RESUMEN

Mannheimia haemolytica is the main etiological bacterial agent in ruminant respiratory disease. M. haemolytica secretes leukotoxin, lipopolysaccharides, and proteases, which may be targeted to treat infections. We recently reported the purification and in vivo detection of a 110 kDa Zn metalloprotease with collagenase activity (110-Mh metalloprotease) in a sheep with mannheimiosis, and this protease may be an important virulence factor. Due to the increase in the number of multidrug-resistant strains of M. haemolytica, new alternatives to antibiotics are being explored; one option is lactoferrin (Lf), which is a multifunctional iron-binding glycoprotein from the innate immune system of mammals. Bovine apo-lactoferrin (apo-bLf) possesses many properties, and its bactericidal and bacteriostatic effects have been highlighted. The present study was conducted to investigate whether apo-bLf inhibits the secretion and proteolytic activity of the 110-Mh metalloprotease. This enzyme was purified and sublethal doses of apo-bLf were added to cultures of M. haemolytica or co-incubated with the 110-Mh metalloprotease. The collagenase activity was evaluated using zymography and azocoll assays. Our results showed that apo-bLf inhibited the secretion and activity of the 110-Mh metalloprotease. Molecular docking and overlay assays showed that apo-bLf bound near the active site of the 110-Mh metalloprotease, which affected its enzymatic activity.

Asunto(s)

Lactoferrina , Mannheimia haemolytica , Metaloproteasas , Proteolisis , Lactoferrina/metabolismo , Lactoferrina/farmacología , Metaloproteasas/metabolismo , Metaloproteasas/antagonistas & inhibidores , Animales , Apoproteínas/metabolismo , Apoproteínas/química , Simulación del Acoplamiento Molecular , Ovinos , Bovinos , Colagenasas/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/química , Zinc/metabolismo

RESUMEN

Parkinson's disease (PD) is the second most prevalent neurodegenerative disorder worldwide, and the therapeutic is focused on several approaches including the inhibition of fibril formation by small compounds, avoiding the formation of cytotoxic oligomers. Thus, we decided to explore the capacity of compounds carrying catechol moieties to inhibit the progression of α-synuclein. Overall, the compounds rosmarinic acid (1), carnosic acid (2), carnosol (3), epiisorosmanol (4), and rosmanol (5) avoid the progression of fibril formation assessed by Thiofavine T (ThT), and atomic force microscopy images showed that morphology is influenced for the actions of compounds over fibrillization. Moreover, ITC experiments showed a Kd varying from 28 to 51 µM, the ΔG showed that the reaction between compounds and α-syn is spontaneous, and ΔH is associated with an exothermic reaction, suggesting the interactions of hydrogen bonds among compounds and α-syn. Docking experiments reinforce this idea showing the intermolecular interactions are mostly hydrogen bonding within the sites 2, 9, and 3/13 of α-synuclein, and compounds 1 and 5. Thus, compound 1, rosmarinic acid, interestingly interacts better with site 9 through catechol and Lysines. In cultured Raw 264. 7 cells, the presence of compounds showed that most of them can promote cell differentiation, especially rosmarinic acid, and rosmanol, both preserving tubulin cytoskeleton. However, once we evaluated whether or not the aggregates pre-treated with compounds could prevent the disruption of microtubules of Raw 264.7 cells, only pre-treated aggregates with rosmarinic acid prevented the disruption of the cytoskeleton. Altogether, we showed that especially rosmarinic acid not only inhibits α-syn but stabilizes the remaining aggregates turning them into not-toxic to Raw 264.7 cells suggesting a main role in cell survival and antigen processing in response to external α-syn aggregates.

