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Hemoglobin and myoglobin are generally taken as molecular models of all-α-helical heme-proteins. On the other hand, nitrophorins and nitrobindins (Nb), which are arranged in 8 and 10 β-strands, respectively, represent the molecular models of all-β-barrel heme-proteins. Here, kinetics of the hydroxylamine- (HA-) mediated oxidation of ferrous Mycobacterium tuberculosis, Arabidopsis thaliana, and Homo sapiens nitrobindins (Mt-Nb(II), At-Nb(II), and Hs-Nb(II), respectively), at pH 7.0 and 20.0 °C, are reported. Of note, HA displays antibacterial properties and is a good candidate for the treatment and/or prevention of reactive nitrogen species- (RNS-) linked aging-related pathologies, such as macular degeneration. Under anaerobic conditions, mixing the Mt-Nb(II), At-Nb(II), and Hs-Nb(II) solutions with the HA solutions brings about absorbance spectral changes reflecting the formation of the ferric derivative (i.e., Mt-Nb(III), At-Nb(III), and Hs-Nb(III), respectively). Values of the second order rate constant for the HA-mediated oxidation of Mt-Nb(II), At-Nb(II), and Hs-Nb(II) are 1.1 × 104 M−1 s−1, 6.5 × 104 M−1 s−1, and 2.2 × 104 M−1 s−1, respectively. Moreover, the HA:Nb(II) stoichiometry is 1:2 as reported for ferrous deoxygenated and carbonylated all-α-helical heme-proteins. A comparative look of the HA reduction kinetics by several ferrous heme-proteins suggests that an important role might be played by residues (such as His or Tyr) in the proximity of the heme-Fe atom either coordinating it or not. In this respect, Nbs seem to exploit somewhat different structural aspects, indicating that redox mechanisms for the heme-Fe(II)-to-heme-Fe(III) conversion might differ between all-α-helical and all-β-barrel heme-proteins. [ABSTRACT FROM AUTHOR]

Background: Immune checkpoints are critical regulatory pathways of the immune system which finely tune the response to biological threats. Among them, the CD-28/CTLA-4 and PD-1/PD-L1 axes play a key role in tumour immune escape and are well-established targets of cancer immunotherapy. Summary: The clinical experience accumulated to date provides unequivocal evidence that anti-CTLA-4, PD-1, or PD-L1 monoclonal antibodies, used as monotherapy or in combination regimes, are effective in a variety of advanced/metastatic types of cancer, with improved clinical outcomes compared to conventional chemotherapy. However, the therapeutic success is currently restricted to a limited subset of patients and reliable predictive biomarkers are still lacking. Key Message: The identification and characterization of additional co-inhibitory pathways as novel pharmacological targets to improve the clinical response in refractory patients has led to the development of different immune checkpoint inhibitors, the activities of which are currently under investigation. In this review, we discuss recent literature data concerning the mechanisms of action of next-generation monoclonal antibodies targeting LAG-3, TIM-3, and TIGIT co-inhibitory molecules that are being explored in clinical trials, as single agents or in combination with other immune-stimulating agents. [ABSTRACT FROM AUTHOR]

Haptoglobin (Hp) binding to hemoglobin (Hb) is crucial to prevent extra-erythrocytic Hb-induced damage; in turn, Hp:Hb complexes display heme-based reactivity. Here, the nitrite reductase activity of ferrous human Hb (Hb(II)) complexed with the human Hp phenotypes 1-1 and 2-2 (Hp1-1:Hb(II) and Hp2-2:Hb(II), respectively) is reported to highlight the reactivity of the αβ-dimers of Hb(II) bound to Hp. In the presence of dithionite, values of the apparent second-order rate constant for the NO 2 − -mediated nitrosylation of Hp1-1:Hb(II) and Hp2-2:Hb(II) ( i . e ., k on  = 7.3 M −1  s −1 and 1.2 × 10 1 M −1  s −1 , respectively; pH 7.3 and 20.0 °C) agree with those of sperm whale myoglobin (Mb(II)), of horse heart Mb(II), and of the R-state of Hb(II) ( k on  = 6.0 M −1  s −1 , 2.9 M −1  s −1 , and 6.0 M −1  s −1 , respectively; pH 7.4 and 25.0 °C); in turn, these k on values are higher than that of the T-state of Hb(II) ( k on  = 1.2 × 10 −1 M −1  s −1 ; pH 7.4 and 25.0 °C). Further, the values of k on for the nitrite reductase activity of Hp1-1:Hb(II) and Hp2-2:Hb(II) increase linearly on lowering pH from 7.5 to 5.6 (at 20.0 °C); the value of the slope of Log k on versus pH is −1.10 ± 0.10, reflecting the involvement of one proton in the NO 2 − -mediated nitrosylation of Hp1-1:Hb(II) and Hp2-2:Hb(II). These results indicate that the conformation of the Hb αβ-dimers bound to Hp1-1 and Hp2-2 matches that of the R-state of the Hb tetramer. [ABSTRACT FROM AUTHOR]

