Biology subjects

Silver, P. A.

Publications and source records attributed to Silver, P. A..

At least 19 recordsLinked to original sources

bioRxiv2026

The transition to postpartum lactation establishes a state of maternal antiviral resilience

Influenza A virus (IAV) causes severe disease during late pregnancy, yet how maternal adaptations in host defense shape susceptibility following childbirth remains unknown. Here, we show that the postpartum period represents a state of enhanced maternal antiviral immunity. Across postpartum stages, dams infected before weaning exhibited increased survival and reduced disease following lethal IAV challenge compared with nonpregnant females. Postpartum protection was associated with enhanced viral clearance and increased pulmonary antigen-specific CD8+ T cells and IL-17A-producing gamma-delta T cells. We identify the transition into lactation as a critical determinant of this protection. Protection extended to highly pathogenic avian influenza. Together, these findings reveal that maternal antiviral immunity remains actively shaped after childbirth, establishing the postpartum lactational period as a distinct state of maternal antiviral resilience.

immunology↗

bioRxiv2026

Engineering Biosensors to Enhance Monoterpene Indole Alkaloid Production in Yeast

Monoterpene Indole Alkaloids (MIAs) are a diverse family of plant natural products with various medicinal applications. Although MIAs, such as vinblastine and reserpine, are clinically validated, sourcing of MIAs for clinical use or drug discovery from natural resources or via chemical synthesis is hampered due to their scarcity and chemical complexity. Refactoring MIA biosynthesis pathways in microbial cell factories could offer an alternative, more stable and potentially sustainable manufacturing route for alkaloid medicines and novel therapies. However, reaching commercially attractive titers, rates and yields remains challenging owing to the length and complexity of these metabolic pathways. One critical bottleneck is the low screening throughput and very high cost of the analytical methods used to quantify MIA for optimizing production. In this study, we evolved RamR, a promiscuous bacterial transcription factor to respond to five different MIAs, resulting in highly sensitive and selective sensor variants (EC50<10 M). X-ray crystallography and computational modeling provided insight into the MIA binding of the evolved biosensor variants. The RamR biosensing platform was functionalized in yeast and subsequently applied in a cost-effective semi-throughput screening campaign of a 188-gene overexpression library to identify high-performing cell factory designs for strictosidine, the common precursor for all MIAs. The fluorescent biosensor signal correlated with HPLC quantification (r2 = 0.932) allowing identification of single metabolic engineering hits which when combined yielded a maximum titer of >220 mg/L strictosidine, 3-fold higher than the parental reference strain. This study demonstrates the development of selective biosensors for MIAs and the cost-effective identification of novel metabolic engineering hits for optimizing MIA production in microbial cell factories.

synthetic biology↗

bioRxiv2025

LigifyDB: An interactive platform for predicted small molecule biosensors

Prokaryotic transcription factors (TFs) are used as small molecule biosensors with broad applications in biotechnology, yet only a small fraction from microbial genomes have been characterized. To address this gap, we recently described the bioinformatic method Ligify, which leverages information from genome context and enzyme reaction databases to predict a TFs cognate effector molecule. Here we report Ligify 2.0, a modern web server for Ligify predictions. We systematically evaluate 10,965 small molecules within the Rhea enzyme reaction database for associations to TFs, ultimately generating 13,435 hypothetical interactions between 1,362 small molecules and 3,164 TFs. We then develop an interactive web server (https://ligify.groov.bio) to search and visualize prediction data. Each TF sensor page includes visualizations for chemical ligand structures, interactive TF protein structures, and genome context. Pages also include metadata links, predicted promoter sequences, prediction confidence metrics, and references to relevant literature. A plasmid builder tool enables users to generate custom biosensor circuit designs. Finally, we provide case studies using Ligify 2.0 to identify two TFs from the pathogens Escherichia coli O157:H7 and Mycobacterium abscessus responsive to 4-hydroxybenzoate and Pseudomonas Quinolone Signal, respectively. The Ligify web server aims to facilitate the systematic characterization of biosensors for chemical-control of biological systems. Key pointsO_LILigify 2.0 contains >13,000 predicted transcription factor-small molecule interactions C_LIO_LIA rich web interface provides interactive visualizations and a plasmid design tool C_LIO_LIPredicted ligands for regulators from pathogenic bacteria are experimentally validated C_LI Graphic abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=70 SRC="FIGDIR/small/683484v2_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@1afa575org.highwire.dtl.DTLVardef@97c811org.highwire.dtl.DTLVardef@cfdb93org.highwire.dtl.DTLVardef@58977d_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2025

