 38,000 non-coding genes, and 580 miRNA loci. We further explore the high genetic variation (∼3% heterozygosity) within the species complex. Gene ontology reveals the most variable loci to be associated with pigmentation, development and immunity. The current work sets the stage for further development of genomic resources.
PubMed: 38948846
DOI: 10.1101/2024.06.21.600153