Biology subjects

Khanal, S.

Publications and source records attributed to Khanal, S..

18 recordsLinked to original sources

bioRxiv2026

Hemoglobin C is prone to oxidative denaturation, resulting in red blood cell membrane damage in HbSC disease

Sickle-hemoglobin-C (HbSC) sickle cell disease is characterized by RBC dehydration (xerocytosis), which promotes polymerization of HbS. HbSC causes substantial morbidity despite lower sickling potential than HbSS, suggesting a critical detrimental role of HbC in the disease pathophysiology. We derived HbCC mice by interbreeding our HbSC mice, which demonstrated a similar RBC phenotype of xerocytosis as humans with HbCC. We compared RBCs from HbCC, HbSC, and HbSS mice. Oxidized ferryl (Fe4+)-Hb, and its oxidative-denaturation, which results in hemichrome formation (Heinz-bodies), was most pronounced in HbCC>HbSC>HbSS, despite significantly higher reactive oxygen species in HbSS, illustrating a higher propensity of HbC to denaturation than HbS. RBC deformability followed a similar pattern, with Elongation Index lowest in HbCC<HbSC<HbSS. Next, we determined if RBC from HbSC patients on hydroxyurea showed improved membrane damage. Hydroxyurea treatment reduced Heinz-body formation and improved RBC deformability, despite negligible/modest fetal hemoglobin (HbF) induction, compared to non-hydroxyurea HbSC controls. The antioxidant quercetin showed a similar reduction in Heinz-body burden and improvement in RBC deformability as hydroxyurea, without affecting Hb or HbF concentration, reticulocyte count, or RBC xerocytosis. HbC-driven oxidative denaturation and membrane damage represent important contributors of RBC dysfunction in HbSC disease; hence, oxidative membrane injury could be targeted besides antisickling approaches.

cell biology↗

bioRxiv2026

Subcellular Characterization of the Molecular Determinants of Ebola Virus VP40 Trafficking and Assembly

Ebola virus is a single-stranded negative-sense RNA virus that can cause severe hemorrhagic fevers in humans. Ebola virus, along with other members of the filoviridae family, produce virions with a characteristic filamentous morphology. VP40, the filovirus matrix protein, is responsible for curving the host plasma membrane (PM). Expression of VP40 and the assembly of the matrix layer results in the budding of filamentous particles. VP40 forms cytosolic homodimers via interactions in its N-terminal domain, while interactions in its C-terminal domain drive oligomerization into the 2D-crystalline matrix layer. While VP40 is expressed throughout the host cytosol and assembles on the inner leaflet of the PM, VP40 does not appear to directly bind the PM but instead requires interactions with components of the host secretory machinery. Here, we characterize a series of VP40 mutants targeted to the molecular determinants of Ebola VP40 assembly and traWicking using confocal microscopy and genetically-encoded fluorescent tags. Using this approach, we characterize the subcellular distribution of these mutants, showing novel cellular phenotypes. Several mutants previously characterized as traWicking deficient show aggregation dependent on membrane binding, suggesting a possible route of VP40 traWicking. We then co-expressed these mutants with organelle markers, which provide insights into the aWected parts of the host traWicking pathways. Together, our results provide new insights into molecular interactions that drive the assembly of the Ebola matrix layer.

molecular biology↗

bioRxiv2025

Holographic fingerprinting reveals oligomer-driven phase separation in Bovine Serum Albumin

Biomolecular condensates formed through phase separation are fundamental to cellular organization. Although the physical principles underlying intrinsically disordered proteins are well understood, the molecular determinants of condensate formation in globular proteins remain elusive. Here, we employ Holographic Particle Characterization, a label-free, high-throughput imaging technique, to investigate the self-assembly of Bovine Serum Albumin (BSA), a model globular protein. We show that this technique reliably differentiates amorphous aggregates from liquid-like condensates by their distinct refractive indices and morphologies. Coupled with size-exclusion chromatography, our analysis reveals that BSA phase separation strictly depends on higher-order oligomeric assemblies. Monomeric and dimeric fractions fail to form condensates under identical crowding conditions. Furthermore, the internal packing density of these condensates is highly tunable via pH-driven protonation changes but remains insensitive to ionic screening. These findings support a model of "emergent multivalency," where oligomerization creates a structural scaffold enabling hydrophobically stabilized phase separation, thereby defining a molecular threshold for globular proteins condensation.