The interaction of insulin-degrading enzyme (IDE) with the main intracellular proteasome assemblies (i.e, 30S, 26S and 20S) was analyzed by enzymatic activity, mass spectrometry and native gel electrophoresis. IDE was mainly detected in association with assemblies with at least one free 20S end and biochemical investigations suggest that IDE competes with the 19S in vitro. IDE directly binds the 20S and affects its proteolytic activities in a bimodal fashion, very similar in human and yeast 20S, inhibiting at (IDE) ≤ 30 nM and activating at (IDE) ≥ 30 nM. Only an activating effect is observed in a yeast mutant locked in the “open” conformation (i.e., the α-3ΔN 20S), envisaging a possible role of IDE as modulator of the 20S “open”-”closed” allosteric equilibrium. Protein-protein docking in silico proposes that the interaction between IDE and the 20S could involve the C-term helix of the 20S α-3 subunit which regulates the gate opening of the 20S. [ABSTRACT FROM AUTHOR]

Insulin-degrading enzyme (IDE) is a ubiquitous zinc peptidase of the inverzincin family, which has been initially discovered as the enzyme responsible for insulin catabolism; therefore, its involvement in the onset of diabetes has been largely investigated. However, further studies on IDE unraveled its ability to degrade several other polypeptides, such as β-amyloid, amylin, and glucagon, envisaging the possible implication of IDE dys-regulation in the “aggregopathies” and, in particular, in neurodegenerative diseases. Over the last decade, a novel scenario on IDE biology has emerged, pointing out a multi-functional role of this enzyme in several basic cellular processes. In particular, latest advances indicate that IDE behaves as a heat shock protein and modulates the ubiquitin–proteasome system, suggesting a major implication in proteins turnover and cell homeostasis. In addition, recent observations have highlighted that the regulation of glucose metabolism by IDE is not merely based on its largely proposed role in the degradation of insulinin vivo. There is increasing evidence that improper IDE function, regulation, or trafficking might contribute to the etiology of metabolic diseases. In addition, the enzymatic activity of IDE is affected by metals levels, thus suggesting a role also in the metal homeostasis (metallostasis), which is thought to be tightly linked to the malfunction of the “quality control” machinery of the cell. Focusing on the physiological role of IDE, we will address a comprehensive vision of the very complex scenario in which IDE takes part, outlining its crucial role in interconnecting several relevant cellular processes. [ABSTRACT FROM AUTHOR]

The immunoproteasome is a specialized form of proteasome equipped with modified catalytic subunits that was initially discovered to play a pivotal role in MHC class I antigen processing and immune system modulation. However, over the last years, this proteolytic complex has been uncovered to serve additional functions unrelated to antigen presentation. Accordingly, it has been proposed that immunoproteasome synergizes with canonical proteasome in different cell types of the nervous system, regulating neurotransmission, metabolic pathways and adaptation of the cells to redox or inflammatory insults. Hence, studying the alterations of immunoproteasome expression and activity is gaining research interest to define the dynamics of neuroinflammation as well as the early and late molecular events that are likely involved in the pathogenesis of a variety of neurological disorders. Furthermore, these novel functions foster the perspective of immunoproteasome as a potential therapeutic target for neurodegeneration. In this review, we provide a brain and retina-wide overview, trying to correlate present knowledge on structure-function relationships of immunoproteasome with the variety of observed neuro-modulatory functions. [ABSTRACT FROM AUTHOR]

