Biology subjects

Perez, A.

Publications and source records attributed to Perez, A..

At least 37 records · Page 2Linked to original sources

bioRxiv2025

Hierarchical Extended Linkage Method (HELM)'s Deep Dive into Hybrid Clustering Strategies

Clustering remains a key tool in the analysis of molecular dynamics (MD) simulations, from the preparation of kinetic models to the study of mechanistic pathways and structural determination. It is no surprise then that multiple algorithms are currently used in the MD community, with k-means and hierarchical approaches being arguably the two most popular approaches. The former is very attractive from a purely computational point of view, demanding minimal memory and time resources, but at the price of being able to partition the data in very restrictive ways. Hierarchical strategies, on the other hand, can generate arbitrary partitions, but with steep memory and time requirements due to their need to build a pairwise distance matrix for all the considered conformations/frames. Here we propose a new hybrid paradigm, the Hierarchical Extended Linkage Method (HELM), that retains the efficiency of k-means while incorporating the flexibility of hierarchical methods. The key ingredient is the use of n-ary difference functions as a way to stabilize the k-means results and efficiently build the hierarchy of subsets. We showcase the applicability of this strategy over protein-DNA and protein folding studies, including the complete analysis of simulations with over 1.5 million frames. HELM is freely available in our MDANCE clustering package.

biophysics↗

bioRxiv2025

Pooled CAR-T screening in nonhuman primates identifies designs with enhanced proliferation, trafficking, and persistence

Chimeric antigen receptor T (CAR-T) cell therapy has revolutionized treatment for B-cell malignancies, yet over 60% of patients relapse within one year, often due to insufficient CAR-T persistence. While mouse and primary cell models have been instrumental in advancing CAR-T therapy, they frequently fail to predict clinical outcomes, underscoring the need for more translationally relevant models. To address this limitation, we conducted the first systematic evaluation of CAR structure-function relationships in an immunocompetent nonhuman primate (NHP) model. We engineered an array of 20 CD20-targeted CARs with distinct combinations of hinge, transmembrane, and costimulatory domains. Following ex vivo characterization, we administered pooled autologous CAR-T arrays to three NHPs and tracked CAR abundance longitudinally using a novel digital droplet PCR assay. Ex vivo, CAR-T cells incorporating the MyD88-CD40 costimulatory domain exhibited markedly distinct functional profiles, including increased activation, unique cytokine secretion, tonic signaling, and resistance to exhaustion. In vivo, MyD88-CD40 CARs expanded dramatically, comprising up to 100% of peripheral T cells and significantly outperforming canonical CD28- and 4-1BB-based CARs. This expansion was associated with robust B-cell depletion across all animals. MyD88-CD40 CARs, particularly those with a CD28 hinge and transmembrane domain, demonstrated superior trafficking to secondary lymphoid tissues and persistence through study endpoint, unlike other CARs which waned by day 28. Our findings highlight the value of NHP models for screening CAR designs and identify MyD88-CD40 CARs as candidates with unmatched potency. The unique functional attributes conferred by this domain may provide key insights into features that drive enhanced CAR-T cell activity. Key pointsO_LIWe developed the first pooled CAR-T screening platform in an immunocompetent nonhuman primate model to directly compare CAR designs. C_LIO_LIWe identified MyD88-CD40 costimulatory domain as vastly superior to conventional domains in proliferation, trafficking and persistence. C_LI

immunology↗

bioRxiv2025

PERRC: Protease Engineering with Reactant Residence Time Control

Proteases with engineered specificity hold great potential for targeted therapeutics, protein circuit construction, and biotechnology applications. However, many proteases exhibit broad substrate specificity, limiting their applications. Engineering protease specificity remains challenging because evolving a protease to recognize a new substrate, without counterselecting against its native substrate, often results in high residual activity on the original substrate. To address this, we developed Protease Engineering with Reactant Residence Time Control (PERRC), a platform that exploits the correlation between endoplasmic reticulum (ER) retention sequence strength and ER residence time. PERRC allows precise control over the stringency of protease evolution by adjusting counterselection to selection substrate ratios. Using PERRC, we evolved an orthogonal tobacco etch virus protease variant, TEVESNp, that selectively cleaves a substrate (ENLYFES) that differs by only one amino acid from its parent sequence (ENLYFQS). TEVESNp exhibits a remarkable 65-fold preference for the evolved substrate, marking the first example of an engineered orthogonal protease driven by such a slight difference in substrate recognition. Furthermore, TEVESNp functions as a competent protease for constructing orthogonal protein circuits in bacteria, and molecular dynamic simulations analysis reveals subtle yet functionally significant active site rearrangements. PERRC is a modular dual-substrate display system that facilitates precise engineering of protease specificity.