groovDB in 2026: A community-editable database of small molecule biosensors

The groovDB database (https://groov.bio) was launched in 2022 with the goal of organizing information on prokaryotic ligand-inducible transcription factors (TFs). This class of proteins is important in fundamental areas of microbiology research and for biotechnological applications that develop biosensors for diagnostics, enzyme screening, and real-time metabolite tracking. Uniquely, groovDB contains stringently curated, literature-referenced data on both TF:DNA and TF:ligand interactions. Here, we describe a major technical update to groovDB, making the database community-editable and adding several advanced features. Users can now add new TF entries and update existing entries using a simple online form. New user interface elements display interactive protein structures and DNA-binding motifs. Updated query methods enable database searches via text, chemical similarity, and attribute-filtering. A new data architecture reduces page load time by five-fold. Finally, the number of TF entries has more than doubled and all source code is now open-access. Graphic abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=80 SRC="FIGDIR/small/670880v3_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@17842beorg.highwire.dtl.DTLVardef@65d608org.highwire.dtl.DTLVardef@1c6e2eborg.highwire.dtl.DTLVardef@5bd824_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2025

Continuous accelerated rock weathering by marine bacteria with enhanced siderophore production

Silicate mineral weathering (dissolution) is a scalable strategy for capture and storage of CO2 but is too slow for industrial deployment. Bacteria can accelerate mineral dissolution by secreting siderophores, molecules that solubilize iron released from the mineral that would otherwise passivate the mineral surface. Here, we investigated how to deploy siderophore-producing bacteria at scale to continuously enhance dissolution of the mineral olivine. We demonstrated that natural genetic regulation precludes continuous siderophore production in mineral bioreactors. To overcome this limitation, we engineered the marine bacterium Alteromonas macleodii to enhance its production of siderophores, conferring a 2.6-fold increase in the rate of olivine dissolution. Life Cycle Analysis indicated that renewable feedstocks and minimal replenishment of modified cells are critical to achieve net CO2 removal at scale. With these guidelines, we constructed pilot-scale continuous mineral bioreactors that use unprocessed seawater and a renewable acetate feedstock. In reactors with engineered cells, we directly measured removal of 0.50 g CO2 per day from the air through alkalinity generation, while a control reactor without cells did not generate alkalinity in net. These platforms and bioprocess principles will inform the design of large-scale, sustainable, unit operations for alkaline mineral processing.

systems biology↗

bioRxiv2025

Rational design of selective bispecific EPO-R/CD131 agonists

Erythropoietin (EPO) suppresses apoptosis and promotes survival by signaling through EPO-R/EPO-R on hematopoietic progenitors or EPO-R/CD131 on non-hematopoietic cells. However, EPO signaling through EPO-R/CD131 is controversial and there is no solved structure of a complex. Here, we constructed a structural model of EPO-R/CD131 and designed several anti-EPO-R, anti-CD131 bispecific proteins that selectively activate EPO-R/CD131. Treatment with these fusion proteins is sufficient to activate STAT5 phosphorylation downstream of EPO-R/CD131 without engaging EPO-R/EPO-R. We demonstrated that proteins with a tandem scFv or bispecific antibody format activate EPO-R/CD131, in contrast to an equimolar mixture of the individual scFvs. Finally, we explored the effect of modifications to binding domain arrangement and linker length and found results consistent with our structural model of an EPO-R/CD131 complex. These findings highlight the utility of bispecific scaffolds in the development of cytokine receptor agonists and provide a foundation for the study of EPO-R/CD131 biology and future clinical development. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=61 SRC="FIGDIR/small/648227v2_ufig1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@1b207b0org.highwire.dtl.DTLVardef@116bce3org.highwire.dtl.DTLVardef@1cafb70org.highwire.dtl.DTLVardef@141ae60_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2025

