Biology subjects

Chen, J. S.

Publications and source records attributed to Chen, J. S..

16 recordsLinked to original sources

bioRxiv2025

Design principles of the common Gly-X6-Gly membrane protein building block

Protein behavior in lipid is poorly understood and inadequately represented in current computational models. Design and prediction abilities for bilayer-embedded molecular structures may be improved by characterizing membrane proteins most frequent, favored structural features to glean both context-specific and general principles. We used protein design to proactively interrogate the sequence-structure relationship and stabilizing atomic details of two highly prevalent antiparallel transmembrane (TM) motifs with Small-X6-Small consensus sequences. A fragment-based data-mining and sequence statistical inference method including cross-evolutionary structure-aligned covariance enabled engineering of de novo multi-span TM protein assemblies by successfully encoding Gly-X6-Gly and Ala-X6-Ala building blocks. A highly stable glycine-based designs X-ray structure hosts C-H{middle dot}{middle dot}{middle dot}O=C H-bonding alongside extensive backbone-directed van der Waals packing, idealizing features of this motif in Nature. Data-driven design navigates sequence space to directly inquiry upon how to encode and stabilize vital membrane protein structural elements, facilitating efficacious construction of lipid-embedded architectures of increasing complexity. SignificanceMembrane proteins comprised of -helices pack together within lipid bilayers, establishing stabilities and architectures that brace function and guide evolution. De novo design was used to clarify the consensus sequences and molecular features encoding of one exceedingly common TM helix packing architecture ([~]10% of those in membrane folds). Small-X6-Small residue patterns were proven to reliably drive minimal proteins into these antiparallel TM geometries, revealing glycine mainchain hydrogen bonding or via fully apolar interfaces can encode the motif. Optimizing steric packing was the most decisive feature amongst the synthetic proteins. New membrane-specific design methods and model molecules are validated in route to outlining this important sequence-structure relationship broadly impacting the membrane proteome and revealing generalized structure-energetic imperatives governing interactions in lipid.

biophysics↗

bioRxiv2025

Th1 cells express Tfh effector molecules and associate with B cells at the T-B borderfollowing viral infection

Antibodies protect the host from pathogens by neutralizing viruses, targeting pathogens for phagocytosis and complement deposition, and by activating innate immune cells. Most studies on the cellular mechanisms of antibody induction focus on the role of germinal centers (GCs) - areas within lymph nodes (LNs) and spleen where activated B cells undergo mutation and then selection by T follicular helper (Tfh) cells to produce high affinity antibodies. However, specific and long-lived antibodies can be produced in the absence of GCs and Tfh cells, sometimes referred to as "extrafollicular" (EF) responses. However, EF responses are typically assumed to be short-lived and low affinity. We characterized the CD4+ T cells present in wild type and Tfh cell-deficient mice in lung draining LNs following influenza A virus or SARS-CoV-2 infection to compare the nature of T cell help for activated B cells. We found that a population of helper Th1 cells remains in LNs seven days following viral infection and interacts with IgG2c+ B cells at the T-B border in both WT and Tfh-deficient mice. We propose that Th1 cells promote virus-specific antibody production in parallel with Tfh cells in the GC and that such a response is contiguous with the T-B border and therefore is not an EF response as classically defined.

immunology↗

bioRxiv2024

A novel microporous biomaterial vaccine platform for long-lasting antibody mediated immunity against viral infection.

Current antigen delivery platforms, such as alum and nanoparticles, are not readily tunable, thus may not generate optimal adaptive immune responses. We created an antigen delivery platform by loading lyophilized Microporous Annealed Particle (MAP) with aqueous solution containing target antigens. Upon administration of antigen loaded MAP (VaxMAP), the biomaterial reconstitution forms an instant antigen-loaded porous scaffold area with a sustained release profile to maximize humoral immunity. VaxMAP induced CD4+ T follicular helper (Tfh) cells and germinal center (GC) B cell responses in the lymph nodes similar to Alum. VaxMAP loaded with SARS-CoV-2 spike protein improved the magnitude and duration of anti-receptor binding domain antibodies compared to Alum and mRNA-vaccinated mice. A single injection of Influenza specific HA1-loaded-VaxMAP enhanced neutralizing antibodies and elicited greater protection against influenza virus challenge than HA1-loaded-Alum. Thus, VaxMAP is a platform that can be used to promote adaptive immune cell responses to generate more robust neutralizing antibodies, and better protection upon pathogen challenge.

