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16 pages, 6394 KiB

Open AccessArticle

Insulin-Degrading Enzyme Interacts with Mitochondrial Ribosomes and Respiratory Chain Proteins

by Ayse Yilmaz, Chiara Guerrera, Emmanuelle Waeckel-Énée, Joanna Lipecka, Barbara Bertocci and Peter van Endert

Biomolecules 2023, 13(6), 890; https://doi.org/10.3390/biom13060890 - 26 May 2023

Cited by 1 | Viewed by 1185

Abstract

Insulin-degrading enzyme (IDE) is a highly conserved metalloprotease that is mainly localized in the cytosol. Although IDE can degrade insulin and some other low molecular weight substrates efficiently, its ubiquitous expression suggests additional functions supported by experimental findings, such as a role in [...] Read more.

Insulin-degrading enzyme (IDE) is a highly conserved metalloprotease that is mainly localized in the cytosol. Although IDE can degrade insulin and some other low molecular weight substrates efficiently, its ubiquitous expression suggests additional functions supported by experimental findings, such as a role in stress responses and cellular protein homeostasis. The translation of a long full-length IDE transcript has been reported to result in targeting to mitochondria, but the role of IDE in this compartment is unknown. To obtain initial leads on the function of IDE in mitochondria, we used a proximity biotinylation approach to identify proteins interacting with wild-type and protease-dead IDE targeted to the mitochondrial matrix. We find that IDE interacts with multiple mitochondrial ribosomal proteins as well as with proteins involved in the synthesis and assembly of mitochondrial complex I and IV. The mitochondrial interactomes of wild type and mutant IDE are highly similar and do not reveal any likely proteolytic IDE substrates. We speculate that IDE could adopt similar additional non-proteolytic functions in mitochondria as in the cytosol, acting as a chaperone and contributing to protein homeostasis and stress responses. Full article

(This article belongs to the Special Issue Hemoproteins and Proteolytic Enzymes: Variations on the Theme of Functional Modulation: A Themed Issue in Honor of Professor Massimo Coletta)

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13 pages, 2495 KiB

Open AccessArticle

Electronic Circular Dichroism Detects Conformational Changes Associated with Proteasome Gating Confirmed Using AFM Imaging

by Alessandro D’Urso, Roberto Purrello, Alessandra Cunsolo, Danilo Milardi, Caterina Fattorusso, Marco Persico, Maria Gaczynska, Pawel A. Osmulski and Anna Maria Santoro

Biomolecules 2023, 13(4), 704; https://doi.org/10.3390/biom13040704 - 20 Apr 2023

Viewed by 1346

Abstract

Many chronic diseases, including cancer and neurodegeneration, are linked to proteasome dysregulation. Proteasome activity, essential for maintaining proteostasis in a cell, is controlled by the gating mechanism and its underlying conformational transitions. Thus, developing effective methods to detect gate-related specific proteasome conformations could [...] Read more.

Many chronic diseases, including cancer and neurodegeneration, are linked to proteasome dysregulation. Proteasome activity, essential for maintaining proteostasis in a cell, is controlled by the gating mechanism and its underlying conformational transitions. Thus, developing effective methods to detect gate-related specific proteasome conformations could be a significant contribution to rational drug design. Since the structural analysis suggests that gate opening is associated with a decrease in the content of α-helices and β-sheets and an increase in random coil structures, we decided to explore the application of electronic circular dichroism (ECD) in the UV region to monitor the proteasome gating. A comparison of ECD spectra of wild type yeast 20S proteasome (predominantly closed) and an open-gate mutant (α3ΔN) revealed an increased intensity in the ECD band at 220 nm, which suggests increased contents of random coil and β-turn structures. This observation was further supported by evaluating ECD spectra of human 20S treated with low concentration of SDS, known as a gate-opening reagent. Next, to evaluate the power of ECD to probe a ligand-induced gate status, we treated the proteasome with H2T4, a tetracationic porphyrin that we showed previously to induce large-scale protein conformational changes upon binding to h20S. H2T4 caused a significant increase in the ECD band at 220 nm, interpreted as an induced opening of the 20S gate. In parallel, we imaged the gate-harboring alpha ring of the 20S with AFM, a technique that we used previously to visualize the predominantly closed gate in latent human or yeast 20S and the open gate in α3ΔN mutant. The results were convergent with the ECD data and showed a marked decrease in the content of closed-gate conformation in the H2T4-treated h20S. Our findings provide compelling support for the use of ECD measurements to conveniently monitor proteasome conformational changes related to gating phenomena. We predict that the observed association of spectroscopic and structural results will help with efficient design and characterization of exogenous proteasome regulators. Full article

