Biology subjects

Lopez, J.

Publications and source records attributed to Lopez, J..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

Abundance measurements reveal the balance between lysis and lysogeny in the human gut microbiome

The human gut contains diverse communities of bacteriophage, whose interactions with the broader microbiome and potential roles in human health are only beginning to be uncovered. Here, we combine multiple types of data to quantitatively estimate gut phage population dynamics and lifestyle characteristics in human subjects. Unifying results from previous studies, we show that an average human gut contains a low ratio of phage particles to bacterial cells ([~]1:100), but a much larger ratio of phage genomes to bacterial genomes ([~]4:1), implying that most gut phage are effectively temperate (e.g., integrated prophage, phage-plasmids, etc.). By integrating imaging and sequencing data with a generalized model of temperate phage dynamics, we estimate that phage induction and lysis occurs at a low average rate ([~]0.001-0.01 per bacterium per day), imposing only a modest fitness burden on their bacterial hosts. Consistent with these estimates, we find that the phage composition of a diverse synthetic community in gnotobiotic mice can be quantitatively predicted from bacterial abundances alone, while still exhibiting phage diversity comparable to native human microbiomes. These results provide a foundation for interpreting existing and future studies on links between the gut virome and human health.

microbiology↗

bioRxiv2024

High-resolution, high-throughput analysis of Drosophila geotactic behavior

Drosophila innate response to gravity, geotaxis, has been previously used to assess the impact of aging and disease on motor performance. Despite its rich history, fly geotaxis continues to be largely measured manually and assessed through simplistic metrics. The manual nature of this assay introduces substantial experimental variability while simplistic metrics provide limited analytic insights into the behavior. To address these shortcomings, we have constructed a fully automated, programable apparatus, and developed a multi-object tracking software capable of following sub-second movements of individual flies, thus allowing reproducible, detailed, and quantitative analysis of geotactic behavior. The apparatus triggers and monitors geotaxis of 10 fly cohorts simultaneously, with each cohort consisting of up to 7 flies. The tracking program isolates cohorts and records individual fly coordinate outputs allowing for simultaneous multi-group, multifly tracks per experiment, greatly improving throughput and resolution. The algorithm tracks individual flies during the entire run with [~]97% accuracy, yielding detailed climbing curve, speed, and movement direction with 1/30 second resolution. Our tracking also allows the construction of multi-variable metrics and the detection of transitory movement phenotypes, such as slips and falls, which have thus far been neglected in geotaxis studies due to limited spatio-temporal resolution. Through a combination of automation and robust tracking, the platform is therefore poised to advance Drosophila geotaxis assay into a comprehensive assessment of locomotor behavior.

neuroscience↗

bioRxiv2024

Data-driven analysis identifies female-specific social modulation deficit after chronic social defeat stress

BackgroundChronic social defeat stress is a widely used depression model in male mice. Several proposed adaptations extend this model to females with variable, often marginal effects. We examine the if widely used male-defined metrics of stress are suboptimal in females and reveal sex-specific adaptations. MethodsUsing a data-driven method we comprehensively classified social interaction behavior in 761 male and female mice after chronic social witness/defeat stress, examining social modulation of behavioral frequencies and associations with conventional metrics (i.e., social interaction (SI) ratio). ResultsSocial stress induces distinct behavioral adaptation patterns in males and females. SI ratio leads to underpowered analyses in females with limited utility to differentiate susceptibility/resilience. Data-driven analyses reveal failure of social adaptation in stressed female mice that is captured in attenuated velocity change from no target to target tests ({Delta}Velocity) and validate this in three female social stress models. Combining SI ratio and {Delta}Velocity optimally differentiates susceptibility/ resilience in females and this metric reveals resilient-specific adaptation in a resilience-associated neural circuit in female mice. ConclusionsWe demonstrate that psychological or physical social defeat stress induces similar deficits in females that is qualitatively distinct from male deficits and inadequately sampled by male-defined metrics. We identify modulation of locomotion as a robust and easily implementable metric for rigorous research in female mice. Overall, our findings highlight the need to critically evaluate sex differences in behavior and implement sex-based considerations in preclinical model design.

neuroscience↗

bioRxiv2024

Coral long-term recovery after bleaching: implications for sexual reproduction and physiology.