biochemistry↗

bioRxiv2025

HEIP1 orchestrates pro-crossover protein activity during mammalian meiosis

Meiotic crossovers are needed to produce genetically balanced gametes. In mammals, crossover formation is mediated by a conserved set of pro-crossover proteins via mechanisms that remain unclear. Here, we characterize a mammalian pro-crossover factor HEIP1. In mouse HEIP1 is essential for crossing over and fertility of both sexes. HEIP1 promotes crossing over by orchestrating the recruitment of other pro-crossover proteins, including the MutS{gamma} complex (MSH4- MSH5) and E3 ligases (HEI10, RNF212, and RNF212B), that are required to mature crossover sites and recruit the crossover-specific resolution complex MutL{gamma}. Moreover, HEIP1 directly interacts with HEI10, suggesting a direct role in controlling the recruitment of pro-crossover E3 ligases. During early stages of meiotic prophase I, HEIP1 interacts with the chromosome axes, independently of recombination, before relocalizing to the central region of the synaptonemal complex. We propose that HEIP1 is a new conserved master regulator of crossover proteins that controls different crossover maturation steps. Significance StatementCrossovers are essential to produce gametes by promoting the proper segregation of the homologous chromosomes. But, if misregulated, they can lead to genetic disorders, miscarriage and infertility. Their formation depends on the conserved pro-crossover factors, which repertoire is expanding and mode of action tightly regulated. This study highlights how the HEIP1 protein organizes pro-crossover protein activities in the mouse. Our findings show that HEIP1, by interacting early with chromosomes independently of recombination initiation, and orchestrating the recruitment of pro-crossover factors, including the MutSg complex and the RING proteins HEI10, RNF212 and RNF212B, is pivotal in this regulation. Our work is of significance to unravel crossover control during meiotic recombination, a conserved mechanism essential for gametes formation.

genetics↗

bioRxiv2025

Two mutations in the same MYC-bHLH transcription factor cause segregation of purple coloration of stolons and seed heads in Zoysia japonica x Zoysia matrella F2 and F1 populations

Anthocyanins play diverse roles in plants, including attracting pollinators and protecting cells from oxidative damage. In zoysiagrass, a warm season turfgrass, their accumulation in seed heads and stolons can decrease the aesthetic appeal. In this study, a high-density genetic map with [~]8000 single nucleotide polymorphism (SNP) markers organized into 20 linkage groups was generated in a Zoysia japonica acc. Meyer x Zoysia matrella acc. PI 231146 F2 population. Using this genetic map, a large-effect quantitative trait locus (QTL) for anthocyanin variation in stolons and seed heads was mapped to chromosome 12 (PP locus). Variant analysis of a candidate gene for PP, Zjn_sc00004.1.g07010.1.sm.mk, which encodes a MYC-bHLH transcription factor that regulates anthocyanin biosynthesis, revealed a SNP at an exon-intron boundary in Meyer that led to intron retention. Interestingly, an F1 population derived from the same parents segregated for seed head color but uniformly displayed purple stolons. Seed head color in the F1 population co-mapped with the PP locus which, combined with genotypic and yeast two-hybrid analyses, revealed that a SNP in PI 231146 leading to an Ala163Ser substitution in the MYB-interacting N-terminal domain of the same MYC-bHLH transcription factor was likely causal. The Ala163Ser substitution affected interaction of MYC-bHLH with MYB in a MYB-dependent manner. The identified mutations can be exploited to develop cultivars with green seed heads and stolons. The high-marker-density interspecific Z. japonica x Z. matrella F2 genetic map also provides a robust tool for identifying genomic regions and genes of agronomic interest that differentiate the two species.