Somatostatin is a cyclic peptide, released in the gastrointestinal system and the central nervous system, where it is involved in the regulation of cognitive and sensory functions, motor activity and sleep. It is a substrate of insulin-degrading enzyme (IDE), as well as a modulator of its activity and expression. In the present study, we have investigated the modulatory role of somatostatin on IDE activity at 37 °C and pH 7.3 for various substrates [i.e. insulin, β-amyloid (Aβ)1-40 and bradykinin], aiming to quantitatively characterize the correlation between the specific features of the substrates and the regulatory mechanism. Functional data indicate that somatostatin, in addition to the catalytic site of IDE (being a substrate), is also able to bind to two additional exosites, which play different roles according to the size of the substrate and its binding mode to the IDE catalytic cleft. In particular, one exosite, which displays high affinity for somatostatin, regulates only the interaction of IDE with larger substrates (such as insulin and Aβ1-40) in a differing fashion according to their various modes of binding to the enzyme. A second exosite, which is involved in the regulation of enzymatic processing by IDE of all substrates investigated (including a 10-25 amino acid long amyloid-like peptide, bradykinin and somatostatin itself, which had been studied previously), probably acts through the alteration of an 'open-closed' equilibrium. [ABSTRACT FROM AUTHOR]

Nitrobindins (Nbs) are all-β-barrel heme proteins spanning from bacteria to Homo sapiens. They inactivate reactive nitrogen species by sequestering NO, converting NO to HNO2, and promoting peroxynitrite isomerization to NO3−. Here, the nitrite reductase activity of Nb(II) from Mycobacterium tuberculosis (Mt-Nb(II)), Arabidopsis thaliana (At-Nb(II)), Danio rerio (Dr-Nb(II)), and Homo sapiens (Hs-Nb(II)) is reported. This activity is crucial for the in vivo production of NO, and thus for the regulation of blood pressure, being of the utmost importance for the blood supply to poorly oxygenated tissues, such as the eye retina. At pH 7.3 and 20.0 °C, the values of the second-order rate constants (i.e., kon) for the reduction of NO2− to NO and the concomitant formation of nitrosylated Mt-Nb(II), At-Nb(II), Dr-Nb(II), and Hs-Nb(II) (Nb(II)-NO) were 7.6 M−1 s−1, 9.3 M−1 s−1, 1.4 × 101 M−1 s−1, and 5.8 M−1 s−1, respectively. The values of kon increased linearly with decreasing pH, thus indicating that the NO2−-based conversion of Nb(II) to Nb(II)-NO requires the involvement of one proton. These results represent the first evidence for the NO2 reductase activity of Nbs(II), strongly supporting the view that Nbs are involved in NO metabolism. Interestingly, the nitrite reductase reactivity of all-β-barrel Nbs and of all-α-helical globins (e.g., myoglobin) was very similar despite the very different three-dimensional fold; however, differences between all-α-helical globins and all-β-barrel Nbs suggest that nitrite reductase activity appears to be controlled by distal steric barriers, even though a more complex regulatory mechanism can be also envisaged. [ABSTRACT FROM AUTHOR]

Human serum heme–albumin (HSA–heme–Fe) displays reactivity and spectroscopic properties similar to those of heme proteins. Here, the nitrite reductase activity of ferrous HSA–heme–Fe [HSA–heme–Fe(II)] is reported. The value of the second-order rate constant for the reduction of $$ {\text{NO}}_{2}^{ - } $$ NO 2 - to NO and the concomitant formation of nitrosylated HSA–heme–Fe(II) (i.e., kon) is 1.3 M −1 s −1 at pH 7.4 and 20 °C. Values of kon increase by about one order of magnitude for each pH unit decrease between pH 6.5 to 8.2, indicating that the reaction requires one proton. Warfarin inhibits the HSA–heme–Fe(II) reductase activity, highlighting the allosteric linkage between the heme binding site [also named the fatty acid (FA) binding site 1; FA1] and the drug-binding cleft FA2. The dissociation equilibrium constant for warfarin binding to HSA–heme–Fe(II) is (3.1 ± 0.4) × 10 −4 M at pH 7.4 and 20 °C. These results: (1) represent the first evidence for the $$ {\text{NO}}_{2}^{ - } $$ NO 2 - reductase activity of HSA–heme–Fe(II), (2) highlight the role of drugs (e.g., warfarin) in modulating HSA(–heme–Fe) functions, and (3) strongly support the view that HSA acts not only as a heme carrier but also displays transient heme-based reactivity. [ABSTRACT FROM AUTHOR]