This study examined the long-term impacts of coral bleaching on the reproduction and physiology of Montipora capitata, a dominant reef-building coral in Hawaii. We monitored bleached and non-bleached colonies during and after a natural coral bleaching event in 2014 and analyzed reproductive traits and transcriptomic signatures eight months later. Our study shows that non-bleached and bleached colonies successfully produced gametes. Colonies that bleached had smaller oocytes, and development was slower than in colonies that did not bleach. Corals with different vulnerabilities to bleaching exhibited distinct transcriptomic responses eight months after a bleaching event. Those more prone to bleaching showed suppression of transcripts associated with sperm motility, calcification, and immunity. We found distinct transcriptomic signatures between fringing and patch reefs, suggesting local adaptation and/or acclimatization. To conserve coral reefs and better understand how they will be affected by future heat stress, we need to track which colonies survive and examine how their physiological and reproductive processes are impacted in the short- and long-term. This is critical as consecutive bleaching events become more frequent, and corals have less time to recover. Our study provides valuable molecular and reproductive data that can be used for conservation and management purposes. This information can help us identify signs of coral vulnerability and resilience to bleaching, project how future bleaching events will affect coral reproduction, determine which traits are most at risk, and assess which sites are more likely to be compromised.

physiology↗

bioRxiv2024

Spatial architecture of CD8+ T cells and DC subsets is critical for the response to immune checkpoint inhibitors in melanoma

BackgroundDendritic cells (DCs) are promising targets for cancer immunotherapies owing to their central role in the initiation and the control of immune responses. Their functions encompass a wide range of mechanisms mediated by different DC subsets. Several studies have identified human tumor- associated DC (TA-DC) populations through limited marker-based technologies, such as immunostaining or flow cytometry. However, tumor infiltration, spatial organization and specific functions in response to immunotherapy of each DC subset remain to be defined. MethodsHere, we implemented a multiplexed immunofluorescence analysis pipeline coupled with bio-informatic analyses to decipher the tumor DC landscape and its spatial organization within melanoma patients lesions, and its association with patients response to immune checkpoint inhibitors (ICI). For this aim, we analyze a cohort of 41 advanced melanoma patients treated with anti- PD1 alone or associated with anti-CTLA4. Distance and cell network analyses were performed to gain further insight into the spatial organization of tumor-associated DCs. A Digital Spatial Profiling analysis further characterized ecosystem of tumor-infiltrating DCs. ResultsPlasmacytoid DCs (pDCs) were the most abundant DC population, followed by conventional cDC1 and mature DCs, present in equal proportions. In contrast to CD8+ T cell frequency, and despite varying densities, all DC subsets were associated with a favorable response to ICI. Distance and cell network analyses demonstrated that tumor-infiltrating DCs were largely organized in dense areas with high homotypic connections, except for cDC1 that exhibited a more scattered distribution. We identified four patterns of ecosystems with distinct preferential interactions between DC subsets. Significantly, the proximity and interactions between CD8+ T cells and cDC1 were positively associated with patients response to ICI. ConclusionsOur study unravels the complex spatial organization of DC subsets and their interactions in melanoma patient lesions, shedding light on their pivotal role in shaping the response to ICI. Our discoveries regarding the spatial arrangement of cDC1, especially with CD8+ T cells, provide valuable clues for improving immunotherapeutic strategies in melanoma patients. What is already known on this topicDendritic cells (DCs) are promising targets for cancer immunotherapies owing to their central role in the initiation and the control of immune responses. Although conventional type 1 dendritic cells (cDC1) were proposed to contribute to immunotherapy response, their precise functions and interactions with other immune populations in human cancers are largely unknown. What this study addsThis study provides a precise characterization of the spatial distribution and organization of tumor- infiltrating DCs in a large cohort of advanced melanoma patients, and in correlation with response to immunotherapy. While DCs are organized in dense areas with high homotypic connections, cDC1 exhibit a more scattered distribution and form heterotypic aggregates with other DC subsets. More importantly, a close connection between cDC1 and CD8 T cell is uniquely correlated with the patients response to immunotherapy. How this study might affect research, practice or policyThis study improves our understanding of CD8-DC spatial organization within the tumor microenvironment and will have a broad spectrum of implications in the design of anti-tumor immune-activating compounds and the design of biomarkers of response to immunotherapy for melanoma patients.

