Brain insulin resistance links the failure of energy metabolism with cognitive decline in both type 2 Diabetes Mellitus (T2D) and Alzheimer's disease (AD), although the molecular changes preceding ...overt brain insulin resistance remain unexplored. Abnormal biliverdin reductase-A (BVR-A) levels were observed in both T2D and AD and were associated with insulin resistance. Here, we demonstrate that reduced BVR-A levels alter insulin signaling and mitochondrial bioenergetics in the brain. Loss of BVR-A leads to IRS1 hyper-activation but dysregulates Akt-GSK3β complex in response to insulin, hindering the accumulation of pGSK3βS9 into the mitochondria. This event impairs oxidative phosphorylation and fosters the activation of the mitochondrial Unfolded Protein Response (UPRmt). Remarkably, we unveil that BVR-A is required to shuttle pGSK3βS9 into the mitochondria. Our data sheds light on the intricate interplay between insulin signaling and mitochondrial metabolism in the brain unraveling potential targets for mitigating the development of brain insulin resistance and neurodegeneration.
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•Insulin signaling alterations impairs mitochondrial metabolism in T2D brain.•BVR-A loss alters mitochondrial bioenergetics in the brain in response to insulin.•UPRmt is activated to face insulin signaling-mitochondria axis alterations.•BVR-A transports GSK3βS9 into the mitochondria to regulate energy metabolism.•BVR-A loss compromise the insulin response, resembling an insulin resistance state.
Wingless/integrase-11 (WNT)/β-catenin pathway is a crucial upstream regulator of a huge array of cellular functions. Its dysregulation is correlated to neoplastic cellular transition and cancer ...proliferation. Members of the Dishevelled (DVL) family of proteins play an important role in the transduction of WNT signaling by contacting its cognate receptor, Frizzled, via a shared PDZ domain. Thus, negative modulators of DVL1 are able to impair the binding to Frizzled receptors, turning off the aberrant activation of the WNT pathway and leading to anti-cancer activity. Through structure-based virtual screening studies, we identified racemic compound RS4690 (1), which showed a promising selective DVL1 binding inhibition with an EC50 of 0.74 ± 0.08 μM. Molecular dynamic simulations suggested a different binding mode for the enantiomers. In the in vitro assays, enantiomer (S)-1 showed better inhibition of DVL1 with an EC50 of 0.49 ± 0.11 μM compared to the (R)-enantiomer. Compound (S)-1 inhibited the growth of HCT116 cells expressing wild-type APC with an EC50 of 7.1 ± 0.6 μM and caused a high level of ROS production. These results highlight (S)-1 as a lead compound for the development of new therapeutic agents against WNT-dependent colon cancer.
Cytochrome cd1 nitrite reductase (cd1 NIR) is a haem-containing enzyme responsible for the reduction of nitrite into nitric oxide (NO), a key step in the bacterial anaerobic respiratory process of ...denitrification. The active site of cytochrome cd1 contains the unique d1-haem cofactor, from which NO must be released. In general, reduced haems bind NO tightly relative to oxidised haems. The mechanism of NO release employed by cd1 NiRs has been recently outlined, highlighting the essential role of the peculiar d1-haem, whose presence in these enzymes was largely unexplained. Briefly, we have shown that cd1 NiRs can release quite rapidly the NO bound to the reduced d1 haem. This finding challenged the classical idea that NO is often an “irreversible” inhibitor of reduced hemoproteins and prompted new studies on the reactivity of ferrous d1 haem with NO, indicating that the d1 haem has evolved to have low affinity for NO, as compared to other ferrous haems.
The heme-containing periplasmic nitrite reductase (cd1 NIR) is responsible for the production of nitric oxide (NO) in denitrifying bacterial species, among which are several animal and plant ...pathogens. Heme NIRs are homodimers, each subunit containing one covalently bound c-heme and one d1-heme. The reduction of nitrite to NO involves binding of nitrite to the reduced protein at the level of d1-heme, followed by dehydration of nitrite to yield NO and release of the latter. The crucial rate-limiting step in the catalytic mechanism is thought to be the release of NO from the d1-heme, which has been proposed, but never demonstrated experimentally, to occur when the iron is in the ferric form, given that the reduced NO-bound derivative was presumed to be very stable, as in other hemeproteins. We have measured for the first time the kinetics of NO binding and release from fully reduced cd1 NIR, using the enzyme from Pseudomonas aeruginosa and its site-directed mutant H369A. Quite unexpectedly, we found that NO dissociation from the reduced d1-heme is very rapid, several orders of magnitude faster than that measured for b-type heme containing reduced hemeproteins. Because the rate of NO dissociation from reduced cd1 NIR, measured in the present report, is faster than or comparable with the turnover number, contrary to expectations this event may well be on the catalytic cycle and not necessarily rate-limiting. This finding also provides a rationale for the presence in cd1 NIR of the peculiar d1-heme cofactor, which has probably evolved to ensure fast product dissociation.