immunology↗

bioRxiv2024

The conformational landscape of human transthyretin revealed by cryo-EM

Transthyretin (TTR) is a natively tetrameric thyroxine transporter found in blood and cerebrospinal fluid whose misfolding and aggregation causes transthyretin amyloidosis. A rational drug design campaign identified the small molecule tafamidis (Vyndaqel/Vyndamax) as an effective stabilizer of the native TTR fold, and this aggregation inhibitor is regulatory agency-approved for the treatment of TTR amyloidosis. Despite 50 years of structural studies on TTR and this triumph of structure-based drug design, there remains a notable dearth of structural information available to understand ligand binding allostery and amyloidogenic TTR unfolding intermediates. We used single-particle cryo-electron microscopy (cryo-EM) to investigate the conformational landscape of this 55 kiloDalton tetramer in the absence and presence of one or two ligands, revealing inherent asymmetries in the tetrameric architecture and previously unobserved conformational states. These findings provide critical mechanistic insights into negatively cooperative ligand binding and the structural pathways responsible for TTR amyloidogenesis. This study underscores the capacity of cryo-EM to provide new insights into protein structures that have been historically considered too small to visualize and to identify pharmacological targets suppressed by the confines of the crystal lattice, opening uncharted territory in structure-based drug design.

molecular biology↗

bioRxiv2022

Vascular tortuosity quantification as an outcome metric of the oxygen-induced retinopathy model of ischemic retinopathy

The murine oxygen-induced retinopathy (OIR) model is one of the most widely used animal models of ischemic retinopathy, mimicking hallmark pathophysiology of initial vaso-obliteration (VO) resulting in ischemia that drives neovascularization (NV). In addition to NV and VO, human ischemic retinopathies including Retinopathy of Prematurity (ROP) are characterized by increased vascular tortuosity. Vascular tortuosity is an indicator of disease severity, need to treat, and treatment response in ROP. Current literature investigating novel therapeutics in the OIR model report their effects on NV and VO, but no standardized quantification of vascular tortuosity exists to date despite this metrics relevance to human disease in clinics. The current proof-of-concept study applied a computer-based image analysis algorithm capable of calculating standardized measurements of vascular tortuosity. Quantification of vascular tortuosity correlated with disease activity in OIR analogously to that observed in infants with ROP. Treatment of OIR mice with anti-Vascular Endothelial Growth Factor (aflibercept) rescued vascular tortuosity in the model. Altogether, these data demonstrated that vascular tortuosity is a quantifiable feature of the OIR model and may be used as an outcome measurement in future studies investigating new treatment modalities for retinal ischemia.

neuroscience↗

bioRxiv2022

Pneumatic Controlled Nano-Sieve for Efficient Capture and Release of Nanoparticles

A pneumatic controlled nano-sieve device is demonstrated for the efficient capture and release of 15 nm quantum dots. This device consists of a 200 nm deep glass channel and a PDMS-based pneumatic pressure layer to enhance target capture. The fluid motion inside the nano-sieve is studied by computational fluidic dynamics (CFD) and microfluidic experiments, enabling efficient target capture with a flow rate as high as 100 L/min. In addition, micro-grooves are fabricated inside the nano-sieve to create low flow rate regions, which further improves the target capture efficiency. A velocity contour plot is constructed with CFD, revealing the flow rate is lowest at the top and bottom of the micro-grooves. This phenomenon is supported by the observed nanoparticle clusters surrounding the micro-grooves. By changing the morphology and pneumatic pressure, this device will also facilitate rapid capture and release of various biomolecules.

bioengineering↗

bioRxiv2021

High-affinity, neutralizing antibodies to SARS-CoV-2 can be made in the absence of T follicular helper cells

T follicular helper (Tfh) cells are the conventional drivers of protective, germinal center (GC)-based antiviral antibody responses. However, loss of Tfh cells and GCs has been observed in patients with severe COVID-19. As T cell-B cell interactions and immunoglobulin class switching still occur in these patients, non-canonical pathways of antibody production may be operative during SARS-CoV-2 infection. We found that both Tfh-dependent and -independent antibodies were induced against SARS-CoV-2 as well as influenza A virus. Tfh-independent responses were mediated by a population we call lymph node (LN)-Th1 cells, which remain in the LN and interact with B cells outside of GCs to promote high-affinity but broad-spectrum antibodies. Strikingly, antibodies generated in the presence and absence of Tfh cells displayed similar neutralization potency against homologous SARS-CoV-2 as well as the B.1.351 variant of concern. These data support a new paradigm for the induction of B cell responses during viral infection that enables effective, neutralizing antibody production to complement traditional GCs and even compensate for GCs damaged by viral inflammation. One-Sentence SummaryComplementary pathways of antibody production mediate neutralizing responses to SARS-CoV-2.