(This article belongs to the Special Issue Hemoproteins and Proteolytic Enzymes: Variations on the Theme of Functional Modulation: A Themed Issue in Honor of Professor Massimo Coletta)

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16 pages, 3321 KiB

Open AccessEditor’s ChoiceArticle

The Role of the Hydrogen Bond Network in Maintaining Heme Pocket Stability and Protein Function Specificity of C. diphtheriae Coproheme Decarboxylase

by Federico Sebastiani, Chiara Baroni, Gaurav Patil, Andrea Dali, Maurizio Becucci, Stefan Hofbauer and Giulietta Smulevich

Biomolecules 2023, 13(2), 235; https://doi.org/10.3390/biom13020235 - 25 Jan 2023

Cited by 4 | Viewed by 3591

Abstract

Monoderm bacteria accumulate heme b via the coproporphyrin-dependent biosynthesis pathway. In the final step, in the presence of two molecules of H₂O₂, the propionate groups of coproheme at positions 2 and 4 are decarboxylated to form vinyl groups by [...] Read more.

Monoderm bacteria accumulate heme b via the coproporphyrin-dependent biosynthesis pathway. In the final step, in the presence of two molecules of H₂O₂, the propionate groups of coproheme at positions 2 and 4 are decarboxylated to form vinyl groups by coproheme decarboxylase (ChdC), in a stepwise process. Decarboxylation of propionate 2 produces an intermediate that rotates by 90° inside the protein pocket, bringing propionate 4 near the catalytic tyrosine, to allow the second decarboxylation step. The active site of ChdCs is stabilized by an extensive H-bond network involving water molecules, specific amino acid residues, and the propionate groups of the porphyrin. To evaluate the role of these H-bonds in the pocket stability and enzyme functionality, we characterized, via resonance Raman and electronic absorption spectroscopies, single and double mutants of the actinobacterial pathogen Corynebacterium diphtheriae ChdC complexed with coproheme and heme b. The selective elimination of the H-bond interactions between propionates 2, 4, 6, and 7 and the polar residues of the pocket allowed us to establish the role of each H-bond in the catalytic reaction and to follow the changes in the interactions from the substrate to the product. Full article

(This article belongs to the Special Issue Hemoproteins and Proteolytic Enzymes: Variations on the Theme of Functional Modulation: A Themed Issue in Honor of Professor Massimo Coletta)

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22 pages, 2442 KiB

Open AccessEditor’s ChoiceArticle

Structural Basis of Sequential and Concerted Cooperativity

by Veronica Morea, Francesco Angelucci, Jeremy R. H. Tame, Enrico Di Cera and Andrea Bellelli

Biomolecules 2022, 12(11), 1651; https://doi.org/10.3390/biom12111651 - 07 Nov 2022

Cited by 4 | Viewed by 2316

Abstract

Allostery is a property of biological macromolecules featuring cooperative ligand binding and regulation of ligand affinity by effectors. The definition was introduced by Monod and Jacob in 1963, and formally developed as the “concerted model” by Monod, Wyman, and Changeux in 1965. Since [...] Read more.

Allostery is a property of biological macromolecules featuring cooperative ligand binding and regulation of ligand affinity by effectors. The definition was introduced by Monod and Jacob in 1963, and formally developed as the “concerted model” by Monod, Wyman, and Changeux in 1965. Since its inception, this model of cooperativity was seen as distinct from and not reducible to the “sequential model” originally formulated by Pauling in 1935, which was developed further by Koshland, Nemethy, and Filmer in 1966. However, it is difficult to decide which model is more appropriate from equilibrium or kinetics measurements alone. In this paper, we examine several cooperative proteins whose functional behavior, whether sequential or concerted, is established, and offer a combined approach based on functional and structural analysis. We find that isologous, mostly helical interfaces are common in cooperative proteins regardless of their mechanism. On the other hand, the relative contribution of tertiary and quaternary structural changes, as well as the asymmetry in the liganded state, may help distinguish between the two mechanisms. Full article

(This article belongs to the Special Issue Hemoproteins and Proteolytic Enzymes: Variations on the Theme of Functional Modulation: A Themed Issue in Honor of Professor Massimo Coletta)

