N-Gemini peptides: cytosolic protease resistance via N-terminal dimerization of unstructured peptides.
Ontology highlight
ABSTRACT:
SUBMITTER: Fayer EL
PROVIDER: S-EPMC6034174 | biostudies-literature | 2017 Dec
REPOSITORIES: biostudies-literature
Publications
N-Gemini peptides: cytosolic protease resistance via N-terminal dimerization of unstructured peptides.
Fayer Effrat L EL Gilliland William M WM Ramsey J Michael JM Allbritton Nancy L NL Waters Marcey L ML
Chemical communications (Cambridge, England) 20171201 2
Herein we describe a synthetically simple strategy for increasing the lifetime of unstructured peptides in cytosolic environment via dimerization at the N-terminus to block threading into the catalytic cleft of cytosolic proteases. We establish this approach with kinase substrates, allowing for phosphorylation in cells as a demonstration of protease resistance. ...[more]
Similar Datasets
Project description:Aggregation of amyloid beta (A?) into oligomers and fibrils is believed to play an important role in the development of Alzheimer's disease (AD). To gain further insight into the principles of aggregation, we have investigated the induction of ?-sheet secondary conformation from disordered native peptide sequences through lipidation, in 1-2% hexafluoroisopropanol (HFIP) in phosphate buffered saline (PBS). Several parameters, such as type and number of lipid chains, peptide sequence, peptide length and net charge, were explored keeping the ratio peptide/HFIP constant. The resulting lipoconjugates were characterized by several physico-chemical techniques: Circular Dichroism (CD), Attenuated Total Reflection InfraRed (ATR-IR), Thioflavin T (ThT) fluorescence, Dynamic Light Scattering (DLS), solid-state Nuclear Magnetic Resonance (ssNMR) spectroscopy and Electron Microscopy (EM). Our data demonstrate the generation of ?-sheet aggregates from numerous unstructured peptides under physiological pH, independent of the amino acid sequence. The amphiphilicity pattern and hydrophobicity of the scaffold were found to be key factors for their assembly into amyloid-like structures.
| S-EPMC4160191 | biostudies-other
Project description:Organelle transport requires dynamic cytoskeleton remodeling, but whether cytoskeletal dynamics are, in turn, regulated by organelles remains elusive. Here, we demonstrate that late endosomes, a type of prelysosomal organelles, facilitate actin-cytoskeleton remodeling via cytosolic translocation of immature protease cathepsin D (cathD) during microglia migration. After cytosolic translocation, late endosome-derived cathD juxtaposes actin filaments at the leading edge of lamellipodia. Suppressing cathD expression or blocking its cytosolic translocation impairs the maintenance but not the initiation of lamellipodial extension. Moreover, immature cathD balances the activity of the actin-severing protein cofilin to maintain globular-actin (G-actin) monomer pool for local actin recycling. Our study identifies cathD as a key lysosomal molecule that unconventionally contributes to actin cytoskeleton remodeling via cytosolic translocation during adenosine triphosphate-evoked microglia migration.
| S-EPMC7725477 | biostudies-literature
Project description:HslVU is an ATP-dependent proteolytic complex present in certain bacteria and in the mitochondrion of some primordial eukaryotes, including deadly parasites such as Leishmania. It is formed by the dodecameric protease HslV and the hexameric ATPase HslU, which binds via the C-terminal end of its subunits to HslV and activates it by a yet unclear allosteric mechanism. We undertook the characterization of HslV from Leishmania major (LmHslV), a trypanosomatid that expresses two isoforms for HslU, LmHslU1 and LmHslU2. Using a novel and sensitive peptide substrate, we found that LmHslV can be activated by peptides derived from the C-termini of both LmHslU1 and LmHslU2. Truncations, Ala- and D-scans of the C-terminal dodecapeptide of LmHslU2 (LmC12-U2) showed that five out of the six C-terminal residues of LmHslU2 are essential for binding to and activating HslV. Peptide cyclisation with a lactam bridge allowed shortening of the peptide without loss of potency. Finally, we found that dodecapeptides derived from HslU of other parasites and bacteria are able to activate LmHslV with similar or even higher efficiency. Importantly, using electron microscopy approaches, we observed that the activation of LmHslV was accompanied by a large conformational remodeling, which represents a yet unidentified layer of control of HslV activation.
