An inhibitory domain of E12 transcription factor prevents DNA binding in E12 homodimers but not in E12 heterodimers.

The kappa E2 sequence binding proteins, E12 and E47, are generated by alternative splicing of the E2A gene, giving closely related basic and helix-loop-helix structures crucial for DNA binding and dimerization. Measurements of dimerization constants and binding strengths to the optimal DNA sequence (the kappa E2 site or its near relatives) showed that E47 homodimers and MyoD heterodimers with E12 or E47 dimerized and bound avidly, but E12 homodimerized efficiently and bound to DNA poorly; MyoD homodimerized poorly and bound strongly. An inhibitory domain N-terminal to the basic region of E12 prevents E12 homodimers but not E12/MyoD heterodimers from binding to DNA. Thus, E47 binds to DNA both as a heterodimer with MyoD and as a homodimer, while E12 and MyoD bind to DNA efficiently only as heterodimers.     

Insulin-regulated aminopeptidase: analysis of peptide substrate and inhibitor binding to the catalytic domain

Peptide inhibitors of insulin-regulated aminopeptidase (IRAP) accelerate spatial learning and facilitate memory retention and retrieval by binding competitively to the catalytic site of the enzyme and inhibiting its catalytic activity. IRAP belongs to the M1 family of Zn2+-dependent aminopeptidases characterized by a catalytic domain that contains two conserved motifs, the HEXXH(X)18E Zn2+-binding motif and the GXMEN exopeptidase motif. To elucidate the role of GXMEN in binding peptide substrates and competitive inhibitors, site-directed mutagenesis was performed on the motif. Non-conserved mutations of residues G428, A429 and N432 resulted in mutant enzymes with altered catalytic activity, as well as divergent changes in kinetic properties towards the synthetic substrate leucine beta-naphthylamide. The affinities of the IRAP inhibitors angiotensin IV, Nle1-angiotensin IV, and LVV-hemorphin-7 were selectively decreased. Substrate degradation studies using the in vitro substrates vasopressin and Leu-enkephalin showed that replacement of G428 by either D, E or Q selectively abolished the catalysis of Leu-enkephalin, while [A429G]IRAP and [N432A]IRAP mutants were incapable of cleaving both substrates. These mutational studies indicate that G428, A429 and N432 are important for binding of both peptide substrates and inhibitors, and confirm previous results demonstrating that peptide IRAP inhibitors competitively bind to its catalytic site.     

Binding mode of SARS-CoV-2 fusion peptide to human cellular membrane

Infection of human cells by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) relies on its binding to a specific receptor and subsequent fusion of the viral and host cell membranes. The fusion peptide (FP), a short peptide segment in the spike protein, plays a central role in the initial penetration of the virus into the host cell membrane, followed by the fusion of the two membranes. Here, we use an array of molecular dynamics simulations that take advantage of the highly mobile membrane mimetic model to investigate the interaction of the SARS-CoV2 FP with a lipid bilayer representing mammalian cellular membranes at an atomic level and to characterize the membrane-bound form of the peptide. Six independent systems were generated by changing the initial positioning and orientation of the FP with respect to the membrane, and each system was simulated in five independent replicas, each for 300 ns. In 73% of the simulations, the FP reaches a stable, membrane-bound configuration, in which the peptide deeply penetrated into the membrane. Clustering of the results reveals three major membrane-binding modes (binding modes 1–3), in which binding mode 1 populates over half of the data points. Taking into account the sequence conservation among the viral FPs and the results of mutagenesis studies establishing the role of specific residues in the helical portion of the FP in membrane association, the significant depth of penetration of the whole peptide, and the dense population of the respective cluster, we propose that the most deeply inserted membrane-bound form (binding mode 1) represents more closely the biologically relevant form. Analysis of FP-lipid interactions shows the involvement of specific residues, previously described as the “fusion-active core residues,” in membrane binding. Taken together, the results shed light on a key step involved in SARS-CoV2 infection, with potential implications in designing novel inhibitors.     