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Review

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28 pages, 12876 KiB

Open AccessReview

The Insulin-Degrading Enzyme from Structure to Allosteric Modulation: New Perspectives for Drug Design

by Grazia Raffaella Tundo, Giuseppe Grasso, Marco Persico, Oleh Tkachuk, Francesco Bellia, Alessio Bocedi, Stefano Marini, Mariacristina Parravano, Grazia Graziani, Caterina Fattorusso and Diego Sbardella

Biomolecules 2023, 13(10), 1492; https://doi.org/10.3390/biom13101492 - 07 Oct 2023

Viewed by 1534

Abstract

The insulin-degrading enzyme (IDE) is a Zn²⁺ peptidase originally discovered as the main enzyme involved in the degradation of insulin and other amyloidogenic peptides, such as the β-amyloid (Aβ) peptide. Therefore, a role for the IDE in the cure of diabetes and [...] Read more.

The insulin-degrading enzyme (IDE) is a Zn²⁺ peptidase originally discovered as the main enzyme involved in the degradation of insulin and other amyloidogenic peptides, such as the β-amyloid (Aβ) peptide. Therefore, a role for the IDE in the cure of diabetes and Alzheimer’s disease (AD) has been long envisaged. Anyway, its role in degrading amyloidogenic proteins remains not clearly defined and, more recently, novel non-proteolytic functions of the IDE have been proposed. From a structural point of view, the IDE presents an atypical clamshell structure, underscoring unique enigmatic enzymological properties. A better understanding of the structure–function relationship may contribute to solving some existing paradoxes of IDE biology and, in light of its multifunctional activity, might lead to novel therapeutic approaches. Full article

(This article belongs to the Special Issue Hemoproteins and Proteolytic Enzymes: Variations on the Theme of Functional Modulation: A Themed Issue in Honor of Professor Massimo Coletta)

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21 pages, 2841 KiB

Open AccessReview

Ligand-Based Regulation of Dynamics and Reactivity of Hemoproteins

by Emily Samuela Turilli-Ghisolfi, Marta Lualdi and Mauro Fasano

Biomolecules 2023, 13(4), 683; https://doi.org/10.3390/biom13040683 - 17 Apr 2023

Cited by 2 | Viewed by 1485

Abstract

Hemoproteins include several heme-binding proteins with distinct structure and function. The presence of the heme group confers specific reactivity and spectroscopic properties to hemoproteins. In this review, we provide an overview of five families of hemoproteins in terms of dynamics and reactivity. First, [...] Read more.

Hemoproteins include several heme-binding proteins with distinct structure and function. The presence of the heme group confers specific reactivity and spectroscopic properties to hemoproteins. In this review, we provide an overview of five families of hemoproteins in terms of dynamics and reactivity. First, we describe how ligands modulate cooperativity and reactivity in globins, such as myoglobin and hemoglobin. Second, we move on to another family of hemoproteins devoted to electron transport, such as cytochromes. Later, we consider heme-based reactivity in hemopexin, the main heme-scavenging protein. Then, we focus on heme–albumin, a chronosteric hemoprotein with peculiar spectroscopic and enzymatic properties. Eventually, we analyze the reactivity and dynamics of the most recently discovered family of hemoproteins, i.e., nitrobindins. Full article

(This article belongs to the Special Issue Hemoproteins and Proteolytic Enzymes: Variations on the Theme of Functional Modulation: A Themed Issue in Honor of Professor Massimo Coletta)

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15 pages, 1699 KiB

Open AccessEditor’s ChoiceReview

Heme Scavenging and Delivery: The Role of Human Serum Albumin

by Giovanna De Simone, Romualdo Varricchio, Tommaso Francesco Ruberto, Alessandra di Masi and Paolo Ascenzi

Biomolecules 2023, 13(3), 575; https://doi.org/10.3390/biom13030575 - 22 Mar 2023

Cited by 5 | Viewed by 3424

Abstract

Heme is the reactive center of several metal-based proteins that are involved in multiple biological processes. However, free heme, defined as the labile heme pool, has toxic properties that are derived from its hydrophobic nature and the Fe-atom. Therefore, the heme concentration must [...] Read more.