genetics↗

bioRxiv2025

De Novo Protocell Membrane Formation Fueled by Primitive Metabolites

Lipid membranes define cell boundaries, acting as gatekeepers for transport and signaling. A central principle in biology is that all cellular membranes descend from a common ancestral membrane, as they cannot be generated in the absence of preexisting lipid structures. It is thus unclear how the first protocell membranes originated from membrane-less precursors. Here we demonstrate the de novo generation of lipid bilayers in the absence of any preexisting membranes, membrane-bound proteins, or lipid nanostructure templates. Using acetate as a two-carbon precursor, lipid tails are constructed by soluble enzymes and spontaneously conjugate to cysteine backbones, forming diacyl lipids that assemble into vesicles. Pore-forming peptides facilitate precursor transport into vesicles, allowing the continuous generation of new lipids. Formation of glycolipid membranes creates compartments that can maintain proton gradients. The de novo formation of membrane bilayers using primitive chemical building blocks may have been an intermediary step between primitive prebiotic biochemistry and the emergence of cellular life.

synthetic biology↗

bioRxiv2025

Processing of Genomic RNAs by Dicer in Bat Cells Limits SARS-CoV-2 Replication

Bats are reservoirs for numerous viruses that cause serious diseases in other animals and humans. Several mechanisms are proposed to contribute to the tolerance of bats to these pathogens. This study investigates the response of bat cells to double-stranded RNA generated by SARS-CoV-2 replication. Here, we found the involvement of Dicer in the processing of viral genomic RNAs during SARS-CoV-2 infection. Examining RNA sequencing of infected cells, small-interfering RNA (siRNA)-like fragments were found derived from viral RNAs. Depletion of Dicer showed a reduction in these RNAs and an increase in viral loads suggesting unlike other mammals, bats may use Dicer to limit viral replication. This prompted the exploration of key dsRNA sensors in bat cells. Our analysis showed significant upregulation of OAS1 and MX1 in response to dsRNA, while PKR levels remained low, suggesting alternative dsRNA-response mechanisms are present that eschew the common PKR-based system. These results further show how bats employ distinct strategies for antiviral defense that may contribute to tolerating viral infections. They suggest the involvement of Dicer in antiviral mechanisms in bats, a function not observed in other mammals. This highlights a mechanism for bat originating viruses to evolve features that in other animals could cause extreme antiviral responses such as is seen with SARS-CoV-2. GRAPHICAL SUMMARY O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=152 SRC="FIGDIR/small/632425v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@1755d68org.highwire.dtl.DTLVardef@475cb8org.highwire.dtl.DTLVardef@1185813org.highwire.dtl.DTLVardef@13e847a_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2024

Tuning and functionalization of logic gates for time resolved population programming

In order to increase our command over genetically engineered bacterial populations in bioprocessing and therapy, synthetic regulatory circuitry needs to enable the temporal programming of a number of consecutive functional tasks without external interventions. In this context, we have engineered a genetic circuit encoding an autonomous but chemically tunable timer in Escherichia coli, based on the concept of a transcription factor cascade mediated by the cytoplasmic dilution of repressors. As proof-of-concept, we used this circuit to impose a time-resolved two-staged synthetic pathway composed of a production-followed-by-lysis program, via a single input. Moreover, via a recombinase step, this synchronous timer was further engineered into an asynchronous timer in which the generational distance of differentiating daughter cells spawning off from a stem-cell like mother cell becomes a predictable driver and proxy for timer dynamics. Using this asynchronous timer circuit, a temporally defined population heterogeneity can be programmed in bacterial populations. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=116 SRC="FIGDIR/small/593743v2_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@bfccf4org.highwire.dtl.DTLVardef@28ce89org.highwire.dtl.DTLVardef@10fa754org.highwire.dtl.DTLVardef@b2a666_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2023

Pyruvate Oxidation Sustains B Cell Antigen-Specific Activation to Exacerbate MASH

