Biology subjects

Sanchez, J. G.

Publications and source records attributed to Sanchez, J. G..

8 recordsLinked to original sources

bioRxiv2023

ProstT5: Bilingual Language Model for Protein Sequence and Structure

Adapting large language models (LLMs) to protein sequences spawned the development of powerful protein language models (pLMs). Concurrently, AlphaFold2 broke through in protein structure prediction. Now we can systematically and comprehensively explore the dual nature of proteins that act and exist as three-dimensional (3D) machines and evolve as linear strings of one-dimensional (1D) sequences. Here, we leverage pLMs to simultaneously model both modalities by combining 1D sequences with 3D structure in a single model. We encode protein structures as token sequences using the 3Di-alphabet introduced by the 3D-alignment method Foldseek. This new foundation pLM extracts the features and patterns of the resulting "structure-sequence" representation. Toward this end, we built a non-redundant dataset from AlphaFoldDB and fine-tuned an existing pLM (ProtT5) to translate between 3Di and amino acid sequences. As a proof-of-concept for our novel approach, dubbed Protein structure-sequence T5 (ProstT5), we showed improved performance for subsequent prediction tasks, and for "inverse folding", namely the generation of novel protein sequences adopting a given structural scaffold ("fold"). Our work showcased the potential of pLMs to tap into the information-rich protein structure revolution fueled by AlphaFold2. ProstT5 paves the way to develop new tools integrating the vast resource of 3D predictions, and opens new research avenues in the post-AlphaFold2 era. Our model is freely available for all at https://github.com/mheinzinger/ProstT5.

bioinformatics↗

bioRxiv2023

A Pillar and Perfusion Plate Platform for Robust Human Organoid Culture and Analysis

Human organoids have potential to revolutionize in vitro disease modeling by providing multicellular architecture and function that are similar to those in vivo. This innovative and evolving technology, however, still suffers from assay throughput and reproducibility to enable high-throughput screening (HTS) of compounds due to cumbersome organoid differentiation processes and difficulty in scale-up and quality control. Using organoids for HTS is further challenged by lack of easy-to-use fluidic systems that are compatible with relatively large organoids. Here, we overcome these challenges by engineering "microarray three-dimensional (3D) bioprinting" technology and associated pillar and perfusion plates for human organoid culture and analysis. High-precision, high-throughput stem cell printing and encapsulation techniques were demonstrated on a pillar plate, which was coupled with a complementary deep well plate and a perfusion well plate for static and dynamic organoid culture. Bioprinted cells and spheroids in hydrogels were differentiated into liver and intestine organoids for in situ functional assays. The pillar/perfusion plates are compatible with standard 384-well plates and HTS equipment, and thus may be easily adopted in current drug discovery efforts.

bioengineering↗

bioRxiv2022

Cell response to extracellular matrix energy dissipation outweighs rigidity sensing

The mechanical properties of the extracellular matrix (ECM) determine cell differentiation, proliferation and migration through mechanoresponsive proteins including YAP. However, how different mechanical signals cooperate, synergize or compete to steer cell behavior remains poorly understood. Here, we have examined competition between the two major ECM mechanical cues, i.e. rigidity, which activates cell mechanosensing, and viscous energy dissipation, which reduces stiffness blunting cell mechanotransduction. To trigger competition, we have engineered protein hydrogels allowing concomitant modulation of stiffness and viscosity by mechanisms characteristic of native ECM. Culturing cells on these hydrogels, we have found that substrate energy dissipation attenuates YAP mechanosensing prevailing over stiffness cues. Hampered YAP activation on more dissipative substrates correlates with faster actin flow and smaller focal adhesions. Mechanistically, inhibition of actomyosin contractility reverses the outcome of the competition between rigidity and energy dissipation. Our results highlight the dominating contribution of substrate viscosity to the biology of the cell.

bioengineering↗

bioRxiv2022

Enteroendocrine cells protect the stem cell niche by regulating crypt metabolism in response to nutrients

Background and aimsThe intestinal stem cell niche is exquisitely sensitive to changes in diet, with high fat diet, caloric restriction, and fasting resulting in altered crypt metabolism and intestinal stem cell function. Unlike cells on the villus, cells in the crypt are not immediately exposed to the dynamically changing contents of the lumen. We hypothesized that enteroendocrine cells (EECs), which sense environmental cues and in response release hormones and metabolites, are essential for relaying the nutrient status of the animal to cells deep in the crypt. MethodsWe used the tamoxifen-inducible VillinCreERT2 mouse model to deplete EECs (Neurog3fl/fl) from adult intestinal epithelium and we generated human intestinal organoids from wild-type and NEUROG3-null human pluripotent stem cells. We used indirect calorimetry, 1H-NMR metabolomics, mitochondrial live imaging, and the Seahorse bioanalyzer to assess metabolism. Intestinal stem cell activity was measured by proliferation and enteroid-forming capacity. Transcriptional changes were assessed using 10X Genomics single-cell sequencing. ResultsLoss of EECs resulted in increased energy expenditure in mice, an abundance of active mitochondria, and a shift of crypt metabolism to fatty acid oxidation. Crypts from mouse and human intestinal organoids lacking EECs displayed increased intestinal stem cell activity and failed to activate phospho-S6 ribosomal protein, a marker for activity of the master metabolic regulator mammalian target of rapamycin (mTOR). These phenotypes were similar to those observed when wild-type mice were deprived of nutrients. ConclusionsDeletion of EECs recapitulated a fasting phenotype despite normal levels of ingested nutrients. These data suggest that EECs are required to relay nutritional information to the stem cell niche and are essential regulators of intestinal metabolism.