Simple Summary: Immunoproteasome plays a key role in the generation of antigenic peptides. Immune checkpoints therapy is a front-line treatment of advanced/metastatic tumors, and to improve its efficacy, a broader knowledge of the dynamics of antigen repertoire processing by cancer cells is mandatory. The scope of this review is to offer a picture of the role of immunoproteasome in antigen presentation to fuel the hypothesis of novel therapeutic interventions based on the modulation of this proteolytic complex and immune checkpoints. Immunoproteasome is a noncanonical form of proteasome with enzymological properties optimized for the generation of antigenic peptides presented in complex with class I MHC molecules. This enzymatic property makes the modulation of its activity a promising area of research. Nevertheless, immunotherapy has emerged as a front-line treatment of advanced/metastatic tumors providing outstanding improvement of life expectancy, even though not all patients achieve a long-lasting clinical benefit. To enhance the efficacy of the currently available immunotherapies and enable the development of new strategies, a broader knowledge of the dynamics of antigen repertoire processing by cancer cells is needed. Therefore, a better understanding of the role of immunoproteasome in antigen processing and of the therapeutic implication of its modulation is mandatory. Studies on the potential crosstalk between proteasome modulators and immune checkpoint inhibitors could provide novel perspectives and an unexplored treatment option for a variety of cancers. [ABSTRACT FROM AUTHOR]

Abstract: Insulin-degrading enzyme (IDE) is an interesting pharmacological target for Alzheimer''s disease (AD), since it hydrolyzes β-amyloid, producing non-neurotoxic fragments. It has also been shown that the somatostatin level reduction is a pathological feature of AD and that it regulates the neprilysin activity toward β-amyloid. In this work, we report for the first time that IDE is able to hydrolyze somatostatin [k cat (s−1)=0.38 (±0.05); K m (M)=7.5 (±0.9)×10−6] at the Phe6–Phe7 amino acid bond. On the other hand, somatostatin modulates IDE activity, enhancing the enzymatic cleavage of a novel fluorogenic β-amyloid through a decrease of the K m toward this substrate, which corresponds to the 10–25 amino acid sequence of the Aβ(1–40). Circular dichroism spectroscopy and surface plasmon resonance imaging experiments show that somatostatin binding to IDE brings about a concentration-dependent structural change of the secondary and tertiary structure(s) of the enzyme, revealing two possible binding sites. The higher affinity binding site disappears upon inactivation of IDE by ethylenediaminetetraacetic acid, which chelates the catalytic Zn2+ ion. As a whole, these features suggest that the modulatory effect is due to an allosteric mechanism: somatostatin binding to the active site of one IDE subunit (where somatostatin is cleaved) induces an enhancement of IDE proteolytic activity toward fluorogenic β-amyloid by another subunit. Therefore, this investigation on IDE–somatostatin interaction contributes to a more exhaustive knowledge about the functional and structural aspects of IDE and its pathophysiological implications in the amyloid deposition and somatostatin homeostasis in the brain. [Copyright &y& Elsevier]

A recently published paper applies cryo-electron microscopy (EM) studies and biochemical/genetic approaches for the elucidation of the mechanisms linking nucleotide binding by ATPases, proteasome conformation dynamics, and gate opening of the 20S core particle. These insights potentially represent a milestone in our understanding of the structural dynamics of the 26S proteasome. [ABSTRACT FROM AUTHOR]

