Biology subjects

Papavasiliou, F. N.

Publications and source records attributed to Papavasiliou, F. N..

14 recordsLinked to original sources

bioRxiv2025

RNA modifications on Adenosine co-Regulate Macrophage Function

Macrophages are a highly plastic innate immune cell subset that depends on environmental cues to activate, execute and resolve inflammatory responses. This plasticity of function is mirrored by the diversity of RNA modifications that dynamically decorate macrophage transcripts. Here, using the mouse macrophage line RAW 264.7 (RAW), we addressed the combinatorial effect of two major mRNA modifications: adenosine to inosine (A-to-I) deamination by ADAR1 and adenosine N6-methylation (m6A) by METTL3. Using both short-read and single molecule sequencing on RAW macrophages with genetic deletions of ADAR1 or METTL3, we identified transcripts that were modified by both enzymes, with specific functional outcomes on macrophage activation. While m6A levels remained relatively stable even in the absence of ADAR1, loss of METTL3 led to a global reduction in A-to-I editing levels. This interrelation was most apparent when m6A sites were distant from sites of deamination, suggesting a possible function of m6A in ADAR1-mediated editing. Using a dual reporter cell line where guided ADAR1 recruitment can be measured via eGFP reactivation, we observed that m6A modification of ADAR-engager guide RNAs substantially improved targeted RNA editing. Overall, we report the first example of an interdependence between modifications, which can also be therapeutically exploited.

immunology↗

bioRxiv2023

Neoepitope formation through the generation of RNA-derived "editopes"

Increasing the quantity and immunogenicity of neoantigens in tumors is essential for advancing immunotherapy. However, engineering neoantigens remains challenging due to the need for precise, tumor-specific antigen modification without affecting normal cells. To tackle this challenge, we developed Short Precise-Encodable ADAR Recruiting (SPEAR) ADAR-engagers, an approach that uses short guide RNAs to engage the endogenous RNA editor ADAR1 and direct it to regions of mRNA targets known to encode MHC-presented peptides. By precisely editing adenosine-to-inosine (A-to-I) in these contexts, we effectively mutate specific epitopes into neoepitopes (which we now term "editopes"). As proof of concept, we targeted the known antigen MART-1 (Melanoma-Associated Antigen Recognized by T cells-1), and demonstrated that guided ADAR1 editing can generate immunogenic epitopes that activate T cells and promote tumor cell elimination. Building on this concept, we developed a computational pipeline to identify tumor-specific somatic mutations suitable for SPEAR-mediated editing. This strategy enables selective neoantigen generation in cancer cells, effectively increasing their apparent tumor mutational burden and potentially enhancing their susceptibility to immunotherapy.

cancer biology↗

bioRxiv2023

A Structural Classification of the Variant Surface Glycoproteins of the African Trypanosome

Long-term immune evasion by the African trypanosome is achieved through repetitive cycles of surface protein replacement with antigenically distinct versions of the dense Variant Surface Glycoprotein (VSG) coat. Thousands of VSG genes and pseudo-genes exist in the parasite genome that, together with genetic recombination mechanisms, allow for essentially unlimited immune escape from the hosts adaptive immune system. The diversity space of the "VSGnome" at the protein level was thought to be limited to a few related folds whose structures were determined more than 30 years ago. However, recent progress has shown that the VSGs possess significantly more architectural variation than had been appreciated. Here we combine experimental X-ray crystallography with deep-learning structural prediction using Alphafold to produce models of hundreds of VSG proteins. We classify the VSGnome into groups based on protein architecture and oligomerization state, contextualize recent bioinformatics clustering schemes, and extensively map VSG-diversity space. We demonstrate that in addition to the structural variability and post-translational modifications observed thus far, VSGs are also characterized by variations in oligomerization state and possess inherent flexibility and alternative conformations, lending additional variability to what is exposed to the immune system. Finally, these additional experimental structures and the hundreds of Alphafold predictions confirm that the molecular surfaces of the VSGs remain distinct from variant to variant, supporting the hypothesis that protein surface diversity is central to the process of antigenic variation used by this organism during infection.

microbiology↗

bioRxiv2022

Structural and Immunological Similarities Between the Metacyclic and Bloodstream Form Variant Surface Glycoproteins of the African Trypanosome

