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Abstract Unrepaired DNA damage encountered by the cellular replication machinery can stall DNA replication, ultimately leading to cell death. In the DNA damage tolerance pathway translesion synthesis (TLS), replication stalling is alleviated by the recruitment of specialized polymerases to synthesize short stretches of DNA near a lesion. Although TLS promotes cell survival, most TLS polymerases are low-fidelity and must be tightly regulated to avoid harmful mutagenesis. The gram-negative bacterium Escherichia coli has served as the model organism for studies of the molecular mechanisms of bacterial TLS. However, it is poorly understood whether these same mechanisms apply to other bacteria. Here, we use in vivo single-molecule fluorescence microscopy to investigate the TLS polymerase Pol Y1 in the model gram-positive bacterium Bacillus subtilis. We find significant differences in the localization and dynamics of Pol Y1 in comparison to its E. coli homolog, Pol IV. Notably, Pol Y1 is constitutively enriched at or near sites of replication in the absence of DNA damage through interactions with the DnaN clamp; in contrast, Pol IV has been shown to be selectively enriched only upon replication stalling. These results suggest key differences in the roles and mechanisms of regulation of TLS polymerases across different bacterial species. 

Abstract The misfolding and aggregation of superoxide dismutase 1 (SOD1) and its mutants has been implicated in amyotrophic lateral sclerosis (ALS). In this study, we have created three peptide conjugates with the antioxidant pentacyclic terpene celastrol and examined their interactions with SOD1 and its mutants A4V and G93A. The peptides YYIVS, MPDAHL, and GSGGL are derived from natural sources and are known for their inherent antioxidant properties. Docking studies revealed that most conjugates showed strong binding with the metal binding and electrostatic loops as well as the β1, β5, and β6 hydrophobic core of SOD1. The conjugates were synthesized and self‐assembled into nanoassemblies. Surface plasmon resonance studies further confirmed the binding interactions of the nanoassemblies with the SOD1 proteins. The nanoassemblies were found to internalize into HEK293T cells. The HEK 293T cells were then transfected with GFP fused WT (Wild Type), A4V and G93A SOD1 mutants. Flow cytometry revealed that treatment with celastrol‐peptide nanoassemblies, affected the fluorescence of the SOD1 protein, implying their role in modulating SOD1, particularly for the mutants. N–Acetyl–Leu–Leu–Norleucinal (ALLN) induced SOD1 aggregation was also affected upon treatment with the nanoassemblies. These results suggest that the nanoassemblies may potentially modulate the activity and structure of SOD1. 

Alpha-synuclein (ASyn) is a protein that is known to play a critical role in Parkinson’s disease (PD) due to its propensity for misfolding and aggregation. Furthermore, this process leads to oxidative stress and the formation of free radicals that cause neuronal damage. In this study, we have utilized a biomimetic approach to design new peptides derived from marine natural resources. The peptides were designed using a peptide scrambling approach where antioxidant moieties were combined with fibrillary inhibition motifs in order to design peptides that would have a dual targeting effect on ASyn misfolding. Of the 20 designed peptides, 12 were selected for examining binding interactions through molecular docking and molecular dynamics approaches, which revealed that the peptides were binding to the pre-NAC and NAC (non-amyloid component) domain residues such as Tyr39, Asn65, Gly86, and Ala85, among others. Because ASyn filaments derived from Lewy body dementia (LBD) have a different secondary structure compared to pathogenic ASyn fibrils, both forms were tested computationally. Five of those peptides were utilized for laboratory validation based on those results. The binding interactions with fibrils were confirmed using surface plasmon resonance studies, where EQALMPWIWYWKDPNGS, PYYYWKDPNGS, and PYYYWKELAQM showed higher binding. Secondary structural analyses revealed their ability to induce conformational changes in ASyn fibrils. Additionally, PYYYWKDPNGS and PYYYWKELAQM also demonstrated antioxidant properties. This study provides insight into the binding interactions of varying forms of ASyn implicated in PD. The peptides may be further investigated for mitigating fibrillation at the cellular level and may have the potential to target ASyn. 