synthetic biology↗

bioRxiv2025

A novel peptidoglycan deacetylase modulates daughter cell separation in E. coli

Peptidoglycan hydrolases facilitate bacterial cell wall growth by creating space for insertion of new material and allowing physical separation of daughter cells. In Escherichia coli, three peptidoglycan amidases, AmiA, AmiB and AmiC, cleave septal peptidoglycan during cell division. The LytM-domain proteins EnvC and NlpD activate these amidases either from inside the cell or the outer membrane: EnvC binds to the cytoplasmic membrane-anchored divisome components FtsEX, and NlpD and ActS are outer membrane lipoproteins. Here we report the identification of a novel periplasmic deacetylase called SddA that removes acetyl groups from denuded peptidoglycan glycan strands, the products of amidases. SddA is a substrate for the periplasmic protease Prc, suggesting regulation via protein degradation. The sddA gene is co-expressed with the gene encoding EnvC, linking SddA function to amidase activation. Consistent with this link, the deletion of sddA alleviates phenotypes associated with lack of amidase activation, while overexpression of sddA alleviates phenotypes related to a defective Tol-Pal system and causes cell chaining due to reduced septum peptidoglycan cleavage unless envC is co-expressed. We present a model according to which SddA modulates the activation of the septum-splitting amidases during cell division. AUTHOR SUMMARYBacteria surround their cell membrane by the essential peptidoglycan (cell wall) layer to prevent bursting open due to their turgor. During cell division, bacteria produce a septum at midcell, which must be cleaved for daughter cells to separate. Here, we report the identification of a new enzyme, SddA, that modifies a particular type of peptidoglycan material, denuded glycan chains released during the splitting of septal peptidoglycan for daughter cell separation, in the Gram-negative Escherichia coli. We propose a model in which SddA modulates a switch in the septal peptidoglycan splitting, ensuring splitting is activated from the cell membrane in the early stages of cell division and from the outer membrane in the late stages.

microbiology↗

bioRxiv2024

An Update on Active and Passive Surveillance for African Swine Fever in the Dominican Republic

African Swine Fever (ASF) is a viral, hemorrhagic disease of swine that is reportable to the World Organisation for Animal Health. Since 2007, ASF has been expanding globally and caused severe disruption to the global swine industry. In 2021, ASF was detected in the Dominican Republic, prompting an emergency response from local and international officials. Nearly three years later, ASF is still present in the country despite control efforts. This study used data from January 2023-March 2024 from government-mandated sampling of commercial farms, a newly initiated active surveillance program of backyard farms, and passive reports to provide a comprehensive and descriptive assessment of ASF in the Dominican Republic. The attack rate for each region was calculated, and the reproductive ratio (R0) was estimated using the doubling time method. ASF continues to be distributed throughout the Dominican Republic with lower attack rates in central regions and R0 nearing 1. These results suggest that ASF in the country is reaching a stable state that does not resemble an epidemic situation. This may suggest the need to change from an approach of emergency response to one of sustained and progressive control, ultimately for the long-term goal of ASF eradication in the Dominican Republic.

microbiology↗

bioRxiv2024

Transient protein structure guides surface diffusion pathways for electron transport in membrane supercomplexes