cancer biology↗

bioRxiv2023

Sale of critically endangered sharks in the United States

Shark meat is widely available in the United States in grocery stores and seafood markets. The meat is often mislabeled or generically labeled as "shark". The ambiguity of these generic labels makes it challenging to assess the conservation implications of this practice and for consumers to avoid species with high mercury concentrations. For this study we purchased and DNA barcoded 30 shark products purchased in the United States to determine their species identity and conservation status. These samples consisted of 19 filets sold in grocery stores, seafood markets, and Asian specialty markets (mostly in North Carolina) and 11 ordered online as "jerky". 70% of samples were "soft mislabeled" (i.e., labeled generically as shark but not as a specific species). Of the nine samples labeled to species, eight were mislabeled (e.g., spinner shark labeled as mako shark). Only one sample was correctly labeled. All 30 samples were identified as shark and came from 11 different species, including three species listed by the IUCN as Critically Endangered: great hammerhead, scalloped hammerhead, and tope. The first two species have been found to contain very high levels of mercury, illustrating the implications of seafood mislabeling for human health. The widespread availability of shark meat in U.S. grocery stores is surprising given the dramatic decline of shark populations globally. Moreover, the fact that nearly all shark meat is either mislabeled or not labeled to species amplifies the problem. Accurate, verified product labels for shark meat would benefit consumers and shark conservation efforts, and should be a priority for the seafood industry.

genetics↗

bioRxiv2022

High Sensitivity Top-down Proteomics Captures Single Muscle Cell Heterogeneity in Large Proteoforms

Single-cell proteomics has emerged as a powerful method to characterize cellular phenotypic heterogeneity and the cell-specific functional networks underlying biological processes. However, significant challenges remain in single-cell proteomics for the analysis of proteoforms arising from genetic mutations, alternative splicing, and post-translational modifications. Herein, we have developed a highly sensitive functionally integrated top-down proteomics method for the comprehensive analysis of proteoforms from single cells. We applied this method to single muscle fibers (SMFs) to resolve their heterogeneous functional and proteomic properties at the single cell level. Notably, we have detected single-cell heterogeneity in large proteoforms (>200 kDa) from the SMFs. Using SMFs obtained from three functionally distinct muscles, we found fiber-to-fiber heterogeneity among the sarcomeric proteoforms which can be related to the functional heterogeneity. Importantly, we reproducibly detected multiple isoforms of myosin heavy chain (~223 kDa), a motor protein that drives muscle contraction, with high mass accuracy to enable the classification of individual fiber types. This study represents the first "single-cell" top-down proteomics analysis that captures single muscle cell heterogeneity in large proteoforms and establishes a direct relationship between sarcomeric proteoforms and muscle fiber types, highlighting the potential of top-down proteomics for uncovering the molecular underpinnings of cell-to-cell variation in complex systems. Significance StatementSingle-cell technologies are revolutionizing biology and molecular medicine by allowing direct investigation of the biological variability among individual cells. Top-down proteomics is uniquely capable of dissecting biological heterogeneity at the intact protein level. Herein, we develop a highly sensitive single-cell top-down proteomics method to reveal diverse molecular variations in large proteins (>200 kDa) among individual single muscle cells. Our results both reveal and characterize the differences in protein post-translational modifications and isoform expression possible between individual muscle cells. We further integrate functional properties with proteomics and accurately measure myosin isoforms for individual muscle fiber type classification. Our study highlights the potential of top-down proteomics for understanding how single-cell protein heterogeneity contributes to cellular functions.

