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16 pages, 1716 KiB

Open AccessArticle

Designing Cyclic-Constrained Peptides to Inhibit Human Phosphoglycerate Dehydrogenase

by Xiaoyu Qing, Qian Wang, Hanyu Xu, Pei Liu and Luhua Lai

Molecules 2023, 28(17), 6430; https://doi.org/10.3390/molecules28176430 - 4 Sep 2023

Cited by 1 | Viewed by 1675

Abstract

Although loop epitopes at protein-protein binding interfaces often play key roles in mediating oligomer formation and interaction specificity, their binding sites are underexplored as drug targets owing to their high flexibility, relatively few hot spots, and solvent accessibility. Prior attempts to develop molecules [...] Read more.

Although loop epitopes at protein-protein binding interfaces often play key roles in mediating oligomer formation and interaction specificity, their binding sites are underexplored as drug targets owing to their high flexibility, relatively few hot spots, and solvent accessibility. Prior attempts to develop molecules that mimic loop epitopes to disrupt protein oligomers have had limited success. In this study, we used structure-based approaches to design and optimize cyclic-constrained peptides based on loop epitopes at the human phosphoglycerate dehydrogenase (PHGDH) dimer interface, which is an obligate homo-dimer with activity strongly dependent on the oligomeric state. The experimental validations showed that these cyclic peptides inhibit PHGDH activity by directly binding to the dimer interface and disrupting the obligate homo-oligomer formation. Our results demonstrate that loop epitope derived cyclic peptides with rationally designed affinity-enhancing substitutions can modulate obligate protein homo-oligomers, which can be used to design peptide inhibitors for other seemingly intractable oligomeric proteins. Full article

(This article belongs to the Special Issue Peptide and Protein Chemistry: Design, Synthesis, and Applications)

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20 pages, 8039 KiB

Open AccessArticle

Antiviral Evaluation of New Synthetic Bioconjugates Based on GA-Hecate: A New Class of Antivirals Targeting Different Steps of Zika Virus Replication

by Paulo Ricardo da Silva Sanches, Ricardo Sanchez Velazquez, Mariana Nogueira Batista, Bruno Moreira Carneiro, Cintia Bittar, Giuditta De Lorenzo, Paula Rahal, Arvind H. Patel and Eduardo Maffud Cilli

Molecules 2023, 28(13), 4884; https://doi.org/10.3390/molecules28134884 - 21 Jun 2023

Cited by 2 | Viewed by 1533

Abstract

Re-emerging arboviruses represent a serious health problem due to their rapid vector-mediated spread, mainly in urban tropical areas. The 2013–2015 Zika virus (ZIKV) outbreak in South and Central America has been associated with cases of microcephaly in newborns and Guillain–Barret syndrome. We previously [...] Read more.

Re-emerging arboviruses represent a serious health problem due to their rapid vector-mediated spread, mainly in urban tropical areas. The 2013–2015 Zika virus (ZIKV) outbreak in South and Central America has been associated with cases of microcephaly in newborns and Guillain–Barret syndrome. We previously showed that the conjugate gallic acid—Hecate (GA-FALALKALKKALKKLKKALKKAL-CONH₂)—is an efficient inhibitor of the hepatitis C virus. Here, we show that the Hecate peptide is degraded in human blood serum into three major metabolites. These metabolites conjugated with gallic acid were synthesized and their effect on ZIKV replication in cultured cells was evaluated. The GA-metabolite 5 (GA-FALALKALKKALKKL-COOH) was the most efficient in inhibiting two ZIKV strains of African and Asian lineage at the stage of both virus entry (virucidal and protective) and replication (post-entry). We also demonstrate that GA-metabolite 5 does not affect cell growth after 7 days of continuous treatment. Thus, this study identifies a new synthetic antiviral compound targeting different steps of ZIKV replication in vitro and with the potential for broad reactivity against other flaviviruses. Our work highlights a promising strategy for the development of new antivirals based on peptide metabolism and bioconjugation. Full article

(This article belongs to the Special Issue Peptide and Protein Chemistry: Design, Synthesis, and Applications)

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13 pages, 2028 KiB

Open AccessArticle

Thiocholine-Mediated One-Pot Peptide Ligation and Desulfurization

by Sae Suzuki, Yuya Nakajima, Naoki Kamo, Akihisa Osakabe, Akimitsu Okamoto, Gosuke Hayashi and Hiroshi Murakami

Molecules 2023, 28(9), 3655; https://doi.org/10.3390/molecules28093655 - 22 Apr 2023

Cited by 4 | Viewed by 2854

Abstract

Thiol catalysts are essential in native chemical ligation (NCL) to increase the reaction efficiency. In this paper, we report the use of thiocholine in chemical protein synthesis, including NCL-based peptide ligation and metal-free desulfurization. Evaluation of thiocholine peptide thioester in terms of NCL [...] Read more.

