Biomedical subjects

Richard F Helm

Publications and source records attributed to Richard F Helm.

4 recordsLinked to original sources

PubMed2026

An Arabidopsis Protein-Flavonoid Interactome Identifies Peroxiredoxin A as a Candidate for Flavonoid Action in Chloroplasts.

The ability of phytochemicals to act as small molecule effectors of protein function is a largely overlooked dimension of plant biochemistry. This is particularly true for the ubiquitous flavonoids where, despite abundant examples of functional interactions with human proteins, biological activities in plants are primarily attributed to ROS scavenging. We used affinity capture to explore the protein interactome of the flavonoid glycoside, rutin, in Arabidopsis seedlings. Unexpectedly, the 397 high-confidence candidates included numerous proteins associated with chloroplasts, where flavonoids are present at exceedingly low levels. Intriguingly, several identified targets are conserved with known flavonoid-interacting proteins in mammals, where the bioavailability of flavonoids is similarly low. Using one of these, the Arabidopsis plastidial 2-cys peroxiredoxin A, as a test case, this study substantiated the potential of affinity proteomics for identifying novel protein targets of phytochemicals and suggests that flavonoids modulate protein function in plants to a larger extent than previously suspected.

Arabidopsis↗

PubMed2026

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, cryo-field emission scanning electron microscopy (CryoFE-SE), and rheometry. BGM contained high-molecular weight molecules also found in native gastric mucus. Proteomic analysis confirmed that BGM contained MUC5AC, MUC6, MUC1, and other stomach-specific molecules such as pepsin C, trefoil factor 2, and gastrokine. CryoFE-SE showed that both BGM and native mucus had a porous structure and a characteristic honeycomb scaffold. However, the viscosity of the BGM was generally lower than that of native human gastric mucus, and BGM failed to exhibit gelation at low pH. Collectively, these findings demonstrate the potential as well as some limitations of BGM as an accessible model system for human gastric mucus.NEW & NOTEWORTHY We 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 and physical architecture. This work highlights the translational potential of organoid-derived mucus for functional investigations of the human gastric mucus layer.

Humans↗

PubMed2025

Type IV Pili-Associated Secretion of a Biofilm Matrix Protein From Clostridium perfringens That Forms Intermolecular Isopeptide Bonds.

Clostridium perfringens is a gram-positive, anaerobic, spore-forming bacterial pathogen of humans and animals. C. perfringens also produces type IV pili (T4P) and has two complete sets of T4P-associated genes, one of which has been shown to produce surface pili needed for cell adherence. One hypothesis about the second set of T4P genes is that they comprise a type II secretion system (TTSS) like those found in gram-negative bacteria, but for gram-positive bacteria, the TTSS would aid transit across the thick peptidoglycan (PG) layer. The secretome of mutants lacking type IV pilins was examined, and a single protein, BsaC (CPE0517), was identified as being dependent on pilin PilA3 for secretion. The bsaC gene is in an operon with genes encoding a SipW signal peptidase and two putative biofilm matrix proteins, BsaA and BsaB, both of which have remote homology to Bacillus subtilis biofilm protein TasA. Since BsaA forms long oligomers that are secreted, we analyzed BsaA monomer interactions with de novo modeling. These models projected that the monomers formed isopeptide bonds as part of a donor strand exchange process. Mutations in residues predicted to form the isopeptide bonds led to the loss of oligomerization, supporting an exchange and lock mechanism, and isopeptide bonds were detected by mass spectrometry methods. Phylogenetic analysis showed the BsaA family of proteins is widespread among bacteria and archaea, but only a subset is predicted to form isopeptide bonds.

Fimbriae, Bacterial↗

PubMed2025

Mutant gltS alleles enable a Vibrio fischeri D-glutamate auxotroph to grow with lower requirements for exogenous D-glutamate.

D-glu is a key component of peptidoglycan (PG) and is essential for growth in most bacteria. To assess constraints on PG evolution and bacterial requirements for D-glu, we sought to artificially evolve PG biosynthesis, leading to either replacement of D-glu in the PG peptide or alternative pathways to D-glu incorporation. We previously found that suppression of D-glu auxotrophy in a murI racD mutant of Vibrio fischeri grown on lysogeny broth salts (LBS) medium was rare but could be accomplished by mutation of bsrF, with restoration of wild-type PG structure. Here, we selected nine additional prototrophic suppressors of the same murI racD mutant from 1010 colony-forming units (CFU) plated on LBS supplemented with ~2.7 mM D-gln. Each suppressor had a mutation in gltS, which encodes a putative sodium:glutamate symporter. Increased copy numbers of mutant gltS alleles enabled growth on unsupplemented LBS and resulted in PG containing D-glu. Examination of media components suggests that D-gln supplementation had inadvertently added ~14 μM D-glu, and that LBS itself contains ~1.4 μM D-glu. The mutations in gltS enabled growth with similarly low D-glu concentrations, but also increased sensitivity to homocysteic acid, suggesting more promiscuous transport. Surprisingly, we discovered that expression of mutant gltS in the auxotroph leads to incorporation of lysine into PG, in addition to canonical D-glu. When seawater is supplemented with D-glu, this V. fischeri mutant still colonized Euprymna scolopes and triggered PG-induced morphogenesis. Our results shed light on glutamate transport, highlight trade-offs in GltS structure and function, and reveal an unusual PG modification.IMPORTANCED-glu is an important building block in the peptidoglycan (PG) component of the bacterial cell wall, and its endogenous production is considered essential in most bacteria, even when grown in complex media. In Vibrio fischeri, in trans expression of mutant GltS symporters allows D-glu auxotrophic strains to grow on lysogeny broth salts (LBS) medium without exogenous D-glu, although there is a fitness trade-off of increased sensitivity to homocysteic acid. Our finding that LBS contains sufficient D-glu to support robust growth highlights the undervalued importance of D-amino acid transport and the ubiquity of D-amino acids. Moreover, the discovery of D-lysine in the PG peptide is an unusual PG modification that warrants further study.

Aliivibrio fischeri↗