biochemistry↗

bioRxiv2022

The UBP5 histone H2A deubiquitinase counteracts PRC2-mediated repression to regulate Arabidopsis development and stress responses

Polycomb Repressive Complexes (PRCs) control gene expression through the incorporation of H2Aub and H3K27me3. However, there is limited knowledge about PRCs interacting proteins and their interplay with PRCs in epigenome reshaping, which is fundamental to understand gene regulatory mechanisms. Here, we identified UBIQUITIN SPECIFIC PROTEASE 5 (UBP5) as a novel interactor of the PRC2 subunit SWINGER and its associated factor PWO1 in Arabidopsis thaliana. As inferred from the functional analyses of ubp5 CRISPR-Cas9 mutant plants, UBP5 regulates plant development and stress responses, notably by promoting H2A monoubiquitination erasure, leading to transcriptional de-repression. Preferential association of UBP5 at PRC2 recruiting motifs and local H3K27me3 gaining in ubp5 mutant plants further suggest the existence of functional interplays between UBP5 and PRC2 in regulating epigenome dynamics. In summary, UBP5 provides novel insights to disentangle the complex PRC2 interaction network and is a crucial regulator of the pivotal epigenetic repressive marks H2Aub and H3K27me3.

developmental biology↗

bioRxiv2022

Computational pipeline provides mechanistic understanding of Omicron variant of concern neutralizing engineered ACE2 receptor traps

The SARS-CoV-2 Omicron variant, with 15 mutations in Spike receptor binding domain (Spike-RBD), renders virtually all clinical monoclonal antibodies against WT SARS-CoV-2 ineffective. We recently engineered the SARS-CoV-2 host entry receptor, ACE2, to tightly bind WT-Spike-RBD and prevent viral entry into host cells ("receptor traps"). Here we determine cryo-EM structures of our receptor traps in complex with full length Spike. We develop a multi-model pipeline combining Rosetta protein modeling software and cryo-EM to allow interface energy calculations even at limited resolution and identify interface side chains that allow for high affinity interactions between our ACE2 receptor traps and Spike-RBD. Our structural analysis provides a mechanistic rationale for the high affinity (0.53 - 4.2nM) binding of our ACE2 receptor traps to Omicron-RBD confirmed with biolayer interferometry measurements. Finally, we show that ACE2 receptor traps potently neutralize Omicron- and Delta-pseudotyped viruses, providing alternative therapeutic routes to combat this evolving virus.

biochemistry↗

bioRxiv2022

An intranasal lentiviral booster broadens immune recognition of SARS-CoV-2 variants and reinforces the waning mRNA vaccine-induced immunity that it targets to lung mucosa

As the COVID-19 pandemic continues and new SARS-CoV-2 variants of concern emerge, the adaptive immunity initially induced by the first-generation COVID-19 vaccines wains and needs to be strengthened and broadened in specificity. Vaccination by the nasal route induces mucosal humoral and cellular immunity at the entry point of SARS-CoV-2 into the host organism and has been shown to be the most effective for reducing viral transmission. The lentiviral vaccination vector (LV) is particularly suitable for this route of immunization because it is non-cytopathic, non-replicative and scarcely inflammatory. Here, to set up an optimized cross-protective intranasal booster against COVID-19, we generated an LV encoding stabilized Spike of SARS-CoV-2 Beta variant (LV::SBeta-2P). mRNA vaccine-primed and -boosted mice, with waning primary humoral immunity at 4 months post-vaccination, were boosted intranasally with LV::SBeta-2P. Strong boost effect was detected on cross-sero-neutralizing activity and systemic T-cell immunity. In addition, mucosal anti-Spike IgG and IgA, lung resident B cells, and effector memory and resident T cells were efficiently induced, correlating with complete pulmonary protection against the SARS-CoV-2 Delta variant, demonstrating the suitability of the LV::SBeta-2P vaccine candidate as an intranasal booster against COVID-19.