Heme is the reactive center of several metal-based proteins that are involved in multiple biological processes. However, free heme, defined as the labile heme pool, has toxic properties that are derived from its hydrophobic nature and the Fe-atom. Therefore, the heme concentration must be tightly controlled to maintain cellular homeostasis and to avoid pathological conditions. Therefore, different systems have been developed to scavenge either Hb (i.e., haptoglobin (Hp)) or the free heme (i.e., high-density lipoproteins (HDL), low-density lipoproteins (LDL), hemopexin (Hx), and human serum albumin (HSA)). In the first seconds after heme appearance in the plasma, more than 80% of the heme binds to HDL and LDL, and only the remaining 20% binds to Hx and HSA. Then, HSA slowly removes most of the heme from HDL and LDL, and finally, heme transits to Hx, which releases it into hepatic parenchymal cells. The Hx:heme or HSA:heme complexes are internalized via endocytosis mediated by the CD91 and CD71 receptors, respectively. As heme constitutes a major iron source for pathogens, bacteria have evolved hemophores that can extract and uptake heme from host proteins, including HSA:heme. Here, the molecular mechanisms underlying heme scavenging and delivery from HSA are reviewed. Moreover, the relevance of HSA in disease states associated with increased heme plasma concentrations are discussed. Full article

(This article belongs to the Special Issue Hemoproteins and Proteolytic Enzymes: Variations on the Theme of Functional Modulation: A Themed Issue in Honor of Professor Massimo Coletta)

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15 pages, 3340 KiB

Open AccessFeature PaperReview

Modulation of Allosteric Control and Evolution of Hemoglobin

by Maurizio Brunori and Adriana Erica Miele

Biomolecules 2023, 13(3), 572; https://doi.org/10.3390/biom13030572 - 22 Mar 2023

Cited by 1 | Viewed by 1668

Abstract

Allostery arises when a ligand-induced change in shape of a binding site of a protein is coupled to a tertiary/quaternary conformational change with a consequent modulation of functional properties. The two-state allosteric model of Monod, Wyman and Changeux [J. Mol. Biol. 1965; 12, [...] Read more.

Allostery arises when a ligand-induced change in shape of a binding site of a protein is coupled to a tertiary/quaternary conformational change with a consequent modulation of functional properties. The two-state allosteric model of Monod, Wyman and Changeux [J. Mol. Biol. 1965; 12, 88–118] is an elegant and effective theory to account for protein regulation and control. Tetrameric hemoglobin (Hb), the oxygen transporter of all vertebrates, has been for decades the ideal system to test for the validity of the MWC theory. The small ligands affecting Hb’s behavior (organic phosphates, protons, bicarbonate) are produced by the red blood cell during metabolism. By binding to specific sites, these messengers make Hb sensing the environment and reacting consequently. HbI and HbIV from trout and human HbA are classical cooperative models, being similar yet different. They share many fundamental features, starting with the globin fold and the quaternary assembly, and reversible cooperative O₂ binding. Nevertheless, they differ in ligand affinity, binding of allosteric effectors, and stability of the quaternary assembly. Here, we recollect essential functional properties and correlate them to the tertiary and quaternary structures available in the protein databank to infer on the molecular basis of the evolution of oxygen transporters. Full article

(This article belongs to the Special Issue Hemoproteins and Proteolytic Enzymes: Variations on the Theme of Functional Modulation: A Themed Issue in Honor of Professor Massimo Coletta)

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10 pages, 1127 KiB

Open AccessReview

The Sickle-Cell Fiber Revisited

by Marilyn F. Bishop and Frank A. Ferrone

Biomolecules 2023, 13(3), 413; https://doi.org/10.3390/biom13030413 - 22 Feb 2023

Cited by 2 | Viewed by 1489

Abstract

Sickle cell disease is the consequence of a single point mutation on the surface of the β chains of the hemoglobin molecule leading to the formation of rigid polymers that disrupt circulation. It has long been established that the polymers are comprised of [...] Read more.

Sickle cell disease is the consequence of a single point mutation on the surface of the β chains of the hemoglobin molecule leading to the formation of rigid polymers that disrupt circulation. It has long been established that the polymers are comprised of seven pairs of double strands that are twisted replicas of the double strands found in crystals. Here, we review several newer developments that elaborate on that simple model and provide deeper insights into the process. Full article

(This article belongs to the Special Issue Hemoproteins and Proteolytic Enzymes: Variations on the Theme of Functional Modulation: A Themed Issue in Honor of Professor Massimo Coletta)

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