An Fc-SPINK1 fusion protein inhibits pancreatic inflammation in a mouse model

Pancreatitis results from premature activation and impaired inactivation of pancreatic proteases, primarily trypsin, leading to self-digestion, tissue necrosis, fibrosis, and inflammation. SPINK1 is a pancreas-specific inhibitor of trypsin that prevents premature trypsin activation, and could be a candidate therapeutic. However, because of its small size, SPINK1 would be subject to rapid renal clearance, making it ineffective. To construct a long half-life therapeutic inhibitor of trypsin for pancreatitis treatment we fused this protein to the C-terminus of an IgG1 antibody Fc element, increasing the size to [~]78 kDa, thereby exceeding the renal clearance threshold and providing for FcRn-mediated recycling out of cells. A non-glycosylated form of Fc-SPINK1 was expressed in the yeast Pichia pastoris. Fc-SPINK1 inhibits trypsin enzyme activity in vitro. The blood pharmacokinetics in mice are consistent with a three-compartment distribution model and a terminal half-life of [~]3 days. In a caerulein-induced mouse model of pancreatitis, Fc-SPINK1 significantly ameliorated cell death and immune cell infiltration. We developed an automated image analysis technique to quantify pancreatitis-associated loss of tissue cohesion, and found that Fc-SPINK1 also reduced this effect. This study demonstrates the potential of Fc-SPINK1 as a rationally designed therapy for pancreatitis.

pharmacology and toxicology↗

bioRxiv2024

Comparative evaluation of cell-based assay technologies for scoring drug-induced condensation of SARS-CoV-2 nucleocapsid protein

Protein-nucleic acid phase separation has been implicated in many diseases such as viral infections, neurodegeneration, and cancer. There is great interest in identifying condensate modulators (CMODs), which are small molecules that alter the dynamics and functions of phase-separated condensates, as a potential therapeutic modality. Most CMODs were identified in cellular high-content screens (HCS) where micron-scale condensates were characterized by fluorescence microscopy. These approaches lack information on protein dynamics, are limited by microscope resolution, and are insensitive to subtle condensation phenotypes missed by overfit analysis pipelines. Here, we evaluate two alternative cell-based assays: high-throughput single molecule tracking (htSMT) and proximity-based condensate biosensors using NanoBIT (split luciferase) and NanoBRET (bioluminescence resonance energy transfer) technologies. We applied these methods to evaluate condensation of the SARS-CoV-2 nucleocapsid (N) protein under GSK3 inhibitor treatment, which we had previously identified in our HCS campaign to induce condensation with well-defined structure-activity relationships (SAR). Using htSMT, we observed robust changes in N protein diffusion as early as 3 hours post GSK3 inhibition. Proximity-based N biosensors also reliably reported on condensation, enabling the rapid assaying of large compound libraries with a readout independent of imaging. Both htSMT and proximity-based biosensors performed well in a screening format and provided information on CMOD activity that was complementary to HCS. We expect that this expanded toolkit for interrogating phase-separated proteins will accelerate the identification of CMODs for important therapeutic targets.

molecular biology↗

bioRxiv2024

A Coronaviral Pore-Replicase Complex Drives RNA Synthesis in Double Membrane Vesicles

Coronavirus-infected cells contain double-membrane vesicles (DMVs) that are key for viral RNA replication and transcription, perforated by hexameric pores connecting the vesicular lumen to the cytoplasm. How pores form and traverse two membranes, and how DMVs organize RNA synthesis, is unknown. Using structure prediction and functional assays, we show that the non-structural viral membrane protein nsp4 is the key DMV pore organizer, spanning the double membrane and forming most of the pore lining. Nsp4 interacts with nsp3 on the cytoplasmic side and with the viral replicase inside the DMV. Newly synthesized mRNAs exit the DMV into the cytoplasm, passing through a narrow ring of conserved nsp4 residues. Steric constraints imposed by the ring predict that modified nucleobases block mRNA transit, with broad spectrum anti-coronaviral activity.

biochemistry↗

bioRxiv2023

A long-acting prolactin to combat lactation insufficiency

Human infants are born to breastfeed. While 50% of lactating persons struggle to make enough milk, there are no governmentally-approved drugs to enhance lactation1. Here, we engineer a variant of the naturally-occurring driver of lactation, the hormone Prolactin, to increase its serum half-life and produce a viable drug candidate. Our engineered variant, Prolactin-eXtra Long-acting (Prolactin-XL), is comprised of endogenously active human prolactin fused to an engineered human IgG Fc domain designed to overcome the unique drug development challenges specific to the lactating person-infant dyad. Our Prolactin-XL has a serum half-life of 70.9h in mice, 2,625-fold longer than endogenously active prolactin alone (70.9h v. 0.027h). We demonstrate that Prolactin-XL increases milk production and restores growth of pups fed by dams with pharmacologically-ablated lactation. We show that Prolactin-XL-enhanced lactation is accompanied by reversible, lactocyte-driven changes in mammary gland morphology. This work establishes long-acting prolactins as a potentially powerful pharmacologic means to combat insufficient lactation.