Myoglobin (Mb), generally taken as the molecular model of monomeric globular heme-proteins, is devoted: (i) to act as an intracellular oxygen reservoir, (ii) to transport oxygen from the sarcolemma to the mitochondria of vertebrate heart and red muscle cells, and (iii) to act as a scavenger of nitrogen and oxygen reactive species protecting mitochondrial respiration. Here, the first evidence of ·NO inhibition of ferric Mb- (Mb(III)) mediated detoxification of peroxynitrite is reported, at pH 7.2 and 20.0 °C. ·NO binds to Mb(III) with a simple equilibrium; the value of the second-order rate constant for Mb(III) nitrosylation (i.e., ·NOkon) is (6.8 ± 0.7) × 104 M−1 s−1 and the value of the first-order rate constant for Mb(III)-NO denitrosylation (i.e., ·NOkoff) is 3.1 ± 0.3 s−1. The calculated value of the dissociation equilibrium constant for Mb(III)-NO complex formation (i.e., ·NOkoff/·NOkon = (4.6 ± 0.7) × 10−5 M) is virtually the same as that directly measured (i.e., ·NOK = (3.8 ± 0.5) × 10−5 M). In the absence of ·NO, Mb(III) catalyzes the conversion of peroxynitrite to NO3−, the value of the second-order rate constant (i.e., Pkon) being (1.9 ± 0.2) × 104 M−1 s−1. However, in the presence of ·NO, Mb(III)-mediated detoxification of peroxynitrite is only partially inhibited, underlying the possibility that also Mb(III)-NO is able to catalyze the peroxynitrite isomerization, though with a reduced rate (Pkon* = (2.8 ± 0.3) × 103 M−1 s−1). These data expand the multiple roles of ·NO in modulating heme-protein actions, envisaging a delicate balancing between peroxynitrite and ·NO, which is modulated through the relative amount of Mb(III) and Mb(III)-NO. [ABSTRACT FROM AUTHOR]

Haptoglobin (Hp) counterbalances the adverse effects of extra-erythrocytic hemoglobin (Hb) trapping the αβ dimers of Hb. In turn, the Hp:Hb complexes display heme-based reactivity. Here, the kinetics of cyanide and carbon monoxide dissociation from ferrous-ligated Hp:Hb complexes are reported at pH 7.0 and 20.0 °C. Cyanide dissociation from Hp1-1:Hb(II)-CN− and Hp2-2:Hb-CN− has been followed upon the dithionite-mediated conversion of ferric to ferrous-ligated Hp:Hb complexes. Values of kon for the dithionite-mediated reduction of Hp1-1:Hb(III)-CN− and Hp2-2:Hb(III)-CN− are (7.3 ± 1.1) × 106 M−1 s−1 and (6.2 ± 1.0) × 106 M−1 s−1, respectively. Values of the first-order rate constant (i.e., h) for cyanide dissociation from Hp1-1:Hb(II)-CN− and Hp2-2:Hb(II)-CN− are (1.2 ± 0.2) × 10−1 s−1 and (1.3 ± 0.2) × 10−1 s−1, respectively. CO dissociation from Hp:Hb(II)-CO complexes has been followed by replacing CO with NO. Values of the first-order rate constant (i.e., l) for CO dissociation from Hp1-1:Hb(II)-CO are (1.4 ± 0.2) × 10−2 s−1 and (6.2 ± 0.8) × 10−3 s−1, and those from Hp2-2:Hb(II)-CO are (1.3 ± 0.2) × 10−2 s−1 and (7.3 ± 0.9) × 10−3 s−1. Values of kon, h, and l correspond to those reported for the R-state of tetrameric Hb and isolated α and β chains. This highlights the view that the conformation of the Hb αβ-dimers bound to Hp1-1 and Hp2-2 matches that of the R-state of the Hb tetramer. Furthermore, unlike ferric Hb(III), ligated ferrous Hb(II) does not show an assembly-linked structural change. [ABSTRACT FROM AUTHOR]