B cells play a crucial role in the pathogenesis of metabolic dysfunction-associated steatohepatitis (MASH), a severe form of steatotic liver disease that if persistent can lead to cirrhosis, liver failure, and cancer. Chronic inflammation and fibrosis are key features of MASH that determine disease progression and outcomes. Recent advances have revealed that pathogenic B cell-derived cytokines and antibodies promote the development of MASH. However, the mechanisms through which B cells promote fibrosis and the metabolic adaptations underlying their pathogenic responses remain unclear. Here, we report that a subset of mature B cells with heightened cytokine responses accumulate in the liver and promote inflammation in MASH. To meet the increased energetic demand of effector responses, B cells increase their ATP production via oxidative phosphorylation (OXPHOS) fueled by pyruvate oxidation in a B cell receptor (BCR)-specific manner. Blocking pyruvate oxidation completely abrogated the inflammatory capacity of MASH B cells. Accordingly, the restriction of the BCR led to MASH attenuation, including reductions in steatosis, hepatic inflammation, and fibrosis. Mechanistically, BCR restriction decreased B cell maturation, activation, and effector responses in the liver, accompanied by decreased T cell- and macrophage-mediated inflammation. Notably, attenuated liver fibrosis in BCR-restricted mice was associated with lower IgG production and decreased expression of Fc-gamma receptors on hepatic stellate cells. Together, these findings indicate a key role for B cell antigen-specific responses in promoting steatosis, inflammation, and fibrosis during MASH.

physiology↗

bioRxiv2023

Characterization of Differences of Seed Endophytic Microbiome in Conventional and Organic Rice by Amplicon-based Sequencing and Culturing Methods

The seed serves as the primary source of microbial inoculum for plant microbiota, playing a crucial role in establishing microbial populations in plants across subsequent generations, ultimately impacting plant growth and its overall health. Cropping conditions, especially farming practices, can influence the composition and functionality of the seed microbiome. Very little is known about the differences in seed microbiome between organic and conventional production systems. In this study, we characterized the endophytic microbial populations in seeds of rice grown under organic and conventional management practices through culture-dependent and independent analyses. The V4 region of 16S rRNA was used for bacterial taxa identification, and the ITS1 region was used in the identification of fungal taxa. Our results revealed significantly higher Shannon and Simpson indices for bacterial diversity in the conventional farming system whereas the fungal diversity was higher for observed, Shannon, and Simpson indices in the organic farming system. The cultivable endophytic bacteria were isolated and identified by the full-length 16S rRNA gene. There was no difference in culturable endophytic bacterial isolates in rice seeds grown under both conventional and organic farming systems. Among 33 unique isolates tested in vitro, three bacteria Bacillus sp. ST24, Burkholderia sp. OR5, and Pantoea sp. ST25, showed antagonistic activities against Marasmius graminum, Rhizoctonia solani AG4, and R. solani AG11, the fungal pathogens causing rice seedling blight. IMPORTANCEIn this paper, we studied the differences in the endophytic microbial composition of rice seeds grown in conventional and organic farming systems. Our results demonstrate a greater bacterial diversity in conventional farming, while organic farming showcases a higher fungal diversity. Additionally, our research reveals the ability of seed bacterial endophytes to inhibit the growth of three fungal pathogens responsible for causing seedling blight in rice. This study provides valuable insights into the potential use of beneficial seed microbial endophytes for developing a novel microbiome-based strategy in the management rice diseases. Such an approach has the potential to enhance overall plant health and improve crop productivity.

microbiology↗

bioRxiv2023

Deletion of smooth muscle O-GlcNAc transferase prevents development of atherosclerosis in western diet-fed hyperglycemic ApoE-/- mice in vivo

Accumulating evidence highlights protein O-GlcNAcylation as a putative pathogenic contributor of diabetic vascular complications. We previously reported that elevated protein O-GlcNAcylation correlates with increased atherosclerotic lesion formation and VSMC proliferation in response to hyperglycemia. However, the role of O-GlcNAc transferase (OGT), regulator of O-GlcNAc signaling, in evolution of diabetic atherosclerosis remains elusive. The goal of this study was to determine whether smooth muscle OGT (smOGT) plays a direct role in hyperglycemia-induced atherosclerotic lesion formation and SMC de-differentiation. Using tamoxifen-inducible Myh11-CreERT2 and OGTfl/fl mice, we generated smOGTWT and smOGTKO mice, with and without ApoE-null backgrounds. Following STZ-induced hyperglycemia, smOGTWT and smOGTKO mice were kept on standard laboratory diet for study duration. In a parallel study, smOGTWTApoE-/- and smOGTKOApoE-/- were kept on Western diet beginning 8-wks-age. Animals harvested at 14-16-wks-age were used for plasma and tissue collection. Loss of smOGT augmented SM contractile marker expression in aortic vessels of STZ-induced hyperglycemic smOGTKO mice. Consistently, smOGT deletion attenuated atherosclerotic lesion lipid burden (Oil red O), plaque area (H&E), leukocyte (CD45) and smooth muscle cell (ACTA2) abundance in Western diet-induced hyperglycemic smOGTKOApoE-/- mice. This was accompanied with increased SM contractile markers, and reduced inflammatory and proliferative marker expression. Further, smOGT deletion attenuated YY1 and SRF expression (transcriptional regulators of SM contractile genes) in hyperglycemic smOGTKOApoE-/- and smOGTKO mice. These data uncover an atheroprotective outcome of smOGT loss-of-function and suggest a direct regulatory role of OGT-mediated O-GlcNAcylation in VSMC de-differentiation in hyperglycemia.