immunology↗

bioRxiv2020

Cyclooxgenase-2 is induced by SARS-CoV-2 infection but does not affect viral entry or replication

Identifying drugs that regulate severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and its symptoms has been a pressing area of investigation during the coronavirus disease 2019 (COVID-19) pandemic. Nonsteroidal anti-inflammatory drugs (NSAIDs), which are frequently used for the relief of pain and inflammation, could modulate both SARS-CoV-2 infection and the host response to the virus. NSAIDs inhibit the enzymes cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2), which mediate the production of prostaglandins (PGs). PGE2, one of the most abundant PGs, has diverse biological roles in homeostasis and inflammatory responses. Previous studies have shown that NSAID treatment or inhibition of PGE2 receptor signaling leads to upregulation of angiotensin-converting enzyme 2 (ACE2), the cell entry receptor for SARS-CoV-2, thus raising concerns that NSAIDs could increase susceptibility to infection. COX/PGE2 signaling has also been shown to regulate the replication of many viruses, but it is not yet known whether it plays a role in SARS-CoV-2 replication. The purpose of this study was to dissect the effect of NSAIDs on COVID-19 in terms of SARS-CoV-2 entry and replication. We found that SARS-CoV-2 infection induced COX-2 upregulation in diverse human cell culture and mouse systems. However, suppression of COX-2/PGE2 signaling by two commonly used NSAIDs, ibuprofen and meloxicam, had no effect on ACE2 expression, viral entry, or viral replication. Our findings suggest that COX-2 signaling driven by SARS-CoV-2 may instead play a role in regulating the lung inflammation and injury observed in COVID-19 patients. ImportancePublic health officials have raised concerns about the use of nonsteroidal anti-inflammatory drugs (NSAIDs) for treating symptoms of coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). NSAIDs function by inhibiting the enzymes cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2). These enzymes are critical for the generation of prostaglandins, lipid molecules with diverse roles in maintaining homeostasis as well as regulating the inflammatory response. While COX-1/COX-2 signaling pathways have been shown to affect the replication of many viruses, their effect on SARS-CoV-2 infection remains unknown. We found that SARS-CoV-2 infection induced COX-2 expression in both human cell culture systems and mouse models. However, inhibition of COX-2 activity with NSAIDs did not affect SARS-CoV-2 entry or replication. Our findings suggest that COX-2 signaling may instead regulate the lung inflammation observed in COVID-19 patients, which is an important area for future studies.

microbiology↗

bioRxiv2020

Quantification of Cas9 binding and cleavage across diverse guide sequences maps landscapes of target engagement

The RNA-guided nuclease Cas9 has unlocked powerful methods for perturbing both the genome through targeted DNA cleavage and the regulome through targeted DNA binding, but limited biochemical data has hampered efforts to quantitatively model sequence perturbation of target binding and cleavage across diverse guide sequences. We present scalable, sequencing-based platforms for high-throughput filter binding and cleavage, then perform 62,444 quantitative binding and cleavage assays on 35,047 on- and off-target DNA sequences across 90 Cas9 ribonucleoproteins (RNPs) loaded with distinct guide RNAs. We observe that binding and cleavage efficacy, as well as specificity, vary substantially across RNPs; canonically studied guides often have atypically high specificity; sequence context surrounding the target significantly influences Cas9 on-rate; and Cas9 RNPs may sequester targets in nonproductive states that contribute to "proofreading" capability. Finally, we distill our findings into an interpretable biophysical model that predicts changes in binding and cleavage for diverse target sequence perturbations.