Thiol catalysts are essential in native chemical ligation (NCL) to increase the reaction efficiency. In this paper, we report the use of thiocholine in chemical protein synthesis, including NCL-based peptide ligation and metal-free desulfurization. Evaluation of thiocholine peptide thioester in terms of NCL and hydrolysis kinetics revealed its practical utility, which was comparable to that of other alkyl thioesters. Importantly, thiocholine showed better reactivity as a thiol additive in desulfurization, which is often used in chemical protein synthesis to convert Cys residues to more abundant Ala residues. Finally, we achieved chemical synthesis of two differently methylated histone H3 proteins via one-pot NCL and desulfurization with thiocholine. Full article

(This article belongs to the Special Issue Peptide and Protein Chemistry: Design, Synthesis, and Applications)

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11 pages, 2604 KiB

Open AccessArticle

Region Met225 to Ile412 of Bacillus cereus Hemolysin II Is Capable to Agglutinate Red Blood Cells

by Alexey S. Nagel, Natalia V. Rudenko, Polina N. Luchkina, Anna P. Karatovskaya, Anna V. Zamyatina, Zhanna I. Andreeva-Kovalevskaya, Alexander V. Siunov, Fedor A. Brovko and Alexander S. Solonin

Molecules 2023, 28(8), 3581; https://doi.org/10.3390/molecules28083581 - 19 Apr 2023

Cited by 2 | Viewed by 1441

Abstract

Hemolysin II (HlyII) is one of the virulence factors of the opportunistic bacterium Bacillus cereus belonging to the group of β-pore-forming toxins. This work created a genetic construct encoding a large C-terminal fragment of the toxin (HlyIILCTD, M225–I412 according to the numbering of [...] Read more.

Hemolysin II (HlyII) is one of the virulence factors of the opportunistic bacterium Bacillus cereus belonging to the group of β-pore-forming toxins. This work created a genetic construct encoding a large C-terminal fragment of the toxin (HlyIILCTD, M225–I412 according to the numbering of amino acid residues in HlyII). A soluble form of HlyIILCTD was obtained using the SlyD chaperone protein. HlyIILCTD was first shown to be capable of agglutinating rabbit erythrocytes. Monoclonal antibodies against HlyIILCTD were obtained by hybridoma technology. We also proposed a mode of rabbit erythrocyte agglutination by HlyIILCTD and selected three anti-HlyIILCTD monoclonal antibodies that inhibited the agglutination. Full article

(This article belongs to the Special Issue Peptide and Protein Chemistry: Design, Synthesis, and Applications)

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15 pages, 1718 KiB

Open AccessArticle

Selective Grafting of Protease-Resistant Adhesive Peptides on Titanium Surfaces

by Annj Zamuner, Antonella Pasquato, Ignazio Castagliuolo, Monica Dettin and Paola Brun

Molecules 2022, 27(24), 8727; https://doi.org/10.3390/molecules27248727 - 9 Dec 2022

Cited by 3 | Viewed by 1312

Abstract

In orthopedic, dental, and maxillofacial fields, joint prostheses, plates, and screws are widely used in the treatment of problems related to bone tissue. However, the use of these prosthetic systems is not free from complications: the fibrotic encapsulation of endosseous implants often prevents [...] Read more.

In orthopedic, dental, and maxillofacial fields, joint prostheses, plates, and screws are widely used in the treatment of problems related to bone tissue. However, the use of these prosthetic systems is not free from complications: the fibrotic encapsulation of endosseous implants often prevents optimal integration of the prostheses with the surrounding bone. To overcome these issues, biomimetic titanium implants have been developed where synthetic peptides have been selectively grafted on titanium surfaces via Schiff base formation. We used the retro-inverted sequence (DHVPX) from [351–359] human Vitronectin and its dimer (D2HVP). Both protease-resistant peptides showed increased human osteoblast adhesion and proliferation, an augmented number of focal adhesions, and cellular spreading with respect to the control. D2HVP-grafted samples significantly enhance Secreted Phosphoprotein 1, Integrin Binding Sialoprotein, and Vitronectin gene expression vs. control. An estimation of peptide surface density was determined by Two-photon microscopy analysis on a silanized glass model surface labeled with a fluorescent analog. Full article