immunology↗

bioRxiv2021

Formylglycine-generating enzyme-like proteins constitute a novel family of widespread type VI secretion system immunity proteins

Competition is a critical aspect of bacterial life, as it enables niche establishment and facilitates the acquisition of essential nutrients. Warfare between Gram-negative bacteria is largely mediated by the type VI secretion system (T6SS), a dynamic nanoweapon that delivers toxic effector proteins from an attacking cell to adjacent bacteria in a contact-dependent manner. Effector-encoding bacteria prevent self-intoxication and kin cell killing by the expression of immunity proteins, which prevent effector toxicity by specifically binding their cognate effector and occluding its active site. In this study, we investigate Tsi3, a previously uncharacterized T6SS immunity protein present in multiple strains of the human pathogen Acinetobacter baumannii. We show that Tsi3 is the cognate immunity protein of the antibacterial effector of unknown function Tse3. Our bioinformatic analyses indicate that Tsi3 homologs are widespread among Gram-negative bacteria, often encoded within T6SS effector-immunity modules. Surprisingly, we found that Tsi3 homologs possess a characteristic formylglycine-generating enzyme (FGE) domain, which is present in various enzymatic proteins. Our data shows that Tsi3-mediated immunity is dependent on Tse3-Tsi3 protein-protein interactions and that Tsi3 homologs from various bacteria do not protect against Tse3-dependent bacterial killing. Thus, we conclude that Tsi3 homologs are unlikely to be functional enzymes. Collectively, our work identifies FGE domain-containing proteins as important mediators of immunity against T6SS attacks and indicates that the FGE domain can be co-opted as a scaffold in multiple proteins to carry out diverse functions. ImportanceDespite the wealth of knowledge on the diversity of biochemical activities carried out by T6SS effectors, comparably little is known about the various strategies bacteria employ to prevent susceptibility to T6SS-dependent bacterial killing. Our work establishes a novel family of T6SS immunity proteins with a characteristic FGE domain. This domain is present in enzymatic proteins with various catalytic activities. Our characterization of Tsi3 expands the known functions carried out by FGE-like proteins to include defense during T6SS-mediated bacterial warfare. Moreover, it highlights the evolution of FGE domain-containing proteins to carry out diverse biological functions.

microbiology↗

bioRxiv2021

CryoEM and AI reveal a structure of SARS-CoV-2 Nsp2, a multifunctional protein involved in key host processes.

The SARS-CoV-2 protein Nsp2 has been implicated in a wide range of viral processes, but its exact functions, and the structural basis of those functions, remain unknown. Here, we report an atomic model for full-length Nsp2 obtained by combining cryo-electron microscopy with deep learning-based structure prediction from AlphaFold2. The resulting structure reveals a highly-conserved zinc ion-binding site, suggesting a role for Nsp2 in RNA binding. Mapping emerging mutations from variants of SARS-CoV-2 on the resulting structure shows potential host-Nsp2 interaction regions. Using structural analysis together with affinity tagged purification mass spectrometry experiments, we identify Nsp2 mutants that are unable to interact with the actin-nucleation-promoting WASH protein complex or with GIGYF2, an inhibitor of translation initiation and modulator of ribosome-associated quality control. Our work suggests a potential role of Nsp2 in linking viral transcription within the viral replication-transcription complexes (RTC) to the translation initiation of the viral message. Collectively, the structure reported here, combined with mutant interaction mapping, provides a foundation for functional studies of this evolutionary conserved coronavirus protein and may assist future drug design.