biochemistry↗

bioRxiv2019

Remodeling of the C. elegans Non-coding RNA Transcriptome by Heat Shock

Elevated temperatures activate a Heat Shock Response (HSR) to protect cells from the pathological effects of protein mis-folding, cellular mis-organization, organelle dysfunction and altered membrane fluidity. This response includes activation of the conserved transcription factor Heat Shock Factor 1 (HSF-1), which binds Heat Shock Elements (HSEs) in the promoters of genes induced by heat shock (HS). The up-regulation of protein-coding genes (PCGs), such as Heat Shock Proteins (HSPs) and cytoskeletal regulators, is critical for cellular survival during elevated temperatures. While the transcriptional response of PCGs to heat shock has been comprehensively analyzed in a variety of organisms, the effect of this stress on the expression of non-coding RNAs (ncRNAs) has not been systematically examined. Here we show that in Caenorhabditis elegans HS induces up- and down-regulation of specific ncRNAs from multiple classes, including miRNA, piRNA, lincRNA, pseudogene, and repeat elements. Moreover, some ncRNA genes appear to be direct targets of the HSR, as they contain HSF-1 bound HSEs in their promoters and their expression is regulated by this factor during HS. These results demonstrate that multiple ncRNA genes respond to HS, some as direct HSF-1 targets, providing new candidates that may contribute to organismal survival during this stress.

molecular biology↗

bioRxiv2019

Massively parallel kinetic profiling of natural and engineered CRISPR nucleases

Engineered Streptococcus pyogenes (Sp) Cas9s and Acidaminococcus sp. (As) Cas12a (formerly Cpf1) improve cleavage specificity in human cells. However, the fidelity, enzymatic mechanisms, and cleavage products of emerging CRISPR nucleases have not been profiled systematically across partially mispaired off-target DNA sequences. Here, we describe NucleaSeq-- nuclease digestion and deep sequencing--a massively parallel platform that measures cleavage kinetics and captures the time-resolved identities of cleaved products for more than ten thousand DNA targets that include mismatches, insertions, and deletions relative to the guide RNA. The binding specificity of each enzyme is measured on the same DNA library via the chip-hybridized association mapping platform (CHAMP). Using this integrated cleavage and binding platform, we profile four SpCas9 variants and AsCas12a. Engineered Cas9s retain wtCas9-like off-target binding but increase cleavage specificity; Cas9-HF1 shows the most dramatic increase in cleavage specificity. Surprisingly, wtCas12a--reported as a more specific nuclease in cells--has cleavage specificity similar to wtCas9 in vitro. Initial cleavage position and subsequent end-trimming vary across nucleases, guide RNA sequences, and position and base identity of mispairs in target DNAs. Using these large datasets, we develop a biophysical model that reveals mechanistic insights into off-target cleavage activities by these nucleases. More broadly, NucleaSeq enables rapid, quantitative, and systematic comparison of the specificities and cleavage products of engineered and natural nucleases.

molecular biology↗

bioRxiv2018

Molecular mechanism of off-target effects in CRISPR-Cas9

CRISPR-Cas9 is the state-of-the-art technology for editing and manipulating nucleic acids. However, the occurrence of off-target mutations can limit its applicability. Here, all-atom enhanced molecular dynamics (MD) simulations - using Gaussian accelerated MD (GaMD) - are used to decipher the mechanism of off-target binding at the molecular level. GaMD reveals that base pair mismatches in the target DNA at specific distal sites with respect to the Protospacer Adjacent Motif (PAM) induce an extended opening of the RNA:DNA heteroduplex, which leads to newly discovered interactions between the unwound nucleic acids and the protein counterpart. The conserved interactions between the target DNA strand and the L2 loop of the catalytic HNH domain constitute a \"lock\" effectively decreasing the conformational freedom of the HNH domain and its activation for cleavage. Remarkably, depending on their position at PAM distal sites, DNA mismatches leading to off-target cleavages are unable to \"lock\" the HNH domain, thereby identifying the ability to \"lock\" HNH as a key determinant. Consistently, off-target sequences hampering the catalysis have been shown to \"trap\" somehow the HNH domain in an inactive \"conformational checkpoint\" state (Dagdas et al. Sci Adv, 2017). As such, this mechanism identifies the molecular basis underlying off-target cleavages and contributes in clarifying a long-lasting open issue of the CRISPR-Cas9 function. It also poses the foundation for designing novel and more specific Cas9 variants, which could be obtained by magnifying the \"locking\" interactions between HNH and the target DNA in the presence of any incorrect off-target sequence, thus preventing undesired cleavages.

