Biology subjects

Helm, R. F.

Publications and source records attributed to Helm, R. F..

7 recordsLinked to original sources

bioRxiv2025

Physical, chemical, and structural properties of human gastric organoid-derived mucus

The gastric mucus layer protects the epithelium from gastric acid and ingested pathogens. However, studies of human gastric mucus have been limited due to poor accessibility of native human mucus and the abundance of contaminants in these samples. Here, we explored the potential of human gastric organoids as models for mucus production. Immunofluorescence staining confirmed that the organoids produced mucus containing MUC5AC and MUC6. The luminal mucus had viscoelastic properties similar to those of native human gastric mucus, as determined by particle tracking microrheology. To collect organoid-produced gastric mucus, termed bioengineered gastric mucus (BGM), organoids were cultured as monolayers at the air-liquid interface (ALI), and apically-secreted mucus was harvested and analyzed by MUC5AC ELISA, proteomics, CryoFE-SEM, and bulk rheometry. BGM contained high-molecular weight molecules also found in native gastric mucus, including MUC5AC. Proteomic analysis confirmed that BGM contained MUC5AC, MUC6, MUC1, and other stomach-specific molecules such as gastricsin, olfactomedin 4, and gastrokine. CryoFE-SEM showed that both BGM and native mucus had a porous structure and a characteristic honeycomb scaffold. Bulk rheometry confirmed that BGM exhibited shear thinning and predominantly elastic behavior, consistent with native mucus. Collectively, these findings indicate that BGM is an accessible alternative to native gastric mucus that can be produced on-demand for in vitro studies. New and NoteworthyWe demonstrate the structural and functional similarities of organoid-derived gastric mucus and native mucus collected from human patients. The bioengineered gastric mucus mimics its native counterpart in its proteomic profile, physical architecture, and viscoelasticity. This work highlights the translational potential of organoid-derived mucus for functional investigations of the human gastric mucus layer.

cell biology↗

bioRxiv2024

Identification of a new family of peptidoglycan transpeptidases reveals atypical crosslinking is essential for viability in Clostridioides difficile

Clostridioides difficile, the leading cause of antibiotic-associated diarrhea, relies primarily on 3-3 crosslinks created by L,D-transpeptidases (LDTs) to fortify its peptidoglycan (PG) cell wall. This is unusual, as in most bacteria the vast majority of PG crosslinks are 4-3 crosslinks, which are created by penicillin-binding proteins (PBPs). Here we report the unprecedented observation that 3-3 crosslinking is essential for viability in C. difficile. We also report the discovery of a new family of LDTs that use a VanW domain to catalyze 3-3 crosslinking rather than a YkuD domain as in all previously known LDTs. Bioinformatic analyses indicate VanW domain LDTs are less common than YkuD domain LDTs and are largely restricted to Gram-positive bacteria. Our findings suggest that LDTs might be exploited as targets for antibiotics that kill C. difficile without disrupting the intestinal microbiota that is important for keeping C. difficile in check.

microbiology↗

bioRxiv2023

Genetically encoded, noise-tolerant, auxin biosensors in yeast facilitate metabolic engineering and directed evolution

Auxins are important plant growth regulating compounds that are applied in vast quantities to crops across the globe to control weeds and improve crop quality and yield. Auxins are also produced by nearly every kingdom of life and control both organismal behavior as well as inter-kingdom interactions. Improving our understanding of auxin biosynthesis and signaling is critical to both improving crop plants and controlling symbiotic, commensal, and parasitic inter-kingdom relationships, many of which are critical to ecosystems from forests and oceans to the human microbiome. We present a suite of auxin biosensors that will advance our understanding of and ability to engineer auxin perception by plants and auxin production by fungi. We have developed genetically encoded, ratiometric auxin biosensors in the model yeast Saccharomyces cerevisiae, based on the mechanism plants use to perceive auxin. The ratiometric design of these biosensors improves measurements of auxin concentration by reducing clonal and growth phase variation. These biosensors are capable of measuring exogenous auxin in yeast cultures across five orders of magnitude, likely spanning the physiologically relevant range. We implement these biosensors to measure the production of auxin during different growth conditions and phases for S. cerevisiae. Finally, we demonstrate how these biosensors could be used to improve quantitative functional studies and directed evolution of plant auxin perception machinery. These genetically encoded auxin biosensors will enable future studies of auxin biosynthesis, transport, and signaling in a wide range of yeast species, as well as other fungi, and plants. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=68 SRC="FIGDIR/small/533585v1_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@82743dorg.highwire.dtl.DTLVardef@9d5528org.highwire.dtl.DTLVardef@141cbfaorg.highwire.dtl.DTLVardef@b690e2_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2022

CADD from Chlamydia trachomatis is a manganese-dependent oxygenase that employs a self-sacrificing mechanism for the synthesis of p-aminobenzoate