synthetic biology↗

bioRxiv2023

Tardigrade secretory proteins protect biological structures from desiccation

Tardigrades, microscopic animals that survive a broad range of environmental stresses, express a unique set of proteins termed tardigrade-specific intrinsically disordered proteins (TDPs). TDPs are often expressed at high levels in tardigrades upon desiccation, and appear to mediate stress adaptation. Here, we focused on the proteins belonging to the secretory family of tardigrade proteins termed secreted-abundant heat soluble ("SAHS") proteins, and investigated their ability to protect diverse biological structures. Recombinantly expressed SAHS proteins prevented desiccated liposomes from fusion, and enhanced desiccation tolerance of E. coli and Rhizobium tropici upon extracellular application. Molecular dynamics simulation and comparative structural analysis suggest a model by which SAHS proteins may undergo a structural transition upon desiccation, in which removal of water and solutes from a large internal cavity in SAHS proteins destabilizes the beta-sheet structure. These results highlight the potential application of SAHS proteins as stabilizing molecules for preservation of cells.

molecular biology↗

bioRxiv2023

LanTERN: a fluorescent sensor that specifically binds lanthanides

Lanthanides, a series of fifteen f-block elements, are crucial in modern technology, and their purification by conventional chemical means comes at a significant environmental cost. Synthetic biology offers promising solutions. However, the lack of biochemical tools to measure lanthanide binding is a bottleneck to progress. Here, we introduce LanTERN, a lanthanide-responsive fluorescent protein rationally engineered from the lanmodulin-binding protein, LanM. LanTERN was designed based on GCaMP, a genetically encoded calcium indicator that couples the ion binding of four EF hand motifs to increased GFP fluorescence. We engineered seven mutations across the parent constructs four EF hand motifs to switch specificity from calcium to lanthanides. The resulting protein, LanTERN, directly converts the binding of 10 measured lanthanides to 14-fold or greater increased fluorescence. LanTERN development opens new avenues for creating improved lanthanide-binding proteins and bio-sensing systems.

synthetic biology↗

bioRxiv2022

High spontaneous integration rates of end-modified linear DNAs upon mammalian cell transfection

In gene therapy, potential integration of therapeutic transgene into host cell genomes is a serious risk that can lead to insertional mutagenesis and tumorigenesis. Viral vectors are often used as the gene delivery vehicle, but they are prone to undergoing integration events. More recently, non-viral delivery of linear DNAs having modified geometry such as closed-end linear duplex DNA (CELiD) have shown promise as an alternative, due to prolonged transgene expression and less cytotoxicity. However, whether such modified-end linear DNAs can also provide a safe, non-integrating gene transfer remains unanswered. Herein, we provide a systematic comparison of genomic integration frequency upon transfection of cells with expression vectors in the forms of circular plasmid, unmodified linear DNA, CELiD, and Streptavidin-conjugated blocked-end linear DNA. All of these forms of linear DNA resulted in a high fraction of the cells being stably transfected - between 10% and 20% of the initially transfected cells, with CELiDs showing the highest rates of integration. These results indicate that blocking the ends of linear DNA is insufficient to prevent integration. Moreover, our analysis suggest that conventional AAV-based gene therapy may be highly susceptible to integration, which is consistent with recent findings from long-term clinical studies.

bioengineering↗

bioRxiv2022

A screen for modulation of nucleocapsid protein condensation identifies small molecules with anti-coronavirus activity

Biomolecular condensates formed by liquid-liquid phase separation have been implicated in multiple diseases. Modulation of condensate dynamics by small molecules has therapeutic potential, but so far, few condensate modulators have been disclosed. The SARS-CoV-2 nucleocapsid (N) protein forms phase separated condensates that are hypothesized to play critical roles in viral replication, transcription and packaging, suggesting that N condensation modulators might have anti-coronavirus activity across multiple strains and species. Here, we show that N proteins from all seven human coronaviruses (HCoVs) vary in their tendency to undergo phase separation when expressed in human lung epithelial cells. We developed a cell-based high-content screening platform and identified small molecules that both promote and inhibit condensation of SARS-CoV-2 N. Interestingly, these host-targeted small molecules exhibited condensate-modulatory effects across all HCoV Ns. Some have also been reported to exhibit antiviral activity against SARS-CoV-2, HCoV-OC43 and HCoV-229E viral infections in cell culture. Our work reveals that the assembly dynamics of N condensates can be regulated by small molecules with therapeutic potential. Our approach allows for screening based on viral genome sequences alone and might enable rapid paths to drug discovery with value for confronting future pandemics.