biophysics↗

bioRxiv2021

Sentinel cells enable genetic detection of SARS-CoV-2 Spike protein

The COVID-19 pandemic has demonstrated the need for exploring different diagnostic and therapeutic modalities to tackle future viral threats. In this vein, we propose the idea of sentinel cells, cellular biosensors capable of detecting viral antigens and responding to them with customizable responses. Using SARS-CoV-2 as a test case, we developed a live cell sensor (SARSNotch) using a de novo-designed protein binder against the SARS-CoV-2 Spike protein. SARSNotch is capable of driving custom genetically-encoded payloads in immortalized cell lines or in primary T lymphocytes in response to purified SARS-CoV-2 Spike or in the presence of Spike-expressing cells. Furthermore, SARSNotch is functional in a cellular system used in directed evolution platforms for development of better binders or therapeutics. In keeping with the rapid dissemination of scientific knowledge that has characterized the incredible scientific response to the ongoing pandemic, we extend an open invitation for others to make use of and improve SARSNotch sentinel cells in the hopes of unlocking the potential of the next generation of smart antiviral therapeutics.

synthetic biology↗

bioRxiv2021

Beyond single discrete responses: An integrative and multidimensional analysis of behavioral dynamics assisted by Machine Learning

Behavioral systems, understanding it as an emergent system comprising the environment and organism subsystems, include spatial dynamics as a primary dimension in natural settings. Nevertheless, under the standard approaches, the experimental analysis of behavior is based on the single response paradigm and the temporal distribution of discrete responses. Thus, the continuous analysis of spatial behavioral dynamics has been a scarcely studied field. The technological advancements in computer vision have opened new methodological perspectives for the continuous sensing of spatial behavior. With the application of such advancements, recent studies suggest that there are multiple features embedded in the spatial dynamics of behavior, such as entropy, and that they are affected by programmed stimuli (e.g., schedules of reinforcement), at least, as much as features related to discrete responses. Despite the progress, the characterization of behavioral systems is still segmented, and integrated data analysis and representations between discrete responses and continuous spatial behavior are exiguous in the Experimental Analysis of Behavior. Machine Learning advancements, such as t-SNE, variable ranking, provide invaluable tools to crystallize an integrated approach for analyzing and representing multidimensional behavioral data. Under this rationale, the present work: 1) proposes a multidisciplinary approach for the integrative and multilevel analysis of behavioral systems, 2) provides sensitive behavioral measures based on spatial dynamics and helpful data representations to study behavioral systems, and 3) reveals behavioral aspects usually ignored under the standard approaches in the experimental analysis of behavior. To exemplify and evaluate our approach, the spatial dynamics embedded in phenomena relevant to behavioral science, namely water-seeking behavior, and motivational operations, are examined, showing aspects of behavioral systems hidden until now.

animal behavior and cognition↗

bioRxiv2021

Killing of Gram-negative and Gram-positive bacteria by a bifunctional cell wall-targeting T6SS effector

The type VI secretion system (T6SS) is a powerful tool deployed by Gram-negative bacteria to antagonize neighboring organisms. Here, we report that Acinetobacter baumannii ATCC 17978 (Ab17978) secretes D-lysine (D-Lys), increasing the extracellular pH and enhancing the peptidoglycanase activity of the T6SS effector Tse4. This synergistic effect of D-Lys on Tse4 activity enables Ab17978 to outcompete Gram-negative bacterial competitors, demonstrating that bacteria can modify their microenvironment to increase their fitness during bacterial warfare. Remarkably, this lethal combination also results in T6SS-mediated killing of Gram-positive bacteria. Further characterization revealed that Tse4 is a bifunctional enzyme consisting of both lytic transglycosylase and endopeptidase activities, thus representing a novel family of modularly organized T6SS PG degrading effectors with an unprecedented impact in antagonistic bacterial interactions. One sentence summaryBy modulating local environmental pH through D-Lys secretion, Acinetobacter baumannii enhances the activity of a bifunctional cell wall-targeting T6SS effector, increasing its killing activity against Gram-negative and -positive competitors.