Calcium-binding properties of wild-type and EF-hand mutants of S100B in the presence and absence of a peptide derived from the C-terminal negative regulatory domain of p53

S100B is a dimeric Ca(2+)-binding protein that undergoes a 90 +/- 3 degrees rotation of helix 3 in the typical EF-hand domain (EF2) upon the addition of calcium. The large reorientation of this helix is a prerequisite for the interaction between each subunit of S100B and target proteins such as the tumor suppressor protein, p53. In this study, Tb(3+) was used as a probe to examine how binding of a 22-residue peptide derived from the C-terminal regulatory domain of p53 affects the rate of Ca(2+) ion dissociation. In competition studies with Tb(3+), the dissociation rates of Ca(2+) (k(off)) from the EF2 domains of S100B in the absence and presence of the p53 peptide was determined to be 60 and 7 s(-)(1), respectively. These data are consistent with a previously reported result, which showed that that target peptide binding to S100B enhances its calcium-binding affinity [Rustandi et al. (1998) Biochemistry 37, 1951-1960]. The corresponding Ca(2+) association rate constants for S100B, k(on), for the EF2 domains in the absence and presence of the p53 peptide are 1.1 x 10(6) and 3.5 x 10(5) M(-)(1) s(-)(1), respectively. These two association rate constants are significantly below the diffusion control ( approximately 10(9) M(-)(1) s(-)(1)) and likely involve both Ca(2+) ion association and a Ca(2+)-dependent structural rearrangement, which is slightly different when the target peptide is present. EF-hand calcium-binding mutants of S100B were engineered at the -Z position (EF-hand 1, E31A; EF-hand 2, E72A; both EF-hands, E31A + E72A) and examined to further understand how specific residues contribute to calcium binding in S100B in the absence and presence of the p53 peptide.     

In Silico Design of Dual-Binding Site Anti-Cholinesterase Phytochemical Heterodimers as Treatment Options for Alzheimer’s Disease

The number of patients with neurodegenerative diseases, particularly Alzheimer’s disease (AD), continues to grow yearly. Cholinesterase inhibitors (ChEIs) represent the first-line symptomatic drug treatment for mild-to-moderate AD; however, there is an unmet need to produce ChEIs with improved efficacy and reduced side effects. Herein, phytochemicals with reported anti-acetylcholinesterase (AChE) activity were ranked in silico for their anti-AChE potential. Ligands with a similar or higher binding affinity to AChE than galantamine were then selected for the design of novel dual-binding site heterodimeric drugs. In silico molecular docking of heterodimers with the target enzymes, AChE and butyrylcholinesterase (BuChE), were performed, and anti-cholinesterase binding affinities were compared with donepezil. Drug-likeliness properties and toxicity of the heterodimers were assessed using the SwissADME and ProTox-II webservers. Nine phytochemicals displayed similar or higher binding affinities to AChE than galantamine: sanguinarine > huperzine A > chelerythrine > yohimbine > berberine > berberastine > naringenin > akuammicine > carvone. Eleven heterodimeric ligands were designed with phytochemicals separated by four- or five-carbon alkyl-linkers. All heterodimers were theoretically potent AChE and BuChE dual-binding site inhibitors, with the highest affinity achieved with huperzine-4C-naringenin, which displayed 34% and 26% improved affinity to AChE and BuChE, respectively, then the potent ChEI drug, donepezil. Computational pharmacokinetic and pharmacodynamic screening suggested that phytochemical heterodimers would display useful gastrointestinal absorption and with relatively low predicted toxicity. Collectively, the present study suggests that phytochemicals could be garnered for the provision of novel ChEIs with enhanced drug efficacy and low toxicity.     

Surface behavior of peptides from E1 GBV-C protein: Interaction with anionic model membranes and importance in HIV-1 FP inhibition