The biological significance of protein supercomplexes have remained contentious, particularly how they tune the shuttling of charge-carrier redox proteins across cell membranes during biological energy conversion. We employ multiscale modeling and single particle cryo-electron microscopy (cryo-EM) to determine the mechanisms of diffusive electron transfer in mitochondrial supercomplexes, composed of respiratory complexes III and IV (CIII and CIV). Using a combination of bioinformatic and entropy maximization tools, we model an ensemble of structures representing the conformational space of CIIIs disordered QCR6 hinge within the yeast CIII2CIV2 supercomplex. Molecular and Brownian Dynamics simulations of the entire supercomplex reveal a mechanism for electrostatic coupling between these negatively charged hinge conformations, and binding and directional diffusion of the redox proteins on the mitochondrial membrane, which is simulated over the millisecond timescale. Anionic lipids reinforce this conformationally-coupled recognition of the supercomplex by retaining a pool of the redox proteins in the vicinity of the membrane when the hinges are of a critical length. Cryo-EM models reveal a large-scale rearrangement of the {Delta}QCR6 supercomplex, which retains a surprisingly robust electrostatic environment for recognition of the redox protein, despite compromise in the supercomplexs negative charge, still enabling a surface-mediated electron transfer in this CIII2CIV2 variant. Altogether, the evolutionary need of confining electron carriers on the surface of bioenergetic membranes is found to give rise to a refolding-guided diffusion model of the redox proteins, which improves the energy conversion efficiency within the supercomplex by nearly 30%.

biophysics↗

bioRxiv2024

AlphaFold2 knows some protein folding principles

AlphaFold2 (AF2) has revolutionized protein structure prediction. However, a common confusion lies in equating the protein structure prediction problem with the protein folding problem. The former provides a static structure, while the latter explains the dynamic folding pathway to that structure. We challenge the current status quo and advocate that AF2 has indeed learned some protein folding prin- ciples, despite being designed for structure prediction. AF2s high-dimensional parameters encode an imperfect biophysical scoring function. Typically, AF2 uses multiple sequence alignments (MSAs) to guide the search within a narrow re- gion of its learned surface. In our study, we operate AF2 without MSAs or initial templates, forcing it to sample its entire energy landscape -- more akin to an ab initio approach. Among over 7,000 proteins, a fraction fold using sequence alone, highlighting the smoothness of AF2s learned energy surface. Additionally, by combining recycling and iterative predictions, we discover multiple AF2 interme- diate structures in good agreement with known experimental data. AF2 appears to follow a "local first, global later" folding mechanism. For designed proteins with more optimized local interactions, AF2s energy landscape is too smooth to detect intermediates even when it should. Our current work sheds new light on what AF2 has learned and opens exciting possibilities to advance our understanding of protein folding and for experimental discovery of folding intermediates.

biophysics↗

bioRxiv2024

Limited protection against early-life cytomegalovirus infection results from deficiency of cytotoxic CD8T cells

Differential antiviral T cell immunity in early life impacts the clinical outcome of Cytomegalovirus (CMV) infection, but the underlying mechanisms are not well understood. Here, we found delayed enrichment of early-life murine CMV-specific CD8 T cells due to a general deficiency of {beta} T cells. Adoptive transfer of naive adult T cells into neonates did not protect due to a blockade of CD8 but not of CD4 effector T cell differentiation. Early-life deficiency of critical signal 3 cytokines during T cell priming resulted in the appearance of non-cytotoxic CD8 effector T cells whereas the effector phase of adult-primed T cells was not disrupted in neonates. Accordingly, we found an overall low number of antiviral human CD8 T cells in newborns with congenital CMV. Together, this study suggests defective CD8 T cell immunity as an important factor explaining the higher risk for CMV disease in the early-life phase.

immunology↗

bioRxiv2024

MELD-Adapt: On-the-Fly Belief Updating in Integrative Molecular Dynamics

Integrative structural biology synergizes experimental data with computational methods to elucidate the structures and interactions within biomolecules, a task that becomes critical in the absence of high-resolution structural data. A critical step in integrating the data, is knowing the expected accuracy or belief in the dataset. We previously showed that the Modeling Employing Limited Data (MELD) approach succeeds at predicting structures and finding the best interpretation of the data when the initial belief is equal or slightly lower than the real value. But, this initial belief is not known for many data sources. Here we introduce MELD-Adapt, designed to dynamically evaluate and infer the reliability of input data, thereby enhancing the accuracy of structure predictions. We demonstrate the utility of this method across different systems, particularly emphasizing its capability to correct initial assumptions and identify the correct fraction of data to produce reliable structural models. The approach is tested with two benchmark sets: the folding of twelve proteins with coarse physical insights and the binding of peptides with varying affinities to the Extrateterminal (ET) domain. Our findings also show the importance of how data is distributed in the system (locally clustered or globally distributed) and the subtle balance between data, beliefs, and force fields.