Serine hydroxymethyltransferase (SHMT) is a pivotal enzyme in one-carbon metabolism that catalyses the reversible conversion of serine and tetrahydrofolate into glycine and methylenetetrahydrofolate. ...It exists in cytosolic (SHMT1) and mitochondrial (SHMT2) isoforms. Research on one-carbon metabolism in cancer cell lines has shown that SHMT1 preferentially catalyses serine synthesis, whereas in mitochondria SHMT2 is involved in serine breakdown. Recent research has focused on the identification of inhibitors that bind at the folate pocket. We have previously found that a representative derivative of the pyrazolopyran scaffold, namely 2.12, inhibits both SHMT isoforms, with a preference for SHMT1, causing apoptosis in lung cancer cell lines. Here we show that the affinity of 2.12 for SHMT depends on the identity of the amino acid substrate bound to the enzyme. The dissociation constant of 2.12 is 50-fold lower when it binds to SHMT1 enzyme-serine complex, as compared to the enzyme-glycine complex. Evidence is presented for a similar behaviour of compound 2.12 in the cellular environment. These findings suggest that the presence and identity of the amino acid substrate should be considered when designing SHMT inhibitors. Moreover, our data provide the proof-of-concept that SHMT inhibitors selectively targeting the directionality of one-carbon metabolism flux could be designed.
•The 2.12 inhibitor binds with higher affinity to SHMT-Ser than to SHMT-Gly complex.•Differential affinity probably depends on a hydrogen bond between Ser and 2.12.•Preference of 2.12 for SHMT-Ser is also evident in live cells.
The
cd
1 nitrite reductases, which catalyze the reduction of nitrite to nitric oxide, are homodimers of 60 kDa subunits, each containing one heme-
c and one heme-
d
1. Heme-
c is the electron entry ...site, whereas heme-
d
1 constitutes the catalytic center. The 3D structure of
Pseudomonas aeruginosa nitrite reductase has been determined in both fully oxidized and reduced states. Intramolecular electron transfer (ET), between
c and
d
1 hemes is an essential step in the catalytic cycle. In earlier studies of the
Pseudomonas stutzeri enzyme, we observed that a marked negative cooperativity is controlling this internal ET step. In this study we have investigated the internal ET in the wild-type and His369Ala mutant of
P. aeruginosa nitrite reductases and have observed similar cooperativity to that of the
Pseudomonas stutzeri enzyme. Heme-
c was initially reduced, in an essentially diffusion-controlled bimolecular process, followed by unimolecular electron equilibration between the
c and
d
1 hemes (
k
ET = 4.3 s
−1 and
K = 1.4 at 298K, pH 7.0). In the case of the mutant, the latter ET rate was faster by almost one order of magnitude. Moreover, the internal ET rate dropped (by ∼30-fold) as the level of reduction increased in both the WT and the His mutant. Equilibrium standard enthalpy and entropy changes and activation parameters of this ET process were determined. We concluded that negative cooperativity is a common feature among the
cd
1 nitrite reductases, and we discuss this control based on the available 3D structure of the wild-type and the H369A mutant, in the reduced and oxidized states.
Gab2 is a scaffolding protein, overexpressed in many types of cancers, that plays a key role in the formation of signaling complexes involved in cellular proliferation, migration, and ...differentiation. The interaction between Gab2 and the C-terminal SH3 domain of the protein Grb2 is crucial for the activation of the proliferation-signaling pathway Ras/Erk, thus representing a potential pharmacological target. In this study, we identified, by virtual screening, seven potential inhibitor molecules that were experimentally tested through kinetic and equilibrium binding experiments. One compound showed a remarkable effect in lowering the affinity of the C-SH3 domain for Gab2. This inhibitory effect was subsequently validated in cellula by using lung cancer cell lines A549 and H1299. Our results are discussed under the light of previous works on the C-SH3:Gab2 interaction.