Asunto(s)

Cinamatos , Depsidos , Microtúbulos , Ácido Rosmarínico , alfa-Sinucleína , Depsidos/farmacología , Depsidos/química , Depsidos/aislamiento & purificación , Cinamatos/química , Cinamatos/farmacología , Cinamatos/síntesis química , Animales , Ratones , Células RAW 264.7 , Microtúbulos/efectos de los fármacos , Microtúbulos/metabolismo , Estructura Molecular , alfa-Sinucleína/metabolismo , alfa-Sinucleína/antagonistas & inhibidores , Relación Estructura-Actividad , Relación Dosis-Respuesta a Droga , Supervivencia Celular/efectos de los fármacos , Simulación del Acoplamiento Molecular

RESUMEN

Light chain amyloidosis is a conformational disease caused by the abnormal proliferation and deposition of antibody light chains as amyloid fibers in organs and tissues. The effect of Cu(II) binding to the model recombinant protein 6aJL2-R24G was previously characterized in our group, and we found an acceleration of the aggregation kinetics of the protein. In this study, in order to confirm the Cu(II) binding sites, histidine variants of 6aJL2-R24G were prepared and the effects of their interaction with Cu(II) were analyzed by circular dichroism, fluorescence spectroscopy, isothermal calorimetry titrations, and molecular dynamics simulations. Confirming our earlier work, we found that His8 and His99 are the highest affinity Cu(II) binding sites, and that Cu(II) binding to both sites is a cooperative event.

Asunto(s)

Cobre , Histidina , Unión Proteica , Cobre/metabolismo , Cobre/química , Histidina/química , Histidina/metabolismo , Humanos , Sitios de Unión , Simulación de Dinámica Molecular , Cadenas Ligeras de Inmunoglobulina/metabolismo , Cadenas Ligeras de Inmunoglobulina/genética , Cadenas Ligeras de Inmunoglobulina/química , Amiloidosis de Cadenas Ligeras de las Inmunoglobulinas/metabolismo , Amiloidosis de Cadenas Ligeras de las Inmunoglobulinas/genética , Amiloidosis/metabolismo , Amiloidosis/genética , Cinética

RESUMEN

The lens is a transparent, biconvex anatomical structure of the eyes responsible for light transmission and fine focusing on the retina. It is fundamentally constituted by water-soluble proteins called crystallins which are responsible for lens transparency due to their stable and highly organized disposition in the lens fiber cells. Some conformational changes and the subsequent aggregation of crystallins lead to loss of transparency in the lens and are the beginning of cataracts, which is the most frequent cause of reversible blindness in the world. Ultraviolet radiation is considered one of the risk factors for cataract development. The lens is exposed to radiation between 295 and 400 nm. This UV radiation may induce several processes that destroy the crystallins; the most significant is the oxidative stress due to increased free radicals formation. The oxidative stress is directly involved in modifications of the crystallin proteins leading to the formation of high molecular weight aggregates and then the subsequent opacification of the lens, known as cataracts. This review aims to summarize current knowledge about the damage of the lens proteins caused by ultraviolet radiation and its role in developing cataracts.

Asunto(s)

Catarata , Cristalinas , Cristalino , Humanos , Rayos Ultravioleta/efectos adversos , Cristalino/química , Cristalino/metabolismo , Cristalino/efectos de la radiación , Catarata/etiología , Catarata/metabolismo , Cristalinas/análisis , Cristalinas/química , Cristalinas/metabolismo

RESUMEN

OBJECTIVE: Cellular and molecular events occurring in cartilage regions close to injury are poorly investigated, but can possibly compromise the outcome of cell-based cartilage repair. In this study, key functional properties were assessed for cartilage biopsies collected from the central part of traumatic joint lesions (central) and from regions surrounding the defect (peripheral). These properties were then correlated with the quality of the initial cartilage biopsy and the inflammatory state of the joint. DESIGN: Cartilage samples were collected from knee joints of 42 patients with traumatic knee injuries and analyzed for cell phenotype (by reverse transcriptas-polymerase chain reaction), histological quality, cellularity, cell viability, proliferation capacity, and post-expansion chondrogenic capacity of chondrocytes (in pellet culture). Synovium was also harvested and analyzed for the expression of inflammatory cytokines. RESULTS: Cartilage quality and post-expansion chondrogenic capacity were higher in peripheral versus central samples. Differences between these 2 parameters were more pronounced in joints with high inflammatory features characterized by >100-fold difference in the mRNA levels of IL6 and IL8 in the corresponding synovium. Peripheral chondrocytes isolated from good- versus bad-quality biopsies expressed higher levels of collagen II/I and aggrecan/versican and lower levels of MMP13 and ADAMTS5. They also exhibited reduced proliferation and enhanced cartilage-forming capacity. CONCLUSIONS: Chondrocytes at the periphery of traumatic lesions better maintain properties of healthy cartilage compared to those isolated from the center, even when derived from bad-quality tissues harvested from highly inflamed joints. Future studies are necessary to investigate the change of functional properties of peripheral chondrocytes over time.