biophysics↗

bioRxiv2024

Multiple endocrine defects in adult-onset Sprouty1/2/4 triple knockout mice

Genes of the Sprouty family (Spry1-4) are feedback inhibitors of receptor tyrosine kinases, especially of Ret and the FGF receptors. As such, they play distinct and overlapping roles in embryo morphogenesis and are considered to be tumor suppressors in adult life. Genetic experiments in mice have defined in great detail the role of these genes during embryonic development, however their function in adult mice is less clearly established. Here we generate adult-onset, whole body Spry1/2/4 triple knockout mice. Tumor incidence in triple mutant mice is comparable to that of wild type littermates of up to one year of age, indicating that Sprouty loss per se is not sufficient to initiate tumorigenesis. On the other hand, triple knockout mice do not gain weight as they age, show less visceral fat, and have lower plasma glucose levels than wild type littermates, despite showing similar food intake and slightly reduced motor function. They also show alopecia, eyelid inflammation, and mild hyperthyroidism. Finally, triple knockout mice present phosphaturia and hypophosphatemia, suggesting exacerbated signaling downstream of FGF23. In conclusion, triple knockout mice develop a series of endocrine abnormalities but do not show increased tumor incidence.

physiology↗

bioRxiv2024

Protein Retrieval via Integrative Molecular Ensembles (PRIME) through extended similarity indices

Molecular dynamics (MD) simulations are ideally suited to describe conformational ensembles of biomolecules such as proteins and nucleic acids. Microsecond-long simulations are now routine, facilitated by the emergence of graphical processing units. Processing such ensembles on the basis of statistical mechanics can bring insights about different biologically relevant states, their representative structures, states, and even dynamics between states. Clustering, which groups objects based on structural similarity, is typically used to process ensembles, leading to different states, their populations, and the identification of representative structures. For some purposes, such as in protein structure prediction, we are interested in identifying the representative structure that is more similar to the native state of the protein. The traditional pipeline combines hierarchical clustering for clustering and selecting the cluster centroid as representative of the cluster. However, even when the first cluster represents the native basin, the centroid can be several angstroms away in RMSD from the native state - and many other structures inside this cluster could be better choices of representative structures, reducing the need for protein structure refinement. In this study, we developed a module--Protein Retrieval via Integrative Molecular Ensemble (PRIME), that consists of tools to determine the most prevalent states in an ensemble using extended continuous similarity. PRIME is integrated with our Molecular Dynamics Analysis with N -ary Clustering Ensembles (MDANCE) package and can be used as a post-processing tool for arbitrary clustering algorithms, compatible with several MD suites. PRIME was validated with ensembles of different protein and protein complex systems for their ability to reliably identify the most native-like state, which we compare to their experimental structure, and to the traditional approach. Systems were chosen to represent different degrees of difficulty such as folding processes and binding which require large conformational changes. PRIME predictions produced structures that when aligned to the experimental structure were better superposed (lower RMSD). A further benefit of PRIME is its linear scaling - rather than the traditional O(N 2) traditionally associated to comparisons of elements in a set.