(This article belongs to the Special Issue Peptide and Protein Chemistry: Design, Synthesis, and Applications)

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13 pages, 5870 KiB

Open AccessArticle

Degradation-Suppressed Cocoonase for Investigating the Propeptide-Mediated Activation Mechanism

by Nana Sakata, Ayumi Ogata, Mai Takegawa, Yuri Murakami, Misaki Nishimura, Mitsuhiro Miyazawa, Teruki Hagiwara, Shigeru Shimamoto and Yuji Hidaka

Molecules 2022, 27(22), 8063; https://doi.org/10.3390/molecules27228063 - 20 Nov 2022

Cited by 2 | Viewed by 1923

Abstract

Cocoonase is folded in the form of a zymogen precursor protein (prococoonase) with the assistance of the propeptide region. To investigate the role of the propeptide sequence on the disulfide-coupled folding of cocoonase and prococoonase, the amino acid residues at the degradation sites [...] Read more.

Cocoonase is folded in the form of a zymogen precursor protein (prococoonase) with the assistance of the propeptide region. To investigate the role of the propeptide sequence on the disulfide-coupled folding of cocoonase and prococoonase, the amino acid residues at the degradation sites during the refolding and auto-processing reactions were determined by mass spectrometric analyses and were mutated to suppress the numerous degradation reactions that occur during the reactions. In addition, the Lys⁸ residue at the propeptide region was also mutated to estimate whether the entire sequence is absolutely required for the activation of cocoonase. Finally, a degradation-suppressed [K8D,K63G,K131G,K133A]-proCCN protein was prepared and was found to refold readily without significant degradation. The results of an enzyme assay using casein or Bz-Arg-OEt suggested that the mutations had no significant effect on either the enzyme activity or the protein conformation. Thus, we, herein, provide the non-degradative cocoonase protein to investigate the propeptide-mediated protein folding of the molecule. We also examined the catalytic residues using the degradation-suppressed cocoonase. The point mutations at the putative catalytic residues in cocoonase resulted in the loss of catalytic activity without any secondary structural changes, indicating that the mutated residues play a role in the catalytic activity of this enzyme. Full article

(This article belongs to the Special Issue Peptide and Protein Chemistry: Design, Synthesis, and Applications)

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18 pages, 5521 KiB

Open AccessArticle

Antibody for Serine 65 Phosphorylated Ubiquitin Identifies PLK1-Mediated Phosphorylation of Mitotic Proteins and APC1

by Guy Mann, Prasad Sulkshane, Pradeep Sadhu, Tamar Ziv, Michael H. Glickman and Ashraf Brik

Molecules 2022, 27(15), 4867; https://doi.org/10.3390/molecules27154867 - 29 Jul 2022

Viewed by 2518

Abstract

Deciphering the protein posttranslational modification (PTM) code is one of the greatest biochemical challenges of our time. Phosphorylation and ubiquitylation are key PTMs that dictate protein function, recognition, sub-cellular localization, stability, turnover and fate. Hence, failures in their regulation leads to various disease. [...] Read more.

Deciphering the protein posttranslational modification (PTM) code is one of the greatest biochemical challenges of our time. Phosphorylation and ubiquitylation are key PTMs that dictate protein function, recognition, sub-cellular localization, stability, turnover and fate. Hence, failures in their regulation leads to various disease. Chemical protein synthesis allows preparation of ubiquitinated and phosphorylated proteins to study their biochemical properties in great detail. However, monitoring these modifications in intact cells or in cell extracts mostly depends on antibodies, which often have off-target binding. Here, we report that the most widely used antibody for ubiquitin (Ub) phosphorylated at serine 65 (pUb) has significant off-targets that appear during mitosis. These off-targets are connected to polo-like kinase 1 (PLK1) mediated phosphorylation of cell cycle-related proteins and the anaphase promoting complex subunit 1 (APC1). Full article

(This article belongs to the Special Issue Peptide and Protein Chemistry: Design, Synthesis, and Applications)

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Review

Jump to: Research

31 pages, 3715 KiB

Open AccessReview

Conformational Stability and Denaturation Processes of Proteins Investigated by Electrophoresis under Extreme Conditions

by Patrick Masson and Sofya Lushchekina

Molecules 2022, 27(20), 6861; https://doi.org/10.3390/molecules27206861 - 13 Oct 2022

Cited by 16 | Viewed by 9139

Abstract

The functional structure of proteins results from marginally stable folded conformations. Reversible unfolding, irreversible denaturation, and deterioration can be caused by chemical and physical agents due to changes in the physicochemical conditions of pH, ionic strength, temperature, pressure, and electric field or due [...] Read more.