microbiology↗

bioRxiv2021

Full Brain and Lung Prophylaxis against SARS-CoV-2 by Intranasal Lentiviral Vaccination in a New hACE2 Transgenic Mouse Model or Golden Hamsters

Non-integrative, non-cytopathic and non-inflammatory lentiviral vectors are particularly suitable for mucosal vaccination and recently emerge as a promising strategy to elicit sterilizing prophylaxis against SARS-CoV-2 in preclinical animal models. Here, we demonstrate that a single intranasal administration of a lentiviral vector encoding a prefusion form of SARS-CoV-2 spike glycoprotein induces full protection of respiratory tracts and totally avoids pulmonary inflammation in the susceptible hamster model. More importantly, we generated a new transgenic mouse strain, expressing the human Angiotensin Converting Enzyme 2, with unprecedent brain permissibility to SARS-CoV-2 replication and developing a lethal disease in <4 days post infection. Even though the neurotropism of SARS-CoV-2 is now well established, so far other vaccine strategies under development have not taken into the account the protection of central nervous system. Using our highly stringent transgenic model, we demonstrated that an intranasal booster immunization with the developed lentiviral vaccine candidate achieves full protection of both respiratory tracts and brain against SARS-CoV-2.

microbiology↗

bioRxiv2021

The GA4GH Variation Representation Specification (VRS): a Computational Framework for the Precise Representation and Federated Identification of Molecular Variation

Maximizing the personal, public, research, and clinical value of genomic information will require that clinicians, researchers, and testing laboratories exchange genetic variation data reliably. Developed by a partnership among national information resource providers, public initiatives, and diagnostic testing laboratories under the auspices of the Global Alliance for Genomics and Health (GA4GH), the Variation Representation Specification (VRS, pronounced "verse") is an extensible framework for the semantically precise and computable representation of variation that complements contemporary human-readable and flat file standards for variation representation. VRS objects are designed to be semantically precise representations of variation, and leverage this design to enable unique, federated identification of molecular variation. We describe the components of this framework, including the terminology and information model, schema, data sharing conventions, and a reference implementation, each of which is intended to be broadly useful and freely available for community use. The specification, documentation, examples, and community links are available at https://vrs.ga4gh.org/.

bioinformatics↗

bioRxiv2020

Harnessing Ionic Selectivity In Acetyltransferase Chemoproteomic Probes

Chemical proteomics provides a powerful strategy for the high-throughput assignment of enzyme function or inhibitor selectivity. However, identifying optimized probes for an enzyme family member of interest and differentiating signal from background remain persistent challenges in the field. To address this obstacle, here we report a physiochemical discernment strategy for optimizing chemical proteomics based on the Coenzyme A (CoA) cofactor. First, we synthesize a pair of CoA-based Sepharose pulldown resins differentiated by a single negatively charged residue, and find this change alters their capture properties in gel-based profiling experiments. Next, we integrate these probes with quantitative proteomics and benchmark analysis of probe selectivity versus traditional competitive chemical proteomics. This reveals the former is well-suited for the identification of optimized pulldown probes for specific enzyme family members, while the latter may have advantages in discovery applications. Finally, we apply our anionic CoA pulldown probe to evaluate the selectivity of a recently reported small molecule N-terminal acetyltransferase inhibitor. These studies further validate the use of physical discriminant strategies in chemoproteomic hit identification and demonstrate how CoA-based chemoproteomic probes can be used to evaluate the selectivity of small molecule protein acetyltransferase inhibitors, an emerging class of pre-clinical therapeutic agents.

biochemistry↗