Asunto(s)

Cartílago Articular , Condrocitos , Agrecanos/metabolismo , Diferenciación Celular/genética , Condrocitos/metabolismo , Condrogénesis , Humanos

RESUMEN

Cataract formation is a slow accumulative process due to protein aggregates promoted by different factors over time. Zinc and copper ions have been reported to induce the formation of aggregates opaque to light in the human gamma D crystallin (HγD) in a concentration and temperature dependent manner. In order to gain insight into the mechanism of metal-induced aggregation of HγD under conditions that mimic more closely the slow, accumulative process of the disease, we have studied the non-equilibrium process with the minimal metal dose that triggers HγD aggregation. Using a wide variety of biophysics techniques such as turbidimetry, dynamic light scattering, fluorescence, nuclear magnetic resonance and computational methods, we obtained information on the molecular mechanisms for the formation of aggregates. Zn(II) ions bind to different regions at the protein, probably with similar affinities. This binding induces a small conformational rearrangement within and between domains and aggregates via the formation of metal bridges without any detectable unfolded intermediates. In contrast, Cu(II)-induced aggregation includes a lag time, in which the N-terminal domain partially unfolds while the C-terminal domain and parts of the N-terminal domain remain in a native-like conformation. This partially unfolded intermediate is prone to form the high-molecular weight aggregates. Our results clearly show that different external factors can promote protein aggregation following different pathways.

RESUMEN

Light-chain amyloidosis (AL) is one of the most common systemic amyloidoses, and it is characterized by the deposition of immunoglobulin light chain (LC) variable domains as insoluble amyloid fibers in vital organs and tissues. The recombinant protein 6aJL2-R24G contains λ6a and JL2 germline genes and also contains the Arg24 by Gly substitution. This mutation is present in 25% of all amyloid-associated λ6 LC cases, reduces protein stability, and increases the propensity to form amyloid fibers. In this study, it was found that the interaction of 6aJL2-R24G with Cu(II) decreases the thermal stability of the protein and accelerates the amyloid fibril formation, as observed by fluorescence spectroscopy. Isothermal calorimetry titration showed that Cu(II) binds to the protein with micromolar affinity. His99 may be one of the main Cu(II) interaction sites, as observed by nuclear magnetic resonance spectroscopy. The binding of Cu(II) to His99 induces larger fluctuations of the CDR1 and loop C″, as shown by molecular dynamics simulations. Thus, Cu(II) binding may be inducing the loss of interactions between CDR3 and CDR1, making the protein less stable and more prone to form amyloid fibers. This study provides insights into the mechanism of metal-induced aggregation of the 6aJL2-R24G protein and sheds light on the bio-inorganic understanding of AL disease.

RESUMEN

Context: During a period of 6 months, 36 people reported to health authorities in the Department of Antioquia, Colombia, presenting episodes of bleeding in varying magnitude and locations in the body and alterations in coagulation tests, after having taken a falsified dietary supplement. The identification of the first four cases were to the cell-phone line at the Drug and Poison Research Information Center (CIEMTO). The successive presentation of cases with similar manifestations, taking the same product, served to suspect a possible common link.Case details: All of the patients needed hospitalization, the administration of blood products and / or vitamin K to reverse the clinical manifestations, and to stop the oral consumption of the falsified supplement. For each patient there was a full recovery of coagulation and improvement of haemorrhagic manifestations after the first week of management. The Food and Drug administration of Colombia (INVIMA), withdrew the product from the market, alerted the medical community and the general public and conducted an investigation that finally showed warfarin as a the main contaminant in the dietary supplement.Conclusion: This cases series emphasize the importance of the Poison Control Center to detect promptly potential new exposure of hazards to hundreds of products to the population, some of them fraudulent.