In this work, five new peptides derived from natural resources and two peptide bolaamphiphiles were designed. The self-assembling ability of the peptides and the bolaamphiphiles, as well as their predicted antioxidant activity was examined computationally. In particular, replica modeling molecular dynamics studies were carried out at three different temperatures. Results showed that the bolaamphiphiles as well as three of the peptides efficiently formed spherical or fibrous assemblies, particularly at physiological temperatures. In addition, stacking interactions and hydrogen bonds played a critical role in assembly formation. Furthermore, molecular docking studies with extracellular matrix proteins such as the triple helix motif of collagen and the fibronectin (III) motif of tenascin-X displayed binding interactions with the peptides and the bolaamphiphiles. The most optimal peptide bolaamphiphile WMYGGGWMY-CO-NH-(CH2)4-YMWGGGYMW was then synthesized in the laboratory and its ability to form functional scaffolds upon binding to collagen and tenascin-X was examined. The scaffolds were bioprinted with co-cultures of fibroblasts and keratinocytes. The cells not only proliferated over time but also showed strong adherence and spreading within the matrix. Thus, the peptides and the bolaamphiphiles studied in this work, may be potentially developed as scaffold components for tissue regeneration applications. 

In this work, we utilized a biomimetic approach for targeting KATO (III) tumor cells and 3D tumoroids. Specifically, the binding interactions of the bioactive short peptide sequences ACSAG (A-pep) and LPHVLTPEAGAT (L-pep) with the fibroblast growth factor receptor (FGFR2) kinase domain was investigated for the first time. Both peptides have been shown to be derived from natural resources previously. We then created a new fusion trimer peptide ACSAG-LPHVLTPEAGAT-GASCA (Trimer-pep) and investigated its binding interactions with the FGFR2 kinase domain in order to target the fibroblast growth factor receptor 2 (FGFR2), which is many overexpressed in tumor cells. Molecular docking and molecular dynamics simulation studies revealed critical interactions with the activation loop, hinge and glycine-rich loop regions of the FGFR2 kinase domain. To develop these peptides for drug delivery, DOX (Doxorubicin) conjugates of the peptides were created. Furthermore, the binding of the peptides with the kinase domain was further confirmed through surface plasmon resonance studies. Cell studies with gastric cancer cells (KATO III) revealed that the conjugates and the peptides induced higher cytotoxicity in the tumor cells compared to normal cells. Following confirmation of cytotoxicity against tumor cells, the ability of the conjugates and the peptides to penetrate 3D spheroids was investigated by evaluating their permeation in co-cultured spheroids grown with KATO (III) and colon tumor-associated fibroblasts (CAFs). Results demonstrated that Trimer-pep conjugated with DOX showed the highest permeation, while the ACSAG conjugate also demonstrated reasonable permeation of the drug. These results indicate that these peptides may be further explored and potentially utilized to create drug conjugates for targeting tumor cells expressing FGFR2 for developing therapeutics. 

Development of biocomposite scaffolds has gained tremendous attention due to their potential for tissue regeneration. However, most scaffolds often contain animal-derived collagen that may elicit an immunological response, necessitating the development of new biomaterials. Herein, we developed a new collagen-like peptide,(Pro-Ala-His)10 (PAH)10, and explored its ability to be utilized as a functional biomaterial by incorporating it with a newly synthesized peptide-based self-assembled gel. The gel was prepared by conjugating a pectin derivative, galataric acid, with a pro-angiogenic peptide (LHYQDLLQLQY) and further functionalized with a cortistatin-derived peptide, (Phe-Trp-Lys-Thr)4 (FWKT)4, and the bio-ionic liquid choline acetate. The self-assembly of (PAH)10 and its interactions with the galactarate-peptide conjugates were examined using replica exchange molecular dynamics (REMD) simulations. Results revealed the formation of a multi-layered scaffold, with enhanced stability at higher temperatures. We then synthesized the scaffold and examined its physicochemical properties and its ability to integrate with aortic smooth muscle cells. The scaffold was further utilized as a bioink for bioprinting to form three-dimensional cell-scaffold matrices. Furthermore, the formation of actin filaments and elongated cell morphology was observed. These results indicate that the (PAH)10 hybrid scaffold provides a suitable environment for cell adhesion, proliferation and growth, making it a potentially valuable biomaterial for tissue engineering.