Intracellular procollagen folding begins at the protein’s C-terminal propeptide (C-Pro) domain, which initiates triple-helix assembly and defines the composition and chain register of fibrillar collagen trimers. The C-Pro domain is later proteolytically cleaved and excreted from the body, while the mature triple helix is incorporated into the extracellular matrix. The procollagen C-Pro domain possesses a single
Human acidic fibroblast growth factor (hFGF1) is an all beta-sheet protein that is involved in the regulation of key cellular processes including cell proliferation and wound healing. hFGF1 is known to aggregate when subjected to thermal unfolding. In this study, we investigate the equilibrium unfolding of hFGF1 using a wide array of biophysical and biochemical techniques. Systematic analyses of the thermal and chemical denaturation data on hFGF1 variants (Q54P, K126N, R136E, K126N/R136E, Q54P/K126N, Q54P/R136E, and Q54P/K126N/R136E) indicate that nullification of charges in the heparin-binding pocket can significantly increase the stability of wtFGF1. Triple variant (Q54P/K126N/R136E) was found to be the most stable of all the hFGF1 variants studied. With the exception of triple variant, thermal unfolding of wtFGF1 and the other variants is irreversible. Thermally unfolded triple variant refolds completely to its biologically native conformation. Microsecond-level molecular dynamic simulations reveal that a network of hydrogen bonds and salt bridges linked to Q54P, K126N, and R136E mutations, are responsible for the high stability and reversibility of thermal unfolding of the triple variant. In our opinion, the findings of the study provide valuable clues for the rational design of a stable hFGF1 variant that exhibits potent wound healing properties.
more » « less- Award ID(s):
- 1945465
- NSF-PAR ID:
- 10282938
- Publisher / Repository:
- Nature Publishing Group
- Date Published:
- Journal Name:
- Scientific Reports
- Volume:
- 11
- Issue:
- 1
- ISSN:
- 2045-2322
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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N -glycosylation site that is widely conserved in all the fibrillar procollagens across humans and diverse other species. Given that the C-Pro domain is removed once procollagen folding is complete, theN -glycan might be presumed to be important for folding. Surprisingly, however, there is no difference in the folding and secretion ofN -glycosylated versus non-N -glycosylated collagen type-I, leaving the function of theN -glycan unclear. We hypothesized that the collagenN -glycan might have a context-dependent function, specifically, that it could be required to promote procollagen folding only when proteostasis is challenged. We show that removal of theN -glycan from misfolding-prone C-Pro domain variants does indeed cause serious procollagen and ER proteostasis defects. TheN -glycan promotes folding and secretion of destabilized C-Pro variants by providing access to the ER’s lectin-based chaperone machinery. Finally, we show that the C-ProN -glycan is actually critical for the folding and secretion of even wild-type procollagen under ER stress conditions. Such stress is commonly incurred during development, wound healing, and other processes in which collagen production plays a key role. Collectively, these results establish an essential, context-dependent function for procollagen’s previously enigmaticN -glycan, wherein the carbohydrate moiety buffers procollagen folding against proteostatic challenge. -
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Key points Gap junctions formed by different connexins are expressed throughout the body and harbour unique channel properties that have not been fully defined mechanistically.
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Abstract Connexins form intercellular communication channels, known as gap junctions (GJs), that facilitate diverse physiological roles, from long‐range electrical and chemical coupling to coordinating development and nutrient exchange. GJs formed by different connexin isoforms harbour unique channel properties that have not been fully defined mechanistically. Recent structural studies on Cx46 and Cx50 defined a novel and stable open state and implicated the amino‐terminal (NT) domain as a major contributor for isoform‐specific functional differences between these closely related lens connexins. To better understand these differences, we constructed models corresponding to wildtype Cx50 and Cx46 GJs, NT domain swapped chimeras, and point variants at the 9th residue for comparative molecular dynamics (MD) simulation and electrophysiology studies. All constructs formed functional GJ channels, except the chimeric Cx46‐50NT variant, which correlated with an introduced steric clash and increased dynamical behaviour (instability) of the NT domain observed by MD simulation. Single channel conductance correlated well with free‐energy landscapes predicted by MD, but resulted in a surprisingly greater degree of effect. Additionally, we observed significant effects on transjunctional voltage‐dependent gating (
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Abstract Measurements of regional internal body temperatures can yield important information in the diagnosis of immune response‐related anomalies, for precisely managing the effects of hyperthermia and hypothermia therapies and monitoring other transient body processes such as those associated with wound healing. Current approaches rely on permanent implants that require extraction surgeries after the measurements are no longer needed. Emerging classes of bioresorbable sensors eliminate the requirements for extraction, but their use of percutaneous wires for data acquisition leads to risks for infection at the suture site. As an alternative, a battery‐free, wireless implantable device is reported here, which is constructed entirely with bioresorbable materials for monitoring regional internal body temperatures over clinically relevant timeframes. Ultimately, these devices disappear completely in the body through natural processes. In vivo demonstrations indicate stable operation as subcutaneous and intracranial implants in rat models for up to 4 days. Potential applications include monitoring of healing cascades associated with surgical wounds, recovery processes following internal injuries, and the progression of thermal therapies for various conditions.