molecular biology↗

bioRxiv2021

High content screening and computational prediction reveal viral genes that suppress innate immune response

Suppression of the host innate immune response is a critical aspect of viral replication. Upon infection, viruses may introduce one or more proteins that inhibit key immune pathways, such as the type I interferon pathway. However, the ability to predict and evaluate viral protein bioactivity on targeted pathways remains challenging and is typically done on a single virus/gene basis. Here, we present a medium-throughput high-content cell-based assay to reveal the immunosuppressive effects of viral proteins. To test the predictive power of our approach, we developed a library of 800 genes encoding known, predicted, and uncharacterized human viral genes. We find that previously known immune suppressors from numerous viral families such as Picornaviridae and Flaviviridae recorded positive responses. These include a number of viral proteases for which we further confirmed that innate immune suppression depends on protease activity. A class of predicted inhibitors encoded by Rhabdoviridae viruses was demonstrated to block nuclear transport, and several previously uncharacterized proteins from uncultivated viruses were shown to inhibit nuclear transport of the transcription factors NF-{kappa}B and IRF3. We propose that this pathway-based assay, together with early sequencing, gene synthesis, and viral infection studies, could partly serve as the basis for rapid in vitro characterization of novel viral proteins. IMPORTANCEInfectious diseases caused by viral pathogens exacerbate healthcare and economic burdens. Numerous viral biomolecules suppress the human innate immune system, enabling viruses to evade an immune response from the host. Despite our current understanding of viral replications and immune evasion, new viral proteins, including those encoded by uncultivated viruses or emerging viruses, are being unearthed at a rapid pace from large scale sequencing and surveillance projects. The use of medium- and high-throughput functional assays to characterize immunosuppressive functions of viral proteins can advance our understanding of viral replication and possibly treatment of infections. In this study we assembled a large viral gene library from diverse viral families and developed a high content assay to test for inhibition of innate immunity pathways. Our work expands the tools that can rapidly link sequence and protein function, representing a practical step towards early-stage evaluation of emerging and understudied viruses.

systems biology↗

bioRxiv2021

Biocatalytic Formation of Novel Polyesters with para-Hydroxyphenyl groups in the Backbone - Engineering Cupriavidus necator for production of high-performance materials from CO2 and electricity