| S-EPMC6429459 | biostudies-literature
Project description:The C-terminal Eps 15 Homology Domain proteins (EHD1-4) play important roles in regulating endocytic trafficking. EHD2 is the only family member whose crystal structure has been solved, and it contains an unstructured loop consisting of two proline-phenylalanine (PF) motifs: KPFRKLNPF. In contrast, despite EHD2 having nearly 70% amino acid identity with its paralogs, EHD1, EHD3 and EHD4, the latter proteins contain a single KPF or RPF motif, but no NPF motif. In this study, we sought to define the precise role of each PF motif in EHD2's homo-dimerization, binding with the protein partners, and subcellular localization. To test the role of the NPF motif, we generated an EHD2 NPF-to-NAF mutant to mimic the homologous sequences of EHD1 and EHD3. We demonstrated that this mutant lost both its ability to dimerize and bind to Syndapin2. However, it continued to localize primarily to the cytosolic face of the plasma membrane. On the other hand, EHD2 NPF-to-APA mutants displayed normal dimerization and Syndapin2 binding, but exhibited markedly increased nuclear localization and reduced association with the plasma membrane. We then hypothesized that the single PF motif of EHD1 (that aligns with the KPF of EHD2) might be responsible for both binding and localization functions of EHD1. Indeed, the EHD1 RPF motif was required for dimerization, interaction with MICAL-L1 and Syndapin2, as well as localization to tubular recycling endosomes. Moreover, recycling assays demonstrated that EHD1 RPF-to-APA was incapable of supporting normal receptor recycling. Overall, our data suggest that the EHD2 NPF phenylalanine residue is crucial for EHD2 localization to the plasma membrane, whereas the proline residue is essential for EHD2 dimerization and binding. These studies support the recently proposed model in which the EHD2 N-terminal region may regulate the availability of the unstructured loop for interactions with neighboring EHD2 dimers, thus promoting oligomerization.
| S-EPMC4398442 | biostudies-literature
Project description:Bottom-up proteomics is a mainstay in protein identification and analysis. These studies typically employ proteolytic treatment of biological samples to generate suitably sized peptides for tandem mass spectrometric (MS) analysis. In MS, fragmentation of peptides is largely driven by charge localization. Consequently, peptides with basic centers exclusively on their N-termini produce mainly b-ions. Thus, it was long ago realized that proteases that yield such peptides would be valuable proteomic tools for achieving simplified peptide fragmentation patterns and peptide assignment. Work by several groups has identified such proteases, however, structural analysis of these suggested that enzymatic optimization was possible. We therefore endeavored to find enzymes that could provide enhanced activity and versatility while maintaining specificity. Using these previously described proteases as informatic search templates, we discovered and then characterized a thermophilic metalloprotease with N-terminal specificity for arginine and lysine. This enzyme, dubbed Tryp-N, affords many advantages including improved thermostability, solvent and detergent tolerance, and rapid digestion time.
| S-EPMC7842235 | biostudies-literature
Project description:HTLV-1 [HTLV (human T-cell lymphotrophic virus) type 1] is associated with a number of human diseases. HTLV-1 protease is essential for virus replication, and similarly to HIV-1 protease, it is a potential target for chemotherapy. The primary sequence of HTLV-1 protease is substantially longer compared with that of HIV-1 protease, and the role of the ten C-terminal residues is controversial. We have expressed C-terminally-truncated forms of HTLV-1 protease with and without N-terminal His tags. Removal of five of the C-terminal residues caused a 4-40-fold decrease in specificity constants, whereas the removal of an additional five C-terminal residues rendered the protease completely inactive. The addition of the N-terminal His tag dramatically decreased the activity of HTLV-1 protease forms. Pull-down experiments carried out with His-tagged forms, gel-filtration experiments and dimerization assays provided the first unequivocal experimental results for the role of the C-terminal residues in dimerization of the enzyme. There is a hydrophobic tunnel on the surface of HTLV-1 protease close to the C-terminal ends that is absent in the HIV-1 protease. This hydrophobic tunnel can accommodate the extra C-terminal residues of HTLV-1 protease, which was predicted to stabilize the dimer of the full-length enzyme and provides an alternative target site for protease inhibition.
| S-EPMC2778851 | biostudies-literature
Project description:Protein homeostasis is critically important for cell viability. Key to this process is the refolding of misfolded or aggregated proteins by molecular chaperones or, alternatively, their degradation by proteases. In most prokaryotes and in chloroplasts and mitochondria, protein degradation is performed by the caseinolytic protease ClpP, a tetradecamer barrel-like proteolytic complex. Dysregulating ClpP function has shown promise in fighting antibiotic resistance and as a potential therapy for acute myeloid leukemia. Here we use methyl-transverse relaxation-optimized spectroscopy (TROSY)-based NMR, cryo-EM, biochemical assays, and molecular dynamics simulations to characterize the structural dynamics of ClpP from Staphylococcus aureus (SaClpP) in wild-type and mutant forms in an effort to discover conformational hotspots that regulate its function. Wild-type SaClpP was found exclusively in the active extended form, with the N-terminal domains of its component protomers in predominantly ?-hairpin conformations that are less well-defined than other regions of the protein. A hydrophobic site was identified that, upon mutation, leads to unfolding of the N-terminal domains, loss of SaClpP activity, and formation of a previously unobserved split-ring conformation with a pair of 20-Å-wide pores in the side of the complex. The extended form of the structure and partial activity can be restored via binding of ADEP small-molecule activators. The observed structural plasticity of the N-terminal gates is shown to be a conserved feature through studies of Escherichia coli and Neisseria meningitidis ClpP, suggesting a potential avenue for the development of molecules to allosterically modulate the function of ClpP.