During infection of mammalian hosts, African trypanosomes thwart immunity using antigenic variation of the dense Variant Surface Glycoprotein (VSG) coat, accessing a large repertoire of thousands of genes and pseudogenes and switching to antigenically distinct copies. The parasite is transferred to mammalian hosts through the bite of the tsetse fly. In the salivary glands of the fly, the pathogen adopts the metacyclic form and expresses a limited repertoire of VSG genes specific to that developmental stage. It has remained unknown whether the metacyclic VSGs possess distinct properties associated with this particular and discrete phase of the parasite life cycle. We show here using bioinformatic, crystallographic, and immunological analyses of three metacyclic VSGs that they closely mirror the known classes of bloodstream form VSGs both in structure and in the immunological responses they elicit.

microbiology↗

bioRxiv2022

Acute expression of human APOBEC3B in mice causes lethality associated with RNA editing

RNA editing has been described to promote heterogeneity leading to the development of multiple disorders including cancer. The cytosine deaminase APOBEC3B is known to fuel tumor evolution through DNA mutagenesis, but whether it may also function as an RNA editing enzyme has not been studied. Here, we engineered a novel doxycycline-inducible mouse model of human APOBEC3B-overexpression to understand the impact of this enzyme in tissue homeostasis and address a potential role in C-to-U RNA editing. Elevated and sustained levels of APOBEC3B led to rapid alteration of cellular fitness, major organ dysfunction, and ultimately lethality in mice. Importantly, extensive analyses of RNA-sequencing and WES from mouse tissues expressing high APOBEC3B levels reveal frequent UCC-to-UUC RNA editing events mainly localized in a specific hotspot. This work identifies, for the first time, a new function for APOBEC3B in RNA editing and presents a valuable preclinical tool to understand the emerging role of APOBEC3B as a potent driver of cancer and other diseases.

molecular biology↗

bioRxiv2022

An Epitope-Focused Trypanosome-Derived Vaccine Platform Elicits High-Affinity Antibodies and Immunity Against Fentanyl Effects

Poorly antigenic small molecules pose challenges for the production of clinically efficacious antibodies. To address this, we have developed an immunization platform derived from the antigenic surface coat of the African trypanosome. Through sortase-based conjugation of antigens to the trypanosome surface coat protein variant surface glycoprotein (VSG), we created the VAST (VSG-immunogen Array by Sortase Tagging). We used VAST to elicit protective immunity to the effects fentanyl. Immunizing mice with fentanyl-VAST generated serological memory and protection from fentanyl challenge. Employing single-cell RNAseq, we then developed a streamlined method that synergizes with the VAST to identify immunization-elicited memory B cells and the antibodies they encode. All antibodies selected by this method displayed picomolar affinities for fentanyl. Passive immunization protected animals from fentanyl, while crystallography revealed that the mAbs bind fentanyl in an unusually deep pocket suited to small molecules, demonstrating the ability of the VAST to elicit high-quality therapeutic antibodies.

immunology↗

bioRxiv2021

Immunodominant surface epitopes power immune evasion in the African trypanosome

The African trypanosome survives the immune response of its mammalian host by antigenic variation of its major surface antigen (the Variable Surface Glycoprotein, or VSG). Here we describe the antibody repertoires elicited by different VSGs. We show that the repertoires are highly restricted, and are directed predominantly to distinct epitopes on the surface of the VSGs. They are also highly discriminatory: minor alterations within these exposed epitopes confer antigenically-distinct properties to these VSGs and elicit different repertoires. We propose that the patterned and repetitive nature of the VSG coat focuses host immunity to a restricted set of immunodominant epitopes per VSG, eliciting a highly stereotyped response, minimizing cross reactivity between different VSGs and facilitating prolonged immune evasion through epitope variation.

immunology↗

bioRxiv2021

SARS-CoV-2 variant evolution in the United States: High accumulation of viral mutations over time likely through serial Founder Events and mutational bursts.