pathology↗

bioRxiv2023

Centriole Remodeling Evolved into Centriole Degradation in Mouse Sperm

Centrioles are subcellular organelles found at the cilia base with an evolutionarily conserved structure and a shock absorber-like function. In sperm, centrioles are found at the flagellum base and are essential for embryo development in basal animals. Yet, sperm centrioles have evolved diverse forms, sometimes acting like a transmission system, as in cattle, and sometimes becoming dispensable, as in house mice. How the essential sperm centriole evolved to become dispensable in some organisms is unclear. Here, we test the hypothesis that this transition occurred through a cascade of evolutionary changes to the proteins, structure, and function of sperm centrioles and was possibly driven by sperm competition. We found that the final steps in this cascade are associated with a change in the primary structure of the centriolar luminal protein FAM161A in rodents. This information provides the first insight into the molecular mechanisms and adaptive evolution underlying a major evolutionary transition within the internal structure of the mammalian sperm neck. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=65 SRC="FIGDIR/small/532086v3_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@bdbe1eorg.highwire.dtl.DTLVardef@10cc2b9org.highwire.dtl.DTLVardef@c42734org.highwire.dtl.DTLVardef@1e9c750_HPS_FORMAT_FIGEXP M_FIG C_FIG Highlights- Barrel-shaped centrioles are present in all rodents except members of the Muridae taxonomical family. - The FAM161A sequence evolved selectively in rodents and specifically in a murid subgroup. - The evolution of centriole degradation is correlated with the expression of a novel FAM161A isoform. - The ancestral centriole remodeling program involves elongation followed by redistribution. - The mouse centriole degradation program involves elongation followed by elimination.

cell biology↗

bioRxiv2023

Maternal hormones engage a dormant monomorphic aggression circuit, leading to the introduction of an innate behavior in adulthood in females

Aggression, a sexually dimorphic behaviour, is prevalent in males and typically absent in virgin females. Following parturition, however, the transient expression of aggression in adult female mice protects pups from predators and infanticide by male conspecifics. While maternal hormones are known to elicit nursing, their potential role in maternal aggression remains elusive. Here, we show in mice that a molecularly defined subset of ventral premammillary (PMvDAT) neurons, instrumental for intermale aggression, switch from quiescence to a hyperexcitable state during lactation. We identify that the maternal hormones prolactin and oxytocin excite these cells through actions that include T-type Ca2+ channels. Optogenetic manipulation or genetic ablation of PMvDAT neurons profoundly affects maternal aggression, while activation of these neurons impairs the expression of non-aggression-related maternal behaviours. This work identifies a monomorphic neural substrate that can incorporate hormonal cues to enable the transient expression of a dormant behavioural program in lactating females.

neuroscience↗

bioRxiv2022

Interferon receptor gene dosage determines multiple hallmarks of Down syndrome

Trisomy 21 causes Down syndrome, a condition characterized by cognitive impairments, immune dysregulation, and atypical morphogenesis. Using whole blood transcriptome analysis, we demonstrate that specific overexpression of four interferon receptors encoded on chromosome 21 associates with chronic interferon hyperactivity and systemic inflammation in Down syndrome. To define the contribution of interferon receptor overexpression to Down syndrome phenotypes, we used genome editing to correct interferon receptor gene dosage in mice carrying triplication of a large genomic region orthologous to human chromosome 21. Normalization of interferon receptor copy number attenuated lethal antiviral responses, prevented heart malformations, decreased developmental delays, improved cognition and normalized craniofacial anomalies. Therefore, interferon receptor gene dosage determines major hallmarks of Down syndrome, indicating that trisomy 21 elicits an interferonopathy amenable to therapeutic intervention. One-Sentence SummaryCorrection of interferon receptor gene dosage rescues multiple key phenotypes in a mouse model of trisomy 21.