| S-EPMC6048506 | biostudies-literature
Project description:Dimerization of HIV-1 protease (PR) subunits is an essential process for PR's acquisition of proteolytic activity, which plays a critical role in the maturation of HIV-1. Recombinant wild-type PR (PR(WT)) proved to dimerize, as examined with electrospray ionization mass spectrometry; however, two active site interface PR mutants (PR(T26A) and PR(R87K)) remained monomeric. On the other hand, two termini interface PR mutants (PR(1-C95A) and PR(97/99)) took both monomeric and dimeric forms. Differential scanning fluorimetry indicated that PR(1-C95A) and PR(97/99) dimers were substantially less stable than PR(WT) dimers. These data indicate that intermolecular interactions of two monomers occur first at the active site interface, generating unstable or transient dimers, and interactions at the termini interface subsequently occur, generating stable dimers. Darunavir (DRV), an HIV-1 protease inhibitor, inhibits not only proteolytic activity but also PR dimerization. DRV bound to protease monomers in a one-to-one molar ratio, inhibiting the first step of PR dimerization, whereas conventional protease inhibitors (such as saquinavir) that inhibit enzymatic activity but not dimerization failed to bind to monomers. DRV also bound to mutant PRs containing the transframe region-added PR (TFR-PR(D25N) and TFR-PR(D25N-7AA)), whereas saquinavir did not bind to TFR-PR(D25N) or TFR-PR(D25N-7AA). Notably, DRV failed to bind to mutant PR containing four amino acid substitutions (V32I, L33F, I54M, and I84V) that confer resistance to DRV on HIV-1. To our knowledge, the present report represents the first demonstration of the two-step PR dimerization dynamics and the mechanism of dimerization inhibition by DRV, which should help design further, more potent novel PIs.
| S-EPMC4142999 | biostudies-literature
Project description:DNA damage checkpoint pathways operate to prevent cell-cycle progression in response to DNA damage and replication stress. In S. cerevisiae, Mec1-Ddc2 (human ATR-ATRIP) is the principal checkpoint protein kinase. Biochemical studies have identified two factors, the 9-1-1 checkpoint clamp and the Dpb11/TopBP1 replication protein, as potential activators of Mec1/ATR. Here, we show that G1 phase checkpoint activation of Mec1 is achieved by the Ddc1 subunit of 9-1-1, while Dpb11 is dispensable. However, in G2, 9-1-1 activates Mec1 by two distinct mechanisms. One mechanism involves direct activation of Mec1 by Ddc1, while the second proceeds by Dpb11 recruitment mediated through Ddc1 T602 phosphorylation. Two aromatic residues, W352 and W544, localized to two widely separated, conserved motifs of Ddc1, are essential for Mec1 activation in vitro and checkpoint function in G1. Remarkably, small peptides that fuse the two tryptophan-containing motifs together are proficient in activating Mec1.
| S-EPMC2796261 | biostudies-literature
Project description:Gene silencing in budding yeast relies on the binding of the Silent Information Regulator (Sir) complex to chromatin, which is mediated by extensive interactions between the Sir proteins and nucleosomes. Sir3, a divergent member of the AAA+ ATPase-like family, contacts both the histone H4 tail and the nucleosome core. Here, we present the structure and function of the conserved C-terminal domain of Sir3, comprising 138 amino acids. This module adopts a variant winged helix-turn-helix (wH) architecture that exists as a stable homodimer in solution. Mutagenesis shows that the self-association mediated by this domain is essential for holo-Sir3 dimerization. Its loss impairs Sir3 loading onto nucleosomes in vitro and eliminates silencing at telomeres and HM loci in vivo. Replacing the Sir3 wH domain with an unrelated bacterial dimerization motif restores both HM and telomeric repression in sir3? cells. In contrast, related wH domains of archaeal and human members of the Orc1/Sir3 family are monomeric and have DNA binding activity. We speculate that a dimerization function for the wH evolved with Sir3's ability to facilitate heterochromatin formation.
| S-EPMC3567499 | biostudies-literature