Cationic porphyrins exhibit an amazing variety of binding modes and inhibition mechanisms of 20S proteasome. Depending on the spatial distribution of their electrostatic charges, they can occupy different sites on α rings of 20S proteasome by exploiting the structural code responsible for the interaction with regulatory proteins. Indeed, they can act as competitive or allosteric inhibitors by binding at the substrate gate or at the grooves between the α subunits, respectively. Moreover, the substitution of a charged moiety in the peripheral arm with a hydrophobic moiety revealed a "new" 20S functional state with higher substrate affinity and catalytic efficiency. In the present study, we expand our structure–activity relationship (SAR) analysis in order to further explore the potential of this versatile class of 20S modulators. Therefore, we have extended the study to additional macrocyclic compounds, displaying different structural features, comparing their interaction behavior on the 20S proteasome with previously investigated compounds. In particular, in order to evaluate how the introduction of a peptidic chain can affect the affinity and the interacting mechanism of porphyrins, we investigate the MTPyApi, a porphyrin derivatized with an Arg–Pro-rich antimicrobial peptide. Moreover, to unveil the role played by the porphyrin core, this was replaced with a corrole scaffold, a "contracted" version of the tetrapyrrolic ring due to the lack of a methine bridge. The analysis has been undertaken by means of integrated kinetic, Nuclear Magnetic Resonance, and computational studies. Finally, in order to assess a potential pharmacological significance of this type of investigation, a preliminary attempt has been performed to evaluate the biological effect of these molecules on MCF7 breast cancer cells in dark conditions, envisaging that porphyrins may indeed represent a powerful tool for the modulation of cellular proteostasis. [ABSTRACT FROM AUTHOR]

Truncated hemoglobins (trHbs) are widely distributed in bacteria and plants and have been found in some unicellular eukaryotes. Phylogenetic analysis based on protein sequences shows that trHbs branch into three groups, designated N (or I), O (or II), and P (or III). Most trHbs are involved in the O2/NO chemistry and/or oxidation/reduction function, permitting the survival of the microorganism in the host. Here, a detailed comparative analysis of kinetics and/or thermodynamics of (i) ferrous Mycobacterium tubertulosis trHbs N and O (Mt-trHbN and Mt-trHbO, respectively), and Campylobacter jejuni trHb (Cj-trHbP) nitrosylation, (ii) nitrite-mediated nitrosylation of ferrous Mt-trHbN, Mt-trHbO, and Cj-trHbP, and (iii) NO-based reductive nitrosylation of ferric Mt-trHbN, Mt-trHbO, and Cj-trHbP is reported. Ferrous and ferric Mt-trHbN and Cj-trHbP display a very high reactivity towards NO; however, the conversion of nitrite to NO is facilitated primarily by ferrous Mt-trHbN. Values of kinetic and/or thermodynamic parameters reflect specific trHb structural features, such as the ligand diffusion pathways to/from the heme, the heme distal pocket structure and polarity, and the ligand stabilization mechanisms. In particular, the high reactivity of Mt-trHbN and Cj-trHbP reflects the great ligand accessibility to the heme center by two protein matrix tunnels and the E7-path, respectively, and the penta-coordination of the heme-Fe atom. In contrast, the heme-Fe atom of Mt-trHbO the ligand accessibility to the heme center of Mt-trHbO needs large conformational readjustments, thus limiting the heme-based reactivity. These results agree with different roles of Mt-trHbN, Mt-trHbO, and Cj-trHbP in vivo. [ABSTRACT FROM AUTHOR]

Abstract: Functional and structural properties of protoglobin from Methanosarcina acetivorans, whose Cys(101)E20 residue was mutated to Ser (MaPgb*), and of mutants missing either the first 20 N-terminal amino acids (MaPgb*-ΔN20 mutant), or the first 33 N-terminal amino acids [N-terminal loop of 20 amino acids and a 13-residue Z-helix, preceding the globin fold A-helix; (MaPgb*-ΔN20Z mutant)] have been investigated. In keeping with the MaPgb*-ΔN20 mutant crystal structure, here reported at 2.0Å resolution, which shows an increased exposure of the haem propionates to the solvent, the analysis of ligand binding kinetics highlights high accessibility of ligands to the haem pocket in ferric MaPgb*-ΔN20. CO binding to ferrous MaPgb*-ΔN20 displays a marked biphasic behavior, with a fast binding process close to that observed in MaPgb* and a slow carbonylation process, characterized by a rate-limiting step. Conversely, removal of the first 33 residues induces a substantial perturbation of the overall MaPgb* structure, with loss of α-helical content and potential partial collapse of the protein chain. As such, ligand binding kinetics are characterized by very slow rates that are independent of ligand concentration, this being indicative of a high energy barrier for ligand access to the haem, possibly due to localized misfolding. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins. [Copyright &y& Elsevier]