The functional structure of proteins results from marginally stable folded conformations. Reversible unfolding, irreversible denaturation, and deterioration can be caused by chemical and physical agents due to changes in the physicochemical conditions of pH, ionic strength, temperature, pressure, and electric field or due to the presence of a cosolvent that perturbs the delicate balance between stabilizing and destabilizing interactions and eventually induces chemical modifications. For most proteins, denaturation is a complex process involving transient intermediates in several reversible and eventually irreversible steps. Knowledge of protein stability and denaturation processes is mandatory for the development of enzymes as industrial catalysts, biopharmaceuticals, analytical and medical bioreagents, and safe industrial food. Electrophoresis techniques operating under extreme conditions are convenient tools for analyzing unfolding transitions, trapping transient intermediates, and gaining insight into the mechanisms of denaturation processes. Moreover, quantitative analysis of electrophoretic mobility transition curves allows the estimation of the conformational stability of proteins. These approaches include polyacrylamide gel electrophoresis and capillary zone electrophoresis under cold, heat, and hydrostatic pressure and in the presence of non-ionic denaturing agents or stabilizers such as polyols and heavy water. Lastly, after exposure to extremes of physical conditions, electrophoresis under standard conditions provides information on irreversible processes, slow conformational drifts, and slow renaturation processes. The impressive developments of enzyme technology with multiple applications in fine chemistry, biopharmaceutics, and nanomedicine prompted us to revisit the potentialities of these electrophoretic approaches. This feature review is illustrated with published and unpublished results obtained by the authors on cholinesterases and paraoxonase, two physiologically and toxicologically important enzymes. Full article

(This article belongs to the Special Issue Peptide and Protein Chemistry: Design, Synthesis, and Applications)

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18 pages, 3466 KiB

Open AccessReview

Using the Cyclotide Scaffold for Targeting Biomolecular Interactions in Drug Development

by Binu Jacob, Alicia Vogelaar, Enrique Cadenas and Julio A. Camarero

Molecules 2022, 27(19), 6430; https://doi.org/10.3390/molecules27196430 - 29 Sep 2022

Cited by 14 | Viewed by 2910

Abstract

This review provides an overview of the properties of cyclotides and their potential for developing novel peptide-based therapeutics. The selective disruption of protein–protein interactions remains challenging, as the interacting surfaces are relatively large and flat. However, highly constrained polypeptide-based molecular frameworks with cell-permeability [...] Read more.

This review provides an overview of the properties of cyclotides and their potential for developing novel peptide-based therapeutics. The selective disruption of protein–protein interactions remains challenging, as the interacting surfaces are relatively large and flat. However, highly constrained polypeptide-based molecular frameworks with cell-permeability properties, such as the cyclotide scaffold, have shown great promise for targeting those biomolecular interactions. The use of molecular techniques, such as epitope grafting and molecular evolution employing the cyclotide scaffold, has shown to be highly effective for selecting bioactive cyclotides. Full article

(This article belongs to the Special Issue Peptide and Protein Chemistry: Design, Synthesis, and Applications)

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17 pages, 3266 KiB

Open AccessReview

Posttranslational Chemical Mutagenesis Methods to Insert Posttranslational Modifications into Recombinant Proteins

by Omer Harel and Muhammad Jbara

Molecules 2022, 27(14), 4389; https://doi.org/10.3390/molecules27144389 - 8 Jul 2022

Cited by 11 | Viewed by 3273

Abstract

Posttranslational modifications (PTMs) dramatically expand the functional diversity of the proteome. The precise addition and removal of PTMs appears to modulate protein structure and function and control key regulatory processes in living systems. Deciphering how particular PTMs affect protein activity is a current [...] Read more.

Posttranslational modifications (PTMs) dramatically expand the functional diversity of the proteome. The precise addition and removal of PTMs appears to modulate protein structure and function and control key regulatory processes in living systems. Deciphering how particular PTMs affect protein activity is a current frontier in biology and medicine. The large number of PTMs which can appear in several distinct positions, states, and combinations makes preparing such complex analogs using conventional biological and chemical tools challenging. Strategies to access homogeneous and precisely modified proteins with desired PTMs at selected sites and in feasible quantities are critical to interpreting their molecular code. Here, we summarize recent advances in posttranslational chemical mutagenesis and late-stage functionalization chemistry to transfer novel PTM mimicry into recombinant proteins with emphasis on novel transformations. Full article

(This article belongs to the Special Issue Peptide and Protein Chemistry: Design, Synthesis, and Applications)

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