biophysics↗

bioRxiv2024

FtsZ-mediated spatial-temporal control over septal cell wall synthesis

FtsZ, the tubulin-like GTPase, is the central organizer of the bacterial divisome, a macromolecular complex that synthesizes new septal cell wall and degrades old septal cell wall (made of septal peptidoglycan, sPG) to allow cell wall constriction and cytokinesis. In E. coli, it is well accepted that 1) FtsZ recruits all essential divisome proteins to the septum, including the core sPG synthase complex, FtsWI/QLB and its activator, FtsN; 2) FtsWI/QLB must complex with FtsN to produce sPG under the wild-type background; and 3) the Brownian ratcheting by treadmilling FtsZ polymers drives the directional movements of sPG synthase proteins along the septum circumference; and 4) FtsZ is essential for the early stage, but dispensable for the late stage of cell wall constriction. However, it remains unclear how FtsZ spatial-temporally organizes the divisome for robust bacterial cytokinesis throughout cell wall constriction process. Combining theoretical modeling with experiments in E. coli, we show that at the early stage during cell division, the Brownian ratcheting by FtsZ treadmilling acts both as a template to corral FtsWI/QLB and FtsN into close contacts for FtsWI/QLB-FtsN complex formation and as a conveyor to maximally homologize the septal distribution of sPG synthesis activities to avoid uneven cell wall constriction. When the septum constricts progressively, the FtsN septal density increases via binding to denuded sPG; consequently, the denuded PG-bound FtsN serves as the template to activate FtsWI/QLB for continued sPG synthesis, rendering FtsZ dispensable. Our work establishes an overarching framework that FtsZ spatial-temporally controls over septal cell wall constriction. SignificanceBacteria utilize FtsZ, the tubulin-like GTPase, to organize cell wall enzymes during cell division. FtsZ forms treadmilling polymers along the septum circumference and drives the directional movement of cell wall enzymes for robust cell wall constriction. How this role is achieved is unclear. We show that FtsZ treadmilling acts both as a template to corral cell wall enzymes into close contacts for priming and as a conveyor to homologize the septal distribution of cell wall synthesis activities for even septum constriction. These roles evolve at different stages of cell division and are modulated differentially by different bacteria; they likely define an overarching principle for robust cell division across the microbial world.

biophysics↗

bioRxiv2024

Sifting Through the Noise: A Computational Pipeline for Accurate Prioritization of Protein- Protein Binding Candidates in High-Throughput Protein Libraries

Identifying the interactome for a protein of interest is challenging due to the large number of possible binders. High-throughput experimental approaches narrow down possible binding partners, but often include false positives. Furthermore, they provide no information about what the binding region is (e.g. the binding epitope). We introduce a novel computational pipeline based on an AlphaFold2 (AF) Competition Assay (AF-CBA) to identify proteins that bind a target of interest from a pull-down experiment, along with the binding epitope. Our focus is on proteins that bind the Extraterminal (ET) domain of Bromo and Extraterminal domain (BET) proteins, but we also introduce nine additional systems to show transferability to other peptide-protein systems. We describe a series of limitations to the methodology based on intrinsic deficiencies to AF and AF-CBA, to help users identify scenarios where the approach will be most useful. Given the speed and accuracy of the methodology, we expect it to be generally applicable to facilitate target selection for experimental verification starting from high-throughput protein libraries. Table of Contents O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=173 SRC="FIGDIR/small/576374v1_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@b90071org.highwire.dtl.DTLVardef@1cc2f66org.highwire.dtl.DTLVardef@3c1109org.highwire.dtl.DTLVardef@1827353_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2023

The Genomic Legacy of Human Management and sex-biased Aurochs hybridization in Iberian Cattle

Cattle have been a valuable economic resource and cultural icon since prehistory. From the initial expansion of domestic cattle into Europe during the Neolithic period, taurine cattle (Bos taurus) and their wild ancestor, the aurochs (B. primigenius), had overlapping ranges, leading to ample opportunities for mating (whether intended by farmers or not). We performed a bioarchaeological analysis of 24 Bos remains from Iberia dating from the Mesolithic to the Roman period. The archaeogenomic dataset allows us to investigate the extent of domestic-wild hybridization over time, providing insight into the species behavior and human hunting and management practices by aligning changes with cultural and genomic transitions in the archaeological record. Our results show frequent hybridization during the Neolithic and Chalcolithic, likely reflecting a mix of hunting and herding or relatively unmanaged herds, with mostly male aurochs and female domestic cattle involved in hybridization. This is supported by isotopic evidence consistent with ecological niche sharing, with only a few domestic cattle possibly being managed. The proportion of aurochs ancestry remains relatively constant from about 4000 years ago, probably due to herd management and selection against first generation hybrids, coinciding with other cultural transitions. The constant level of wild ancestry ({bsim}20%) continues into modern western European breeds including the Spanish Lidia breed which is bred for its aggressiveness and fighting ability, but does not display elevated levels of aurochs ancestry. This study takes a genomic glance at the impact of human actions and wild introgression in the establishment of cattle as one of the most important domestic species today.

genomics↗

bioRxiv2023

Do perineuronal nets stabilize the engram of a synaptic circuit?