Abstract: Insulin degradation is a finely tuned process that plays a major role in controlling insulin action and most evidence supports IDE (insulin-degrading enzyme) as the primary degradative agent. However, the biomolecular mechanisms involved in the interaction between IDE and its substrates are often obscure, rendering the specific enzyme activity quite difficult to target. On the other hand, biometals, such as copper, aluminum and zinc, have an important role in pathological conditions such as Alzheimer''s disease or diabetes mellitus. The metabolic disorders connected with the latter lead to some metallostasis alterations in the human body and many studies point at a high level of interdependence between diabetes and several cations. We have previously reported (Grasso et al., Chem. Eur. J. 17 (2011) 2752–2762) that IDE activity toward Aβ peptides can be modulated by metal ions. Here, we have investigated the effects of different metal ions on the IDE proteolytic activity toward insulin as well as a designed peptide comprising a portion of the insulin B chain (B20–30), which has a very low affinity for metal ions. The results obtained by different experimental techniques clearly show that IDE is irreversibly inhibited by copper(I) but is still able to process its substrates when it is bound to copper(II). [Copyright &y& Elsevier]

Abstract: Ferrous human serum heme–albumin (HSA–heme–Fe(II)) displays globin-like properties. Here, the effect of ibuprofen and warfarin on kinetics of HSA–heme–Fe(II) nitrosylation is reported. Values of the second-order rate constant for HSA–heme–Fe(II) nitrosylation (k on) decrease from 6.3×106 M−1 s−1 in the absence of drugs, to 4.1×105 M−1 s−1 and 4.8×105 M−1 s−1, in the presence of saturating amounts of ibuprofen and warfarin, respectively, at pH 7.0 and 20.0°C. From the dependence of k on on the drug concentration, values of the dissociation equilibrium constant for ibuprofen and warfarin binding to HSA–heme–Fe(II) (i.e., K =3.2×10−3 M and 2.6×10−4 M, respectively) were determined. The observed allosteric effects could indeed reflect ibuprofen and warfarin binding to the regulatory fatty acid binding site FA2, which brings about an alteration of heme coordination, slowing down HSA–heme–Fe(II) nitrosylation. Present data highlight the allosteric modulation of HSA–heme–Fe(II) reactivity by heterotropic effectors. [Copyright &y& Elsevier]

The single-cell green alga Chlamydomonas reinhardtii harbors twelve truncated hemoglobins (Cr-TrHbs). Cr-TrHb1-1 and Cr-TrHb1-8 have been postulated to be parts of the nitrogen assimilation pathway, and of a NO-dependent signaling pathway, respectively. Here, spectroscopic and reactivity properties of Cr-TrHb1-1, Cr-TrHb1-2, and Cr-TrHb1-4, all belonging to clsss 1 (previously known as group N or group I), are reported. The ferric form of Cr-TrHb1-1, Cr-TrHb1-2, and Cr-TrHb1-4 displays a stable 6cLS heme-Fe atom, whereas the hexa-coordination of the ferrous derivative appears less strongly stabilized. Accordingly, kinetics of azide binding to ferric Cr-TrHb1-1, Cr-TrHb1-2, and Cr-TrHb1-4 are independent of the ligand concentration. Conversely, kinetics of CO or NO2− binding to ferrous Cr-TrHb1-1, Cr-TrHb1-2, and Cr-TrHb1-4 are ligand-dependent at low CO or NO2− concentrations, tending to level off at high ligand concentrations, suggesting the presence of a rate-limiting step. In agreement with the different heme-Fe environments, the pH-dependent kinetics for CO and NO2−binding to ferrous Cr-TrHb1-1, Cr-TrHb1-2, and Cr-TrHb1-4 are characterized by different ligand-linked protonation events. This raises the question of whether the simultaneous presence in C. reinhardtii of multiple TrHb1s may be related to different regulatory roles. [ABSTRACT FROM AUTHOR]