The interaction between a peptide sequence from GB virus C E1 protein (E1P8) and its structural analogs (E1P8-12), (E1P8-13), and (E1P8-21) with anionic lipid membranes (POPG vesicles and POPG, DPPG or DPPC/DPPG (2:1) monolayers) and their association with HIV-1 fusion peptide (HIV-1 FP) inhibition at the membrane level were studied using biophysical methods. All peptides showed surface activity but leakage experiments in vesicles as well as insertion kinetics in monolayers and lipid/peptide miscibility indicated a low level of interaction: neither E1P8 nor its analogs induced the release of vesicular content and the exclusion pressure values (π_e) were clearly lower than the biological membrane pressure (24–30 mN m^− 1) and the HIV-1 FP (35 mN m^− 1). Miscibility was elucidated in terms of the additivity rule and excess free energy of mixing (G^E). E1P8, E1P8-12 and E1P8-21 (but not E1P8-13) induced expansion of the POPG monolayer. The mixing process is not thermodynamically favored as the positive G^E values indicate. To determine how E1 peptides interfere in the action of HIV-1 FP at the membrane level, mixed monolayers of HIV-1 FP/E1 peptides (2:1) and POPG were obtained. E1P8 and its derivative E1P8-21 showed the greatest HIV-1 FP inhibition. The LC-LE phase lipid behavior was morphologically examined via fluorescence microscopy (FM) and atomic force microscopy (AFM). Images revealed that the E1 peptides modify HIV-1 FP–lipid interaction. This fact may be attributed to a peptide/peptide interaction as indicated by AFM results. Finally, hemolysis assay demonstrated that E1 peptides inhibit HIV-1 FP activity.     

Development of a fluorogenic ADAMTS-7 substrate

The extracellular protease ADAMTS-7 has been identified as a potential therapeutic target in atherosclerosis and associated diseases such as coronary artery disease (CAD). However, ADAMTS-7 inhibitors have not been reported so far. Screening of inhibitors has been hindered by the lack of a suitable peptide substrate and, consequently, a convenient activity assay. Here we describe the first fluorescence resonance energy transfer (FRET) substrate for ADAMTS-7, ATS7FP7. ATS7FP7 was used to measure inhibition constants for the endogenous ADAMTS-7 inhibitor, TIMP-4, as well as two hydroxamate-based zinc chelating inhibitors. These inhibition constants match well with IC₅₀ values obtained with our SDS-PAGE assay that uses the N-terminal fragment of latent TGF-β–binding protein 4 (LTBP4S-A) as a substrate. Our novel fluorogenic substrate ATS7FP7 is suitable for high throughput screening of ADAMTS-7 inhibitors, thus accelerating translational studies aiming at inhibition of ADAMTS-7 as a novel treatment for cardiovascular diseases such as atherosclerosis and CAD.     

The changes of DNA double-strand breaks and DNA repair during ovarian reserve formation in mice

DNA double-strand break (DSB) repair is crucial to maintain genomic stability for sufficient ovarian reserve. It remains unknown the changes of DSBs formation and DNA repair in germ cells during ovarian reserve formation in FVB/N mice. We demonstrated germ cell numbers increased significantly (all P < 0.05) from E11.5 to E13.5 and decreased significantly (all P> 0.05) until P2. OCT4 and SOX2 analyses indicated pluripotency peaks at E13.5 then decreases significantly (all P 0.05) until P2. γH2AX analyses revealed DSB formation significantly (P < 0.05) increased from E13.5 until P2. RAD51 and DMC1 data revealed homologous recombination (HR) pathway repair of DSBs is persistent active during meiosis (E13.5- P2) (all P> 0.05). 53BP1 and KU70 data indicate the non-homologous end-joining pathway (NHEJ) remains active during meiosis. 53BP1 expression was highest at E13.5 (P < 0.05). KU70 expression was higher in germ cells from E15.5 to P2 (all < P 0.05). PH3 and KI67 analyses revealed germ cell proliferation was not significantly different (all P> 0.05) from E13.5 to P2. Caspase-3 and TUNEL analyses showed germ cells apoptosis was not significantly different (all P > 0.05) from E13.5 to P2. In conclusion, we found both germ cell number and pluripotency peak at E13.5 and decline during meiosis. We demonstrated HR and NHEJ continually repair DSBs during meiosis. RAD51 and DMC1 are continuously expressed during meiosis. 53BP1 is mainly expressed at E13.5. KU70 continually functions from E15.5 to P2. Proliferating and apoptotic cells were rarely detected during meiosis. Results provide a basis for further study of how DSBs and DNA repair affect germ cell development.     