Metabolic reprogramming of tumor cells toward serine catabolism is now recognized as a hallmark of cancer. Serine hydroxymethyltransferase (SHMT), the enzyme providing one‐carbon units by converting ...serine and tetrahydrofolate (H4PteGlu) to glycine and 5,10‐CH2‐H4PteGlu, therefore represents a target of interest in developing new chemotherapeutic drugs. In this study, 13 folate analogues under clinical evaluation or in therapeutic use were in silico screened against SHMT, ultimately identifying four antifolate agents worthy of closer evaluation. The interaction mode of SHMT with these four antifolate drugs (lometrexol, nolatrexed, raltitrexed, and methotrexate) was assessed. The mechanism of SHMT inhibition by the selected antifolate agents was investigated in vitro using the human cytosolic isozyme. The results of this study showed that lometrexol competitively inhibits SHMT with inhibition constant (Ki) values in the low micromolar. The binding mode of lometrexol to SHMT was further investigated by molecular docking. These results thus provide insights into the mechanism of action of antifolate drugs and constitute the basis for the rational design of novel and more potent inhibitors of SHMT.
Serine hydroxymethyltransferase (SHMT) has been repeatedly hailed as the missing target for cancer chemotherapy. Here, we identify lometrexol as one of the most potent antifolate inhibitors of human cytosolic SHMT (Kd=2±1 μM) known to date. The results reported represent an initial step toward the development of more potent and effective SHMT inhibitors.
The ability of many bacteria to form biofilms contributes to their resilience and makes infections more difficult to treat. Biofilm growth leads to the formation of internal oxygen gradients, ...creating hypoxic subzones where cellular reducing power accumulates, and metabolic activities can be limited. The pathogen Pseudomonas aeruginosa counteracts the redox imbalance in the hypoxic biofilm subzones by producing redox-active electron shuttles (phenazines) and by secreting extracellular matrix, leading to an increased surface area-to-volume ratio, which favors gas exchange. Matrix production is regulated by the second messenger bis-(3′,5′)-cyclic-dimeric-guanosine monophosphate (c-di-GMP) in response to different environmental cues. RmcA (Redox modulator of c-di-GMP) from P. aeruginosa is a multidomain phosphodiesterase (PDE) that modulates c-di-GMP levels in response to phenazine availability. RmcA can also sense the fermentable carbon source arginine via a periplasmic domain, which is linked via a transmembrane domain to four cytoplasmic Per-Arnt-Sim (PAS) domains followed by a diguanylate cyclase (DGC) and a PDE domain. The biochemical characterization of the cytoplasmic portion of RmcA reported in this work shows that the PAS domain adjacent to the catalytic domain tunes RmcA PDE activity in a redox-dependent manner, by differentially controlling protein conformation in response to FAD or FADH2. This redox-dependent mechanism likely links the redox state of phenazines (via FAD/FADH2 ratio) to matrix production as indicated by a hyperwrinkling phenotype in a macrocolony biofilm assay. This study provides insights into the role of RmcA in transducing cellular redox information into a structural response of the biofilm at the population level. Conditions of resource (i.e. oxygen and nutrient) limitation arise during chronic infection, affecting the cellular redox state and promoting antibiotic tolerance. An understanding of the molecular linkages between condition sensing and biofilm structure is therefore of crucial importance from both biological and engineering standpoints.
Ancient hemes for ancient catalysts Rinaldo, Serena; Brunori, Maurizio; Cutruzzola, Francesca
Plant signaling & behavior,
02/2008, Letnik:
3, Številka:
2
Journal Article
Recenzirano
Odprti dostop
Tetrapyrroles are essential molecules in living organisms and perform a multitude of functions in all kingdoms. Their synthesis is achieved in cells via a complex biosynthetic machinery which is ...unlikely to be maintained, if unnecessary. Here we propose that ancient hemes, such as the d1-heme of cd1 nitrite reductase or the siroheme of bacterial and plant nitrite and sulphite reductases, are molecular fossils which have survived the evolutionary pressure because their role is strategic for the organism where they are found today. The peculiar NO-releasing propensity of the d1-heme of P. aeruginosa NIR, recently shown by our group is, in our opinion, an example of this strategy. The hypothesis is that the d1-heme structure might be a pre-requisite for the fast rate of NO dissociation from the ferrous form, a property which is crucial to enzymatic activity and cannot be achieved with a more common b-type heme.
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