Human serum albumin (HSA), the most abundant protein in human plasma, could be considered as a prototypic monomeric allosteric protein, since the ligand-dependent conformational adaptability of HSA spreads beyond the immediate proximity of the binding site(s). As a matter of fact, HSA is a major transport protein in the bloodstream and the regulation of the functional allosteric interrelationships between the different binding sites represents a fundamental information for the knowledge of its transport function. Here, kinetics and thermodynamics of the allosteric modulation: (i) of carbon monoxide (CO) binding to ferrous human serum heme-albumin (HSA-heme-Fe(II)) by warfarin (WF), and (ii) of WF binding to HSA-heme-Fe(II) by CO are reported. All data were obtained at pH 7.0 and 25°C. Kinetics of CO and WF binding to the FA1 and FA7 sites of HSA-heme-Fe(II), respectively, follows a multi-exponential behavior (with the same relative percentage for the two ligands). This can be accounted for by the existence of multiple conformations and/or heme-protein axial coordination forms of HSA-heme-Fe(II). The HSA-heme-Fe(II) populations have been characterized by resonance Raman spectroscopy, indicating the coexistence of different species characterized by four-, five- and six-coordination of the heme-Fe atom. As a whole, these results suggest that: (i) upon CO binding a conformational change of HSA-heme-Fe(II) takes place (likely reflecting the displacement of an endogenous ligand by CO), and (ii) CO and/or WF binding brings about a ligand-dependent variation of the HSA-heme-Fe(II) population distribution of the various coordinating species. The detailed thermodynamic and kinetic analysis here reported allows a quantitative description of the mutual allosteric effect of CO and WF binding to HSA-heme-Fe(II). [ABSTRACT FROM AUTHOR]

[Display omitted] • Besides familial inheritance, comorbidity with other diseases and a sedentary lifestyle are risk factors for AD development. • Alzheimer's Diseases (AD) is often associated to dyslipidemia. • Accumulation of amyloid deposits of Aβ peptide in the AD brain is related to abnormal lipid metabolism. • Lipids interfere with amyloid clearance by affecting proteasome and autophagy activities Alzheimer's Diseases (AD) is characterized by the accumulation of amyloid deposits of Aβ peptide in the brain. Besides genetic background, the presence of other diseases and an unhealthy lifestyle are known risk factors for AD development. Albeit accumulating clinical evidence suggests that an impaired lipid metabolism is related to Aβ deposition, mechanistic insights on the link between amyloid fibril formation/clearance and aberrant lipid interactions are still unavailable. Recently, many studies have described the key role played by membrane bound Aβ assemblies in neurotoxicity. Moreover, it has been suggested that a derangement of the ubiquitin proteasome pathway and autophagy is significantly correlated with toxic Aβ aggregation and dysregulation of lipid levels. Thus, studies focusing on the role played by lipids in Aβ aggregation and proteostasis could represent a promising area of investigation for the design of valuable treatments. In this review we examine current knowledge concerning the effects of lipids in Aβ aggregation and degradation processes, focusing on the therapeutic opportunities that a comprehensive understanding of all biophysical, biochemical, and biological processes involved may disclose. [ABSTRACT FROM AUTHOR]

The present study provides new evidence that cationic porphyrins may be considered as tunable platforms to interfere with the structural "key code" present on the 20S proteasome α-rings and, by consequence, with its catalytic activity. Here, we describe the functional and conformational effects on the 20S proteasome induced by the cooperative binding of the tri-cationic 5-(phenyl)-10,15,20-(tri N-methyl-4-pyridyl) porphyrin (Tris-T4). Our integrated kinetic, NMR, and in silico analysis allowed us to disclose a complex effect on the 20S catalytic activity depending on substrate/porphyrin concentration. The analysis of the kinetic data shows that Tris-T4 shifts the relative populations of the multiple interconverting 20S proteasome conformations leading to an increase in substrate hydrolysis by an allosteric pathway. Based on our Tris-T4/h20S interaction model, Tris-T4 is able to affect gating dynamics and substrate hydrolysis by binding to an array of negatively charged and hydrophobic residues present on the protein surface involved in the 20S molecular activation by the regulatory proteins (RPs). Accordingly, despite the fact that Tris-T4 also binds to the α3ΔN mutant, allosteric modulation is not observed since the molecular mechanism connecting gate dynamics with substrate hydrolysis is impaired. We envisage that the dynamic view of the 20S conformational equilibria, activated through cooperative Tris-T4 binding, may work as a simplified model for a better understanding of the intricate network of 20S conformational/functional states that may be mobilized by exogenous ligands, paving the way for the development of a new generation of proteasome allosteric modulators. [ABSTRACT FROM AUTHOR]