Charles A. Parkos

Marine invertebrates, particularly the sea cucumber (Isostichopus badionotus), represent a rich reservoir of bioactive collagen peptides with high biomedical potential. This study employed a combined proteomic and computational polypharmacology approach to identify and optimize low-molecular-weight (LMW) collagen peptides for regenerative and metabolic applications. LC-MS/MS analysis of a 1–3 kDa enzymatic hydrolysate derived exclusively from purified I. badionotus body wall collagen successfully identified Alpha-2 collagen as the primary constituent. Crucially, the analysis revealed the natural presence of both the angiogenic RGD (Arg-Gly-Asp) and osteogenic GPR (Gly-Pro-Arg) motifs. In silico blind molecular docking demonstrated that the native sequences possessed moderate polypharmacological affinities for Integrin α_V β_3, Dipeptidyl peptidase-4 (DPP4), and Cyclooxygenase-2 (COX-2). Rational structural modification yielded optimized variants (Loc1d and Loc2g) with vastly superior thermodynamic binding scores. Ultimately, a dual-motif chimeric peptide, Loc3a (YGPRGDPRG), was designed, achieving unparalleled binding against ACE (−225.12), DPP4 (−217.25), and Integrin α_V β₃ (−183.93). Physicochemical and ADMET profiling confirmed these peptides are exceptionally stable (Loc3a Instability Index = −36.17), non-allergenic, and possess a pharmacokinetic profile uniquely suited for safe, localized topical application. Supported by systems biology protein-protein interaction (PPI) networks, these findings position the rationally designed I. badionotus collagen derivatives as highly promising, multi-target therapeutic candidates for the simultaneous management of chronic wounds, oxidative stress, and metabolic dysregulation.

The gastrointestinal epithelium depends on the apical junctional complex (AJC), composed of tight and adherens junctions, to regulate barrier function. Here, we identify the apical polarity protein Crumbs homolog 3 (CRB3) as an important regulator of AJC assembly and barrier function in intestinal epithelium. Using primary murine colonic epithelial cells (colonoids) from inducible, conditional Crb3 -knockout ( Crb3 ^{ER Δ IEC} ) and control ( Crb3 ^fl/fl ) mice, we show that CRB3 deficiency compromised barrier function that was associated with a hypercontractile perijunctional actomyosin network and impaired AJC assembly. Loss of CRB3 exacerbated proinflammatory cytokine–induced AJC remodeling, leading to increased intestinal permeability. Crb3 ^{ER Δ IEC} cells exhibited increased RhoA activity and junctional tension, which could be reversed by ROCK-II or myosin II inhibition, restoring junctional architecture. Mechanistically, CRB3A interacts with the actin cytoskeletal linker protein, Merlin (NF2) via its FERM-binding domain, and NF2 knockdown phenocopied CRB3 loss, suggesting their cooperative role in AJC assembly. These findings establish CRB3 and NF2 signaling as key regulators of perijunctional actomyosin contractility and AJC organization during both de novo junctional assembly and inflammation-induced remodeling. This work defines a CRB3- and NF2-dependent pathway by which epithelial cells regulate mechanical tension to coordinate barrier assembly during homeostasis and junctional remodeling under inflammatory stress.

The gastrointestinal epithelium serves as a critical barrier separating intestinal lumen contents from the underlying tissue environment. Structure and function of the apical junctional complex (AJC), comprising tight and adherens junctions, are essential for establishing and maintaining a polarized and functional epithelial barrier. In this study, we investigated mechanisms by which an apical polarity protein Crumbs homolog 3 (CRB3) regulates AJC assembly and barrier function in primary murine intestinal epithelial cells. Using primary colonic epithelial cells (colonoids) derived from inducible and conditional Crb3 knockdown (Crb3 ^ERΔIEC ) and control mice (Crb3 ^fl/fl ), we demonstrate that Crb3 loss leads to compromised epithelial barrier function that was associated with hypercontractile perijunctional actomyosin and defective assembly of the AJC. We identified CRB3 associates with the Band 4.1 family of cytoskeletal linker proteins, Merlin (NF2) via FERM (band4.1/ezrin/radixin/moesin) binding domain (FBD) of CRB3. Interestingly, NF2 knockdown in cultured intestinal epithelial cells phenocopied the effect of CRB3 deletion, supporting a coordinated role in AJC formation and barrier assembly. Moreover, increased active Rho was detected in assembling junctions of Crb3-null cells and inhibition of ROCKII and myosin II alleviated the hypercontractile phenotype, highlighting involvement of Rho/ROCK signaling. Additionally, increased vinculin localization at the AJC seen in Crb3-null epithelial cells indicates elevated tension at junctions. Our findings underscore the important role of Crb3 and NF2 in regulating contractility of the perijunctional actomyosin ring, mechanical tension at the AJC and barrier function via Rho/ROCK signaling during junctional assembly in intestinal epithelial cells.