genetics↗

bioRxiv2021

Chemoenzymatic Generation of Phospholipid Membranes Mediated by Type I Fatty Acid Synthase

The de novo formation of lipid membranes from minimal reactive precursors is a major goal in synthetic cell research. In nature, the synthesis of membrane phospholipids is orchestrated by numerous enzymes, including fatty acid synthases and membrane-bound acyltransferases. However, these enzymatic pathways are difficult to fully reproduce in vitro. As such, the reconstitution of phospholipid membrane synthesis from simple metabolic building blocks remains a challenge. Here, we describe a chemoenzymatic strategy for lipid membrane generation that utilizes a soluble bacterial fatty acid synthase (cgFAS I) to synthesize palmitoyl-CoA in situ from acetyl-CoA and malonyl-CoA. The fatty acid derivative spontaneously reacts with a cysteine-modified lysophospholipid by native chemical ligation (NCL), affording a non-canonical amidophospholipid that self-assembles into micron-sized membrane-bound vesicles. To our knowledge, this is the first example of reconstituting phospholipid membrane formation directly from acetyl-CoA and malonyl-CoA precursors. Our results demonstrate that combining the specificity and efficiency of a type I fatty acid synthase with a highly selective bioconjugation reaction provides a biomimetic route for the de novo formation of membrane-bound vesicles.

synthetic biology↗

bioRxiv2020

Transcriptional control of DNA repair networks by CDK7 regulates sensitivity to radiation in Myc-driven Medulloblastoma

Myc-driven Medulloblastoma remains a major therapeutic challenge due to frequent metastasis and a poor 5-year survival rate. Myc gene amplification results in transcriptional dysregulation, proliferation, and survival of malignant cells. To identify therapeutic targets in Myc-amplified medulloblastoma we performed a CRISPR-Cas9 essentiality screen targeting 1140 genes annotated as the druggable genome. CDK7 was identified as a mediator of medulloblastoma tumorigenesis. Using covalent inhibitors and genetic depletion of CDK7 we observe the cessation of tumor growth in xenograft mouse models and increase in apoptotic mechanisms. The results are attributed to repression of a core set of Myc-driven transcriptional programs mediating DNA repair. We further establish that blocking CDK7 activity sensitizes cells to ionizing radiation leading to accrual of DNA damage and extended survival and tumor latency in medulloblastoma xenograft mouse models. Our studies establish a mechanism for selective inhibition of Myc-driven MB by CDK7 inhibition combined with radiation as a viable therapeutic strategy for Myc-amplified medulloblastoma.

cancer biology↗

bioRxiv2018

High-Risk Human Papillomavirus Oncogenes Disrupt the Fanconi Anemia DNA repair Pathway by Impairing Localization and De-ubiquitination of FancD2