Synthetic materials are integral components of consumable and durable goods and are indispensable in the modern world. Polyesters are the most versatile bulk- and specialty-polymers but their production is not sustainable and their fate at end-of-life is of great concern. Bioplastics, though highly regarded, often lag behind conventional plastics due to poor material properties and a competitive market. This has limited the success of sustainable replacements at scale. Enabling the production of bioplastics with superior properties from waste-derived feedstocks could change that. To this end, we created a synthetic entry into the metabolic pathway of bio-polyester synthesis of Cupriavidus necator H16 employing heterologous hydroxyacyl-CoA transferase and mutant PHA synthase. The resulting microbial cell factories enabled the co-polymerization of a range of aliphatic and aromatic hydroxy carboxylates, including a hydroxyphenylic and a hydroxyfuranoic acid, for the first time incorporating aromatic rings in the backbone of biological polyesters. The latter were structurally analogous to synthetic polyesters like PET and PEF, as well as PBST and PBAT. The obtained bio-polyesters underwent characterization for specific physicochemical properties, complemented by the prediction of uncharacterized physicochemical and mechanical properties. In a further advance, the transgenic strain was cultivated in a bio-electrochemical system under autotrophic conditions, enabling synthesis of aromatic bio-polyesters with in situ generated H2+O2, while assimilating CO2. Employing elementary flux-mode analysis, metabolic modeling confirmed the feasibility of producing an extended range of aliphatic, arylatic, and aromatic PHAs de novo from various feedstocks. This comprehensive study paves the way toward sustainable bio-production of advanced high-performance thermoplastics and thermosets. Significance StatementBiomaterials can help the chemical industry transition to a carbon-neutral and circular economy, reducing the accumulation of greenhouse gases and plastic waste by developing biological replacements for fossil carbon-based plastics and implementing end-of-life strategies. Accomplished via the genetic engineering of a microbe that can assimilate carbon dioxide, this work demonstrates the first biocatalytic polymerization and incorporation of aromatic building blocks into the backbone of polyhydroxyalkanoates (PHAs). Employing a bio-electrochemical system for cultivation of the microbes, oxyhydrogen is formed and consumed in situ, thus avoiding explosive gas mixtures. The obtained aromatic PHAs are structurally similar and functionally analogs to synthetic bulk- and high-performance polymers such as PET (polyethylene terephthalate) and PEF (polyethylene furanoate) as well as PBST (polybutylene succinate-co-terephthalate) and PBAT (polybutylene adipate-co-terephthalate). Graphical AbstractThe biosynthesized novel aromatic polyhydroxyalkanoates described in this study are structurally similar to incumbent synthetic polyesters such as poly(ethylene terephthalate) (PET, orange box) and poly(ethylene furan-dicarboxylate) (PEF, yellow box). While PET and PEF may be bio-based to varying degrees, they are always synthesized chemically. In contrast, not only the monomers of poly(hydroxybutyrate-co-phloretate) and poly(hydroxybutyrate-co-hydroxymethylfuranoate) (green box) are fully bio-derived, but also the entire polymers are synthesized biologically. This was demonstrated by employing the autotrophic bacterium Cupriavidus necator H16. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=112 SRC="FIGDIR/small/472320v3_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@20714borg.highwire.dtl.DTLVardef@1ee1c06org.highwire.dtl.DTLVardef@171cee7org.highwire.dtl.DTLVardef@c6bc21_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIFirst-ever biocatalytic formation of polyesters with aromatic rings in the backbone C_LIO_LIMicrobial production of aromatic PHAs in a bio-electrochemical system on CO2 and H2+O2 C_LIO_LIExpression-level of PHA synthase and molecular weight of polyesters are inversely correlated C_LIO_LIIn silico design and pathway analysis of bio-polyesters from low-cost carbon-feedstocks C_LIO_LIPrediction of material properties for novel aromatic co-polyesters and structural analogs C_LI

synthetic biology↗

bioRxiv2021

Natural and designed proteins inspired by extremotolerant organisms can form condensates and attenuate apoptosis in human cells

Many organisms can survive extreme conditions and successfully recover to normal life. This extremotolerant behavior has been attributed in part to repetitive, amphipathic, and intrinsically disordered proteins that are upregulated in the protected state. Here, we assemble a library of approximately 300 naturally-occurring and designed extremotolerance-associated proteins to assess their ability to protect human cells from chemically-induced apoptosis. We show that several proteins from tardigrades, nematodes, and the Chinese giant salamander are apoptosis protective. Notably, we identify a region of the human ApoE protein with similarity to extremotolerance-associated proteins that also protects against apoptosis. This region mirrors the phase separation behavior seen with such proteins, like the tardigrade protein CAHS2. Moreover, we identify a synthetic protein, DHR81, that shares this combination of elevated phase separation propensity and apoptosis protection. Finally, we demonstrate that driving protective proteins into the condensate state increases apoptosis protection, and highlight the ability for DHR81 condensates to sequester caspase-7. Taken together, this work draws a link between extremotolerance-associated proteins, condensate formation, and designing human cellular protection.

synthetic biology↗

bioRxiv2021

Rational engineering of an erythropoietin fusion protein to treat hypoxia

Erythropoietin enhances oxygen delivery and reduces hypoxia-induced cell death, but its pro-thrombotic activity is problematic for use of erythropoietin in treating hypoxia. We constructed a fusion protein that stimulates red blood cell production and neuroprotection without triggering platelet production, a marker for thrombosis. The protein consists of an anti-glycophorin A nanobody and an erythropoietin mutant (L108A). The mutation reduces activation of erythropoietin receptor homodimers that induce erythropoiesis and thrombosis, but maintains the tissue-protective signaling. The binding of the nanobody element to glycophorin A rescues homodimeric erythropoietin receptor activation on red blood cell precursors. In a cell proliferation assay, the fusion protein is active at 10-14 M, allowing an estimate of the number of receptor-ligand complexes needed for signaling. This fusion protein stimulates erythroid cell proliferation in vitro and in mice, and shows neuroprotective activity in vitro. Our erythropoietin fusion protein presents a novel molecule for treating hypoxia.

synthetic biology↗