Susceptibility to inflammatory bowel diseases (IBDs), Crohn’s disease (CD), and ulcerative colitis (UC) is linked with loss of intestinal epithelial barrier integrity and mitochondria dysfunction. Steroidogenic acute regulatory (StAR) protein-related lipid transfer (START) domain-containing protein 7 (STARD7) is a phosphatidylcholine-specific (PC-specific) lipid transfer protein that transports PC from the ER to the mitochondria, facilitating mitochondria membrane stabilization and respiration function. The aim of this study was to define the contribution of STARD7 in the regulation of the intestinal epithelial mitochondrial function and susceptibility to colitis. In silico analyses identified significantly reduced expression of STARD7 in patients with UC, which was associated with downregulation of metabolic function and a more severe disease phenotype. STARD7 was expressed in intestinal epithelial cells, and STARD7 knockdown resulted in deformed mitochondria and diminished aerobic respiration. Loss of mitochondria function was associated with reduced expression of tight junction proteins and loss of intestinal epithelial barrier integrity that could be recovered by AMPK activation. Stard7 ^+/– mice were more susceptible to the development of DSS-induced and Il10 ^–/– spontaneous models of colitis. STARD7 is critical for intestinal epithelial mitochondrial function and barrier integrity, and loss of STARD7 function increases susceptibility to IBD.

Interferon‐gamma (IFNγ) is traditionally recognized for its pro‐inflammatory role during intestinal inflammation. Here, we demonstrate that IFNγ also functions as a pro‐repair molecule by increasing TNFα receptor 2 (TNFR2 protein/ TNFRSF1B gene ) expression on intestinal epithelial cells (IEC) following injury in vitro and in vivo. In silico analyses identified binding sites for the IFNγ signaling transcription factor STAT1 in the promoter region of TNFRSF1B . Scratch‐wounded IEC exposed to IFNγ exhibited a STAT1‐dependent increase in TNFR2 expression. In situ hybridization revealed elevated Tnfrsf1b mRNA levels in biopsy‐induced colonic mucosal wounds, while intraperitoneal administration of IFNγ neutralizing antibodies following mucosal injury resulted in impaired IEC Tnfrsf1b mRNA and inhibited colonic mucosal repair. These findings challenge conventional notions that “pro‐inflammatory” mediators solely exacerbate damage by highlighting latent pro‐repair functions. Moreover, these results emphasize the critical importance of timing and amount in the synthesis and release of IFNγ and TNFα during the inflammatory process, as they are pivotal in restoring tissue homeostasis.

The Aedes aegypti mosquito is a vector of many infectious agents, including flaviviruses such as Zika virus. Components of mosquito saliva have pleomorphic effects on the vertebrate host to enhance blood feeding, and these changes also create a favorable niche for pathogen replication and dissemination. Here, we demonstrate that human CD47, which is known to be involved in various immune processes, interacts with a 34-kilodalton mosquito salivary protein named Nest1. Nest1 is up-regulated in blood-fed female A. aegypti and facilitates Zika virus dissemination in human skin explants. Nest1 has a stronger affinity for CD47 than its natural ligand, signal regulatory protein α, competing for binding at the same interface. The interaction between Nest1 with CD47 suppresses phagocytosis by human macrophages and inhibits proinflammatory responses by white blood cells, thereby suppressing antiviral responses in the skin. This interaction elucidates how an arthropod protein alters the human response to promote arbovirus infectivity.

Thrombospondin-1 (TSP1) is a matricellular protein associated with the regulation of cell migration through direct binding interactions with integrin proteins and by associating with other receptors known to regulate integrin function, including CD47 and CD36. We previously demonstrated that deletion of an epithelial TSP1 receptor, CD47, attenuates epithelial wound repair following intestinal mucosal injury. However, the mechanisms by which TSP1 contributes to intestinal mucosal repair remain poorly understood. Our results show upregulated TSP1 expression in colonic mucosal wounds and impaired intestinal mucosal wound healing in vivo upon intestinal epithelium–specific loss of TSP1 ( Villin ^Cre/+ Thbs1 ^fl/fl or Thbs1 ^ΔIEC mice). We report that exposure to exogenous TSP1 enhanced migration of intestinal epithelial cells in a CD47- and TGF-β1–dependent manner and that deficiency of TSP1 in primary murine colonic epithelial cells resulted in impaired wound healing. Mechanistically, TSP1 modulated epithelial actin cytoskeletal dynamics through suppression of RhoA activity, activation of Rho family small GTPase (Rac1), and changes in filamentous-actin bundling. Overall, TSP1 was found to regulate intestinal mucosal wound healing via CD47 and TGF-β1, coordinate integrin-containing cell–matrix adhesion dynamics, and remodel the actin cytoskeleton in migrating epithelial cells to enhance cell motility and promote wound repair.

Claudin family tight junction proteins form charge- and size-selective paracellular channels that regulate epithelial barrier function. In the gastrointestinal tract, barrier heterogeneity is attributed to differential claudin expression. Here, we show that claudin-23 (CLDN23) is enriched in luminal intestinal epithelial cells where it strengthens the epithelial barrier. Complementary approaches reveal that CLDN23 regulates paracellular ion and macromolecule permeability by associating with CLDN3 and CLDN4 and regulating their distribution in tight junctions. Computational modeling suggests that CLDN23 forms heteromeric and heterotypic complexes with CLDN3 and CLDN4 that have unique pore architecture and overall net charge. These computational simulation analyses further suggest that pore properties are interaction-dependent, since differently organized complexes with the same claudin stoichiometry form pores with unique architecture. Our findings provide insight into tight junction organization and propose a model whereby different claudins combine to form multiple distinct complexes that modify epithelial barrier function by altering tight junction structure.