Since the first case of COVID-19 in December 2019 in Wuhan, China, SARS-CoV-2 has spread worldwide and within a year has caused 2.29 million deaths globally. With dramatically increasing infection numbers, and the arrival of new variants with increased infectivity, tracking the evolution of its genome is crucial for effectively controlling the pandemic and informing vaccine platform development. Our study explores evolution of SARS-CoV-2 in a representative cohort of sequences covering the entire genome in the United States, through all of 2020 and early 2021. Strikingly, we detected many accumulating Single Nucleotide Variations (SNVs) encoding amino acid changes in the SARS-CoV-2 genome, with a pattern indicative of RNA editing enzymes as major mutators of SARS-CoV-2 genomes. We report three major variants through October of 2020. These revealed 14 key mutations that were found in various combinations among 14 distinct predominant signatures. These signatures likely represent evolutionary lineages of SARS-CoV-2 in the U.S. and reveal clues to its evolution such as a mutational burst in the summer of 2020 likely leading to a homegrown new variant, and a trend towards higher mutational load among viral isolates, but with occasional mutation loss. The last quartile of 2020 revealed a concerning accumulation of mostly novel low frequency replacement mutations in the Spike protein, and a hypermutable glutamine residue near the putative furin cleavage site. Finally, the end of the year data revealed the presence of known variants of concern including B.1.1.7, which has acquired additional Spike mutations. Overall, our results suggest that predominant viral sequences are dynamically evolving over time, with periods of mutational bursts and unabated mutation accumulation. This high level of existing variation, even at low frequencies and especially in the Spike-encoding region may be become problematic when superspreader events, akin to serial Founder Events in evolution, drive these rare mutations to prominence. AUTHOR SUMMARYThe pandemic of coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has caused the death of more than 2.29 million people and continues to be a severe threat internationally. Although simple measures such as social distancing, periodic lockdowns and hygiene protocols were immediately put into force, the infection rates were only temporarily minimized. When infection rates exploded again new variants of the virus began to emerge. Our study focuses on a representative set of sequences from the United States throughout 2020 and early 2021. We show that the driving force behind the variants of public health concern, is widespread infection and superspreader events. In particular, we show accumulation of mutations over time with little loss from genetic drift, including in the Spike region, which could be problematic for vaccines and therapies. This lurking accumulated genetic variation may be a superspreader event from becoming more common and lead to variants that can escape the immune protection provided by the existing vaccines.

genetics↗

bioRxiv2021

Nanobody Mediated Macromolecular Crowding Induces Membrane Fission and Remodeling in the African Trypanosome

The dense Variant Surface Glycoprotein (VSG) coat of African trypanosomes represents the primary host-pathogen interface. Antigenic variation prevents clearing of the pathogen by employing a large repertoire of antigenically distinct VSG genes, thus neutralizing the hosts antibody response. To explore the epitope space of VSGs, we generated anti-VSG nanobodies and combined high-resolution structural analysis of VSG-nanobody complexes with binding assays on living cells, revealing that these camelid antibodies bind deeply inside the coat. One nanobody caused rapid loss of cellular motility, possibly due to blockage of VSG mobility on the coat, whose rapid endo-and exocytosis is mechanistically linked to T. brucei propulsion and whose density is required for survival. Electron microscopy studies demonstrated that this loss of motility was accompanied by rapid formation and shedding of nanovesicles and nanotubes, suggesting that increased protein crowding on the dense membrane can be a driving force for membrane fission in living cells.

microbiology↗

bioRxiv2020

The trypanosome Variant Surface Glycoprotein mRNA is stabilized by an essential unconventional RNA-binding protein

Salivarian trypanosomes cause human sleeping sickness and economically important livestock diseases. The "bloodstream forms", which replicate extracellularly in the blood and tissue fluids of mammals, are coated by a monolayer of Variant Surface Glycoprotein (VSG). Switching of the expressed VSG gene is central to parasite pathogenicity because it enables the parasites to evade adaptive immunity via antigenic variation. Adequate levels of VSG expression - 10% of total protein and 7% of mRNA - are attained through very active RNA polymerase I transcription, efficient mRNA processing (trans splicing of a capped leader and polyadenylation), and high mRNA stability. We here show how VSG mRNA stability is maintained. Purification of the VSG mRNA with associated proteins specifically selected CFB2, an F-box mRNA-binding protein that lacks known RNA-binding domains. CFB2 binds to a stabilizing complex (MKT1-PBP1-XAC1-LSM12) that recruits poly(A) binding protein and a specialized cap-binding translation initiation complex, EIF4E6-EIF4G5. The interaction of CFB2 with MKT1 is essential for CFB2s expression-promoting activity, while the F-box auto-regulates CFB2 abundance via interaction with SKP1, a component of the ubiquitination machinery. The results of reporter experiments indicate that CFB2 acts via conserved sequences in the VSG mRNA 3-untranslated region. Depletion of CFB2 leads to highly specific loss of VSG mRNA. VSG expression is essential not only for antigenic variation but also for trypanosome cell division. Correspondingly, depletion of CFB2 causes cell cycle arrest, dramatic morphological abnormalities and trypanosome death.

molecular biology↗

bioRxiv2020

APOBEC2 binds Chromatin and Represses Transcription during Myoblast Differentiation