Persistent expression of high-risk HPV oncogenes is necessary for the development of anogenital and oropharyngeal cancers. Here, we show that E6/E7 expressing cells are hypersensitive to DNA crosslinking agent cisplatin and have defects in repairing DNA interstrand crosslinks (ICL). Importantly, we elucidate how E6/E7 attenuate the Fanconi anemia (FA) DNA crosslink repair pathway. Though E6/E7 activated the pathway by increasing FancD2 monoubiquitination and foci formation, they inhibited the completion of the repair by multiple mechanisms. E6/E7 impaired FancD2 colocalization with double-strand breaks (DSB), which subsequently hindered the recruitment of downstream protein Rad51 to DSB in E6 cells. Further, E6 expression caused delayed FancD2 de-ubiquitination, an important process for effective ICL repair. Delayed FancD2 de-ubiquitination was attributed to the increased chromatin retention of FancD2 hindering USP1 de-ubiquitinating activity, and persistently activated ATR/CHK-1/pS565 FancI signaling. E6 mediated p53 degradation did not hamper the cell cycle specific process of FancD2 modifications but abrogated repair by disrupting FancD2 de-ubiquitination. Further, E6 reduced the expression and foci formation of Palb2, which is a repair protein downstream of FancD2. These findings uncover unique mechanisms by which HPV oncogenes contribute to genomic instability and the response to cisplatin therapies.\n\nAUTHOR SUMMARYHigh-risk human papillomavirus (HPV) causes nearly all cervical and many other anogenital cancers, and oropharyngeal cancers. As cisplatin is the most commonly used drug for cervical and HPV-associated oropharyngeal cancers, it is important to understand how HPV oncogenes disrupt the Fanconi anemia (FA) pathway involved primarily in the repair of cisplatin-induced DNA crosslinks. However, the mechanism by which HPV E6 and E7 attenuate the FA pathway is poorly understood. We demonstrate that E6/E7 expression disrupts crosslink repair and increase cisplatin sensitivity, and attenuate the FA pathway through multiple unique mechanisms. First, E6/E7 causes accumulation of FancD2, a central component of the FA pathway, at the sites away from DNA damage. This results in reduced recruitment of Rad51, another repair protein involved in the pathway. Second, E6 causes delayed FancD2 de-ubiquitination, an important process for effective repair. Third, E6 expressing cells decreases the expression and foci formation of Palb2 repair protein. Together, this work elucidates the mechanisms by which HPV attenuates the repair of DNA crosslinks increasing cisplatin cytotoxicity and efficacy in treating HPV-positive cancers.

cancer biology↗

bioRxiv2017

Identification of putative G-quadruplex forming sequences in three manatee papillomaviruses

The Florida manatee (Trichechus manatus latirotris) is considered a threatened aquatic mammal in United States coastal waters. Over the past decade, the appearance of papillomavirus-induced lesions and viral papillomatosis in manatees has been a concern for those involved in the management and rehabilitation of this species. To date, three manatee papillomaviruses (PVs) have been identified in Florida manatees, one forming cutaneous lesions (TmPV1) and two forming genital lesions (TmPV3 and TmPV4). In this study, we identified DNA sequences with the potential to form G-quadruplex structures in all three PVs. G-quadruplex structures (G4) are guanine-rich nucleic acid sequences capable of forming secondary structures in DNA and RNA. In humans, G4 are known to regulate molecular processes such as transcription and translation. Although G4 have been identified in several viral genomes, including human PVs, no attempt has been made to identify G4 in animal PVs. We found that sequences capable of forming G4 were present on both DNA strands and across coding and non-coding regions on all PVs. The vast majority of the identified sequences would allow the formation of non-canonical structures with only two G-tetrads. The formation of one such structure was supported through biophysical analysis. Computational analysis demonstrated enrichment of G4 sequences on the reverse strand in the E2/E4 region on all manatee PVs and on the forward strand in the E2/E4 region on one genital PV. Several G4 sequences occurred at similar regional locations on all PVs, most notably on the reverse strand in the E2 region. In other cases, G4 were identified at similar regional locations only on PVs forming genital lesions. On all PVs, G4 sequences were located near putative E2 binding sites in the non-coding region. Together, these findings suggest that G4 are likely regulatory elements in manatee PVs.\n\nAuthor summaryG-quadruplex structures (G4) are found in the DNA and RNA of many species and are known to regulate the expression of genes and the synthesis of proteins, among other important molecular processes. Recently, these structures have been identified in several viruses, including the human papillomavirus (PV). As regulatory structures, G4 are of great interest to researchers as drug targets for viral control. In this paper, we identify the first G4 sequences in three PVs infecting a non-human animal, the Florida manatee. Through computational and biophysical analysis, we find that a greater variety of sequence patterns may underlie the formation of these structures than previously identified. The sequences are found in all protein coding regions of the virus and near sites for viral replication in non-coding regions. Furthermore, the distribution of these sequences across the PV genomes supports the notion that sequences are conserved across PV types, suggesting they are under selective pressure. This paper extends previous research on G4 in human PVs with additional evidence for their role as regulators. The G4 sequences we identified also provide potential regulatory targets for researchers interested in controlling this virus in the Florida manatee, a threatened aquatic mammal.

molecular biology↗