biophysics↗

bioRxiv2017

CRISPR-Cas12a target binding unleashes single-stranded DNase activity

CRISPR-Cas12a (Cpf1) proteins are RNA-guided DNA targeting enzymes that bind and cut DNA as components of bacterial adaptive immune systems. Like CRISPR-Cas9, Cas12a can be used as a powerful genome editing tool based on its ability to induce genetic changes in cells at sites of double-stranded DNA (dsDNA) cuts. Here we show that RNA-guided DNA binding unleashes robust, non-specific single-stranded DNA (ssDNA) cleavage activity in Cas12a sufficient to completely degrade both linear and circular ssDNA molecules within minutes. This activity, catalyzed by the same active site responsible for site-specific dsDNA cutting, indiscriminately shreds ssDNA with rapid multiple-turnover cleavage kinetics. Activation of ssDNA cutting requires faithful recognition of a DNA target sequence matching the 20-nucleotide guide RNA sequence with specificity sufficient to distinguish between closely related viral serotypes. We find that target-dependent ssDNA degradation, not observed for CRISPR-Cas9 enzymes, is a fundamental property of type V CRISPR-Cas12 proteins, revealing a fascinating parallel with the RNA-triggered general RNase activity of the type VI CRISPR-Cas13 enzymes.\n\nOne Sentence SummaryCas12a (Cpf1) and related type V CRISPR interference proteins possess non-specific, single-stranded DNase activity upon activation by guide RNA-dependent DNA binding.

biochemistry↗

bioRxiv2017

Enhanced proofreading governs CRISPR-Cas9 targeting accuracy

The RNA-guided CRISPR-Cas9 nuclease from Streptococcus pyogenes (SpCas9) has been widely repurposed for genome editing1-4. High-fidelity (SpCas9-HF1) and enhanced specificity (eSpCas9(1.1)) variants exhibit substantially reduced off-target cleavage in human cells, but the mechanism of target discrimination and the potential to further improve fidelity were unknown5-9. Using single-molecule Forster resonance energy transfer (smFRET) experiments, we show that both SpCas9-HF1 and eSpCas9(1.1) are trapped in an inactive state10 when bound to mismatched targets. We find that a non-catalytic domain within Cas9, REC3, recognizes target mismatches and governs the HNH nuclease to regulate overall catalytic competence. Exploiting this observation, we identified residues within REC3 involved in mismatch sensing and designed a new hyper-accurate Cas9 variant (HypaCas9) that retains robust on-target activity in human cells. These results offer a more comprehensive model to rationalize and modify the balance between target recognition and nuclease activation for precision genome editing.

biochemistry↗

bioRxiv2017

A thermostable Cas9 with increased lifetime in human plasma

CRISPR-Cas9 is a powerful technology that has enabled genome editing in a wide range of species. However, the currently developed Cas9 homologs all originate from mesophilic bacteria, making them susceptible to proteolytic degradation and unsuitable for applications requiring function at elevated temperatures. Here, we show that the Cas9 protein from the thermophilic bacterium Geobacillus stearothermophilus (GeoCas9) catalyzes RNA-guided DNA cleavage over a wide temperature range and has an enhanced protein lifetime in human plasma. GeoCas9 is active at temperatures up to 70{degrees}C, compared to 45{degrees}C for Streptococcus pyogenes Cas9 (SpyCas9), which greatly expands the temperature range for CRISPR-Cas9 applications. By comparing features of two closely related Geobacillus homologs, we created a variant of GeoCas9 that doubles the DNA target sequences that can be recognized by this system. We also found that GeoCas9 is an effective tool for editing mammalian genomes when delivered as a ribonucleoprotein (RNP) complex. Together with an increased lifetime in human plasma, the thermostable GeoCas9 provides the foundation for improved RNP delivery in vivo and expands the temperature range of CRISPR-Cas9.

biochemistry↗

bioRxiv2017

A Conformational Checkpoint Between DNA Binding And Cleavage By CRISPR-Cas9

The Cas9 endonuclease is widely utilized for genome engineering applications by programming its single-guide RNA and ongoing work is aimed at improving the accuracy and efficiency of DNA targeting. DNA cleavage of Cas9 is controlled by the conformational state of the HNH nuclease domain, but the mechanism that governs HNH activation at on-target DNA while reducing cleavage activity at off-target sites remains poorly understood. Using single-molecule FRET, we identified an intermediate state of S. pyogenes Cas9, representing a conformational checkpoint between DNA binding and cleavage. Upon DNA binding, the HNH domain transitions between multiple conformations before docking into its active state. HNH docking requires divalent cations, but not strand scission, and this docked conformation persists following DNA cleavage. Sequence mismatches between the DNA target and guide RNA prevent transitions from the checkpoint intermediate to the active conformation, providing selective avoidance of DNA cleavage at stably bound off-target sites.

biophysics↗