cell biology↗

bioRxiv2022

High fat diet initiates rapid adaptation of the intestine

While the systemic impacts of overnutrition are well-known to cause obesity and metabolic syndrome over the course of months, the immediate adaptive response of the intestinal epithelium to dietary changes remains poorly understood. Here we used physiological metrics and single cell analyses to interrogate the adaptive response of intestinal epithelial cells when moved to a high fat diet (HFD). Within 1 day of HFD exposure, mice exhibited altered feeding behavior, an increase in energy expenditure and an increase in intestinal epithelial proliferation. Single cell transcriptional analysis demonstrated several acute cellular changes on day 1, including a cell-stress response in intestinal crypts, de-granularization of Paneth cells, and a shift towards fatty acid cellular metabolism. By 3 days of HFD, there was an emergence of uncommitted progenitors with a transcriptional profile indicative of a shift from secretory populations and towards an absorptive fate. In enterocytes, genes regulating lipid transport and absorption increased over the first 3 days which paralleled a functional increase in lipid absorption in vivo over the course of 7 days on HFD. These findings demonstrate that intestinal epithelial cell populations respond rapidly to changes in diet through initial changes in cellular function followed by a shift in cellular composition.

cell biology↗

bioRxiv2021

Engineering functional human gastrointestinal organoid tissues using the three primary germ layers separately derived from pluripotent stem cells

The development of human organoid model systems has provided new avenues for patient-specific clinical care and disease modeling. However, all organoid systems are missing important cell types that, in the embryo, get incorporated into organ tissues during development. Based on the concept of how embryonic organs are assembled, we developed an organoid assembly approach starting with cells from the three primary germ layers; enteric neuroglial, mesenchymal, and epithelial precursors, all separately derived from human pluripotent stem cells. From these we generated human gastric tissue containing differentiated glands, surrounded by layers of smooth muscle containing functional enteric neurons that controlled contractions of the engineered tissue. We used this highly tractable system to identify essential roles for the enteric nervous system in the growth and regional identity of the gastric epithelium and mesenchyme and for glandular morphogenesis of the antral stomach. This approach of starting with separately-derived germ layer components was applied to building more complex fundic and esophageal tissue, suggesting this as a new paradigm for tissue engineering.

developmental biology↗

bioRxiv2021

Using Human Induced Pluripotent Stem Cell Derived Organoids to Identify New Pathologies in Patients with PDX1 Mutations

Two patients with mutations in PDX1 presented with pancreatic agenesis, chronic diarrhea and poor weight gain, the causes of which were not identified through routine clinical testing. We generated patient derived organoids as a novel diagnostic strategy and observed that PDX1188delC/188delC antral organoids convert to an intestinal phenotype, while intestinal organoids undergo gastric metaplasia with significant reduction in enteroendocrine cells. This prompted a re-examination of gastric and intestinal biopsies from both PDX1188delC/188delC patients, which recapitulated organoid phenotypes. Antral biopsies had increased parietal cells and lacked G-cells suggesting loss of antral identity. These patients will now be monitored for the progression of metaplasia. This study demonstrates the utility of organoids for patient diagnoses and treatment.

developmental biology↗

bioRxiv2020

Enteroendocrine cells couple nutrient sensing to nutrient absorption by regulating ion transport

The ability to absorb ingested nutrients is an essential function of all metazoans and utilizes a wide array of nutrient transporters found on the absorptive enterocytes of the small intestine. A unique population of patients has previously been identified with severe congenital malabsorptive diarrhea upon ingestion of any enteral nutrition. The intestines of these patients are macroscopically normal, but lack enteroendocrine cells (EECs), suggesting an essential role for this rare population of nutrient-sensing cells in regulating macronutrient absorption. We used human and mouse models of EEC deficiency to identify a new role for the EEC hormone peptide YY in regulating ion-coupled absorption of glucose and dipeptides the small intestine. We found that peptide YY is required in to maintain normal electrophysiology in the presence of vasoactive intestinal polypeptide, a potent stimulator of ion secretion produced by enteric neurons. Administration of peptide YY to EEC-deficient mice restored normal electrophysiology, improved glucose and peptide absorption, diminished diarrhea and rescued postnatal survival. These data suggest that peptide YY is a key regulator of macronutrient absorption in the small intestine and may be a viable therapeutic option to treat patients with malabsorption.

physiology↗