The activation induced cytidine deaminase/apolipoprotein B editing complex (AID/APOBEC) family comprises several nucleic acid editors with roles ranging from antibody diversification to mRNA editing. APOBEC2, an evolutionarily conserved member of this family, has neither an established substrate nor a mechanism of action, however genetic evidence suggests functional relevance in tissues such as muscle. Here, we demonstrate that in muscle, APOBEC2 does not have any of the attributed molecular functions of the AID/APOBEC family, such as RNA editing, DNA demethylation, or DNA mutation. Instead, we show that APOBEC2 occupies chromatin at promoter regions of certain genes, whose expression is repressed during muscle cell differentiation. We further demonstrate that APOBEC2 on one hand binds promoter region DNA directly and in a sequence specific fashion, while on the other it interacts with HDAC transcriptional corepressor complexes. Therefore, APOBEC2, by actively repressing the expression of non-myogenesis pathway genes, plays a key role in enforcing the proper establishment of muscle cell fate.

molecular biology↗

bioRxiv2020

A Parasite Coat Protein Binds Suramin to Confer Drug Resistance

Suramin has been a primary early-stage treatment for African trypanosomiasis for nearly one hundred years. Recent studies revealed that trypanosome strains that express the Variant Surface Glycoprotein VSGsur possess heightened resistance to suramin. We show here that VSGsur binds tightly to suramin, other VSGs do not, and that together with VSG13 it defines a structurally divergent subgroup of these coat proteins. The co-crystal structure of VSGsur with suramin reveals that the chemically symmetric drug binds within a large cavity in the VSG homodimer asymmetrically, primarily through contacts of its central benzene rings. Structure-based, loss-of-contact mutations in VSGsur significantly decrease the affinity to suramin and lead to a loss of the resistance phenotype. Altogether, these data show that the resistance phenotype is dependent on the binding of suramin to VSGsur, establishing that the VSG proteins can possess functionality beyond their role in antigenic variation.

microbiology↗

bioRxiv2020

ADAR1 can drive Multiple Myeloma progression by acting both as an RNA editor of specific transcripts and as a DNA mutator of their cognate genes

RNA editing is an epitranscriptomic modification of emerging relevance to disease development and manifestations. ADAR1, which resides on human chromosome 1q21, is an RNA editor whose over-expression, either by interferon (IFN) induction or through gene amplification, is associated with increased editing and poor outcomes in Multiple Myeloma (MM). Here we explored the role of ADAR1 in the context of MM progression, by focusing on a group of 23 patients in the MMRF CoMMpass Study for which RNAseq and WES datasets exist for matched pre-and post-relapse samples. Our analysis reveals an acquisition of new DNA mutations on disease progression at specific loci surrounding the sites of ADAR associated (A-to-I) RNA editing. These analyses suggest that the RNA editing enzyme ADAR1 can function as a DNA mutator during Multiple Myeloma (MM) progression, and further imply that guide-targeted RNA editing has the capacity to generate specific mutational signatures at predetermined locations. This dual role of RNA editor and DNA mutator might be shared by other deaminases, such as APOBECs, so that DNA mutation might be the result of collateral damage on the genome by an editing enzyme whose primary job is to re-code the cognate transcript toward specific functional outcomes.

genomics↗

bioRxiv2018

Genetic loss of the ubiquitin ligase RNF126, an AID interacting partner and modifier, affects strand targeting during somatic hypermutation of antibody genes

Activation-induced cytidine deaminase (AID) initiates somatic hypermutation (SHM) and class switch recombination (CSR) in B lymphocytes by catalyzing the introduction of deoxyuracil: deoxyguanine mismatches into the DNA of the transcribed Ig locus. Repair pathways then process these mismatches to produce point mutations in the Ig variable region or double-stranded DNA breaks in the switch region followed by deletional recombination. It has been suggested that post-translational modifications on AID mediate a number of these different decisions, ranging from global targeting (Ig vs the genome), local targeting (variable vs switch region; transcribed vs non-transcribed strand) as well as process-appropriate DNA repair. Here we demonstrate that absence of RNF126, an E3 ligase shown to mono-ubiquitylate AID, results in a specific strand targeting defect in SHM, producing substantial G>C bias; strickingly, loss of RNF126 was also associated with tandem indels within the variable region (JH4 intron) but only a slight increase in the types of chromosomal translocations that are characteristic of deregulated AID. Conversely, these findings suggest that mono-ubiquitination of AID, likely in situ, is necessary for the proper removal of the protein from the non-transcribed strand, thus producing both optimal patterns of SHM and also limiting the number of indels within the target locus.

immunology↗