CADD (chlamydia protein associating with death domains) is a p-aminobenzoate synthase involved in a non-canonical route for tetrahydrofolate biosynthesis in the intracellular bacterial pathogen, Chlamydia trachomatis. The previously solved crystal structure revealed a seven-helix bundle architecture similar to heme oxygenase with a diiron active site, making CADD a founding member of the emerging HDO (heme-oxygenase-like diiron oxidase) superfamily. The CADD-dependent route for pAB biosynthesis was shown to use L-tyrosine as a precursor, however, in vitro reactions were not stimulated by the addition of free L-tyrosine or other tyrosine-derived metabolites, leading to the proposal that the enzyme uses an internal active site tyrosine residue as a precursor to pAB. Here, we perform further biochemical characterization of CADD to clarify the details of the unique self-sacrificing reaction. Surprisingly, the pAB synthase reaction was shown to be dependent on manganese as opposed to iron and the data are most consistent with an active dimanganese cofactor analogous to class Ib and class Id ribonucleotide reductases. Experiments with 18O2 demonstrated the incorporation of two oxygen atoms from molecular oxygen into the pAB product, supporting the proposed mechanism requiring two monooxygenase reactions. Mass spectrometry-based proteomic analyses of CADD-derived peptides demonstrated a glycine substitution at Tyr27, a modification that was increased in reactions that produced pAB in vitro. Additionally, Lys152 was found to be deaminated and oxidized to aminoadipic acid. Taken together, our results support the conclusion that CADD is a manganese-dependent oxygenase that uses Tyr27 and possibly Lys152 as sacrificial substrates for pAB biosynthesis.

biochemistry↗

bioRxiv2021

Comparative metabolomics of fruits and leaves in a hyperdiverse lineage suggests fruits are a key incubator of phytochemical diversification

Interactions between plants and leaf herbivores have long been implicated as the major driver of plant secondary metabolite diversity. However, other plant-animal interactions, such as those between fruits and frugivores, may also be involved in phytochemical diversification. Using 12 species of Piper, we conducted untargeted metabolomics and molecular networking with extracts of fruits and leaves. We evaluated organ-specific secondary metabolite composition and compared multiple dimensions of phytochemical diversity across organs, including richness, structural complexity, and variability across samples at multiple scales within and across species. Plant organ identity significantly influenced secondary metabolite composition, both independent of and in interaction with species identity. Leaves and fruit shared a majority of compounds, but fruits contained more unique compounds and had higher total estimated chemical richness. While organ-level chemical richness and structural complexity varied substantially across species, fruit diversity exceeded leaf diversity in more species than the reverse. Furthermore, the variance in chemical composition across samples was higher for fruits than leaves. By documenting a broad pattern of high phytochemical diversity in fruits relative to leaves, this study lays groundwork for incorporating fruit into a comprehensive and integrative understanding of the ecological and evolutionary factors shaping secondary metabolite composition at the whole-plant level.

plant biology↗

bioRxiv2020

Catechol-Containing Compounds are a Broad Class of Amyloid Inhibitors: I. Redox State is a Key Determinant of the Activities of the Inhibitory Activities

Mechanisms of amyloid inhibition remains poorly understood, in part because most protein targets of amyloid assembly are partially unfolded or intrinsically disordered, which hinders detailed structural characterization of protein-inhibitor complexes and structural-based mechanistic elucidation. Herein we employed a small molecule screening approach to identify inhibitors against three prototype amyloidogenic proteins: amylin, A{beta} and tau. One remarkable class of inhibitors identified was catechol-containing compounds and redox-related quinones/anthraquinones. Further mechanistic studies determined that the redox state of the broad class of catechol-containing compounds is a key determinant of the amyloid inhibitor activities.

biochemistry↗

bioRxiv2020

Human Tau Isoform Aggregation and Selective Detection of Misfolded Tau from Post-Mortem Alzheimer's Disease Brains

Tau aggregates are present in a large number of neurodegenerative diseases known as "tauopathies", including Alzheimers disease (AD). As there are six human tau isoforms in brain tissues and both 3R and 4R isoforms have been observed in the neuronal inclusions, we tested whether tau isoforms behave differently in aggregation. We discovered that all six tau isoforms are capable of forming PHF-tau like filaments and the 3R tau isoforms aggregate significantly faster than their 4R counterparts. We further mapped key segments of tau isoforms that contribute to their aggregation kinetics, where it was determined that microtubule binding domains R2 and R3 were the major contributors to tau aggregation. To evaluate the feasibility of using the six recombinant tau isoforms as substrates to amplify misfolded tau, we demonstrated that full-length human tau isoforms can seed and detect misfolded tau from the post-mortem AD brain tissues with high specificity by an ultrasensitive technology termed real-time quaking-induced conversion (RT-QuIC). Mass spectrometric analysis of PHF-tau samples extracted from AD brains identified peptides corresponding to all major forms of human brain tau isoforms along with a consensus hyperphosphorylated peptide near the C-terminus. Together, our findings not only reveal new aggregation kinetic properties of human tau isoforms, support the development of methods to quantitatively measure misfolded human tau isoforms in AD brains, but also uncover the capability of full-length human tau isoforms as substrates for "prion-like" tau seeding by RT-QuIC assays that may be used for new biomarker development for AD and other tauopathy diagnosis.

pathology↗