Simultaneously monitoring DNA binding and helicase-catalyzed DNA unwinding by fluorescence polarization

A new method for helicase-catalyzed DNA unwinding is described. This assay takes advantage of the substantial change in fluorescence polarization (FP) upon helicase binding and DNA unwinding. The low anisotropy value, due to the fast tumbling of the free oligonucleotide in solution, increases abruptly upon binding of helicase to the fluorescein-labeled oligonucleotide. The high anisotropy of the helicase– DNA complex decreases as the fluorescein-labeled oligonucleotide is released from the complex through helicase-catalyzed DNA unwinding. This FP signal can be measured in real time by fluorescent spectroscopy. This assay can simultaneously monitor DNA binding and helicase-catalyzed DNA unwinding. It can also be used to determine the polarity in DNA unwinding mediated by helicase. This FP assay should facilitate the study of the mechanism by which helicase unwinds duplex DNA, and also aid in screening for helicase inhibitors, which are of growing interest as potential anticancer agents.     

Substitution of basic amino acids in the basic region stabilizes DNA binding by E12 homodimers.

The E2A gene encodes two alternatively spliced products, E12 and E47. The two proteins differ in their basic helix-loop-helix motifs (bHLH), responsible for DNA binding and dimerization. Although both E12 and E47 can bind to DNA as heterodimers with tissue-specific bHLH proteins, E12 binds to DNA poorly as homodimers. An inhibitory domain in E12 has previously been found to prevent E12 homodimers from binding to DNA. By measuring the dissociation rates using filter binding and electrophoretic mobility shift assays, we have shown here that the inhibitory domain interferes with DNA binding by destabilizing the DNA-protein complexes. Furthermore, we have demonstrated that substitution of basic amino acids (not other amino acids) in the DNA-binding domain of E12 can increase the intrinsic DNA-binding activity of E12 and stabilize the binding complexes, thus alleviating the repression from the inhibitory domain. This ability of basic amino acids to stabilize DNA-binding complexes may be of biological significance in the case of myogenic bHLH proteins, which all possess two more basic amino acids in their DNA binding domain than E12. To function as heterodimers with E12, the myogenic bHLH proteins may need stronger DNA binding domains.     

Prostaglandin E2 stimulates p53 transactivational activity through specific serine 15 phosphorylation in human synovial fibroblasts. Role in suppression of c/EBP/NF-kappaB-mediated MEKK1-induced MMP-1 expression

Cyclooxygenase-2 (COX-2) overexpression has been linked to cell survival, transformation, and hyperproliferation. We examined the regulation of the tumor suppressor gene p53 and p53 target genes by prostaglandin E(2) (PGE(2)) in human synovial fibroblasts (HSF). PGE(2) induced a time-dependent increase in p53 Ser(15) phosphorylation, with no discernible change in overall p53 levels. PGE(2)-dependent Ser(15) phosphorylation was apparently mediated by activated p38 MAP kinase as SB202190, a p38 kinase inhibitor, blocked the response. Overexpression of a MKK3 construct, but not MKK1, stimulated SB202190-sensitive p53 Ser(15) phosphorylation. PGE(2)-stimulated [phospho-Ser(15)]p53 transactivated a p53 response element (GADD45)-luciferase reporter in transiently transfected HSF (SN7); the effect was compromised by overexpression of a dominant-negative mutant (dnm) of p53 or excess p53S15A expression plasmid but mimicked by a constitutively active p53S15E expression construct. PGE(2), wtp53 expression in the presence of PGE(2), and p53S15E suppressed steady-state levels of MEKK1-induced MMP-1 mRNA, effects nullified with co-transfection of p53 dnm or p53S15A. MEKK1-induced MMP-1 promoter-driven luciferase activity was largely dependent on a c/EBPbeta-NF-kappaB-like enhancer site at -2008 to -1972 bp, as judged by deletion and point mutation analyses. PGE(2), overexpression of p53wt with PGE(2), or p53S15E abolished the MEKK1-induced MMP-1 promoter luciferase activity. Gel-shift/super gel-shift analyses identified c/EBPbeta dimers and c/EBPbeta/NF-kappaB p65 heterodimers as binding species at the apparent site of MEKK1-dependent transactivation. PGE(2)-stimulated [phospho-Ser(15)]p53 abrogated the DNA binding of c/EBPbeta dimers and c/EBPbeta/NF-kappaB p65 heterodimers. Our data suggest that COX-2 prostaglandins may be implicated in p53 function and p53 target gene expression.