Perineuronal nets (PNN), a specialized form of ECM (?), surround numerous neurons in the CNS and allow synaptic connectivity through holes in its structure. We hypothesis that PNNs serve as gatekeepers that guard and protect synaptic territory, and thus may stabilize an engram circuit. We present high-resolution, and 3D EM images of PNN- engulfed neurons showing that synapses occupy the PNN holes, and that invasion of other cellular components are rare. PNN constituents are long-lived and can be eroded faster in an enriched environment, while synaptic proteins have high turnover rate. Preventing PNN erosion by using pharmacological inhibition of PNN-modifying proteases or MMP9 knockout mice allowed normal fear memory acquisition but diminished remote-memory stabilization, supporting the above hypothesis. SignificanceIn this multidisciplinary work, we challenge the hypothesis that the pattern of holes in the perineuronal nets (PNN) hold the code for very-long-term memories. The scope of this work might lead us closer to the understanding of how we can vividly remember events from childhood to death bed. We postulate that the PNN holes hold the code for the engram. To test this hypothesis, we used three independent experimental strategies; high-resolution 3D electron microscopy, Stable Isotop Labeling in Mammals (SILAM) for proteins longevity, and pharmacologically and genetically interruption of memory consolidation in fear conditioning experiments. All of these experimental results did not dispute the PNN hypothesis.

neuroscience↗

bioRxiv2022

GENERALIST: An efficient generative model for protein sequence families

Generative models of protein sequence families are an important tool in the repertoire of protein scientists and engineers alike. However, state-of-the-art generative approaches face inference, accuracy, and overfitting-related obstacles when modeling moderately sized to large proteins and/or protein families with low sequence coverage. To that end, we present a simple to learn, tunable, and accurate generative model, GENERALIST: GENERAtive nonLInear tenSor-factorizaTion for protein sequences. Compared to state-of-the-art methods, GENERALIST accurately captures several high order summary statistics of amino acid covariation. GENERALIST also predicts conservative local optimal sequences which are likely to fold in stable 3D structure. Importantly, unlike other methods, the density of sequences in GENERALIST-modeled sequence ensembles closely resembles the corresponding natural ensembles. GENERALIST will be an important tool to study protein sequence variability.

biophysics↗

bioRxiv2022

A consensus view on the folding mechanism of protein G, L and their mutants

Much of our understanding of folding mechanisms comes from interpretations of experimental {phi} and {psi} value analysis - relating the differences in stability of the transition state ensemble (TSE) and folded state. We introduce a unified approach combining simulations and Bayesian inference to provide atomistic detail for the folding mechanism of protein G, L and their mutants. Protein G and L fold to similar topologies despite low sequence similarity, but differ in their folding pathways. A fast folding redesign of protein G, NuG2, switches folding pathways and folds through a similar pathway with protein L. A redesign of protein L also leads to faster folding, respecting the original folding pathway. Our Bayesian inference approach starts from the same prior on all systems and correctly identifies the folding mechanism for each of the four proteins - a success of the force field and sampling strategy. The approach is computationally efficient and correctly identifies the TSE and intermediate structures along the folding pathway in good agreement with experiments. We complement our findings by using two orthogonal approaches that differ in computational cost and interpretability. Adaptive sampling MD combined with Markov State Model provide a kinetic model that confirms the more complex folding mechanism of protein G and its mutant. Finally, a novel fragment decomposition approach using AlphaFold identifies preferences for secondary structure element combinations that follows the order of events observed in the folding pathways.

biophysics↗

bioRxiv2022

AlphaFold encodes the principles to identify high affinity peptide binders

Machine learning has revolutionized structural biology by solving the problem of predicting structures from sequence information. The community is pushing the limits of interpretability and application of these algorithms beyond their original objective. Already, AlphaFolds ability to predict bound conformations for complexes has surpassed the performance of docking methods, especially for protein-peptide binding. A key question is the ability of these methods to differentiate binding affinities between several peptides that bind the same receptor. We show a novel application of AlphaFold for competitive binding of different peptides to the same receptor. For systems in which the individual structures of the peptides are well predicted, predictions in which both peptides are introduced capture the stronger binder in the bound state, and the other peptide in the unbound form. The speed and robustness of the method will be a game changer to screen large libraries of peptide sequences to prioritize for detailed experimental characterization.

biophysics↗