RESUMEN

Late embryogenesis abundant (LEA) proteins accumulate in plants during adverse conditions and their main attributed function is to confer tolerance to stress. One of the deleterious effects of the adverse environment is the accumulation of metal ions to levels that generate reactive oxygen species, compromising the survival of cells. AtLEA4-5, a member of group 4 of LEAs in Arabidopsis, is an intrinsically disordered protein. It has been shown that their N-terminal region is able to undergo transitions to partially folded states and prevent the inactivation of enzymes. We have characterized metal ion binding to AtLEA4-5 by circular dichroism, electronic absorbance spectroscopy (UV-vis), electron paramagnetic resonance, dynamic light scattering, and isothermal titration calorimetry. The data shows that AtLEA4-5 contains a single binding site for Ni(II), while Zn(II) and Cu(II) have multiple binding sites and promote oligomerization. The Cu(II) interacts preferentially with histidine residues mostly located in the C-terminal region with moderate affinity and different coordination modes. These results and the lack of a stable secondary structure formation indicate that an ensemble of conformations remains accessible to the metal for binding, suggesting the formation of a fuzzy complex. Our results support the multifunctionality of LEA proteins and suggest that the C-terminal region of AtLEA4-5 could be responsible for antioxidant activity, scavenging metal ions under stress conditions while the N-terminal could function as a chaperone.

Asunto(s)

Investigación Biomédica , Centros de Control de Intoxicaciones , Intoxicación/terapia , Colombia/epidemiología , Humanos , Intoxicación/epidemiología , Intoxicación/prevención & control

RESUMEN

BACKGROUND: Notochordal cell conditioned medium (NCCM) derived from non-chondrodystrophic dogs has pro-anabolic and anti-catabolic effects upon nucleus pulposus (NP) cells. Here, for the first time, we assessed the ability of NCCM to influence the production of extracellular matrix and inflammatory proteins by healthy and osteoarthritic human chondrocytes within engineered cartilage tissues. We hypothesized that, similar to its action on NP cells, NCCM exerts metabolic and anti-catabolic effects on human articular chondrocytes and has the potential to significantly counteract inflammatory mediators. METHODS: Chondrocytes from nine non-osteoarthritic patients and from six osteoarthritic (OA) donors at the time of total knee arthroplasty were chondro-differentiated in pellets for 2 weeks. Non-OA pellets were exposed for 72 hours to IL-1ß/TNF-α and then cultured up to 14 days in 2 % FBS-supplemented NCCM or 2 % FBS-supplemented medium (control (ctr)). OA pellets were cultured in NCCM or ctr medium without pro-inflammatory treatment. Tissues after each culture phase were analyzed biochemically (GAG/DNA), (immuno-) histologically (collagen I, II and GAG) and by Western blotting. Supernatants were analyzed by ELISA. RESULTS: Response to NCCM was age and disease dependent with healthy chondrocyte pellets (from donors >55 years of age) recovering their glycosaminoglycan (GAG) contents to baseline levels only with NCCM. OA pellets treated with NCCM significantly increased GAG content (1.8-fold) and levels of hyaluronic acid link protein (HAPLN), fibromodulin and SOX-9. The catabolic proteins (matrix metalloproteinase (MMP)-3 and MMP-13) and pro-inflammatory enzyme levels (cyclooxygenase-2 (COX-2)) were markedly reduced and there was significantly reduced secretion of pro-inflammatory chemokines (IL-6 and IL-8). CONCLUSIONS: NCCM restores cartilage matrix production of end-stage human OA chondrocytes towards a healthy phenotype and suppresses the production of inflammatory mediators. Harnessing the necessary and sufficient factors within NCCM that confers chondroprotection and regenerative effects could lead to a minimally invasive agent for treatment of degenerative and inflammatory joint diseases.

Asunto(s)

Cartílago Articular/fisiología , Diferenciación Celular/efectos de los fármacos , Condrocitos/fisiología , Notocorda , Osteoartritis , Regeneración/efectos de los fármacos , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Animales , Western Blotting , Cartílago Articular/citología , Cartílago Articular/efectos de los fármacos , Condrocitos/citología , Condrocitos/efectos de los fármacos , Medios de Cultivo Condicionados/farmacología , Perros , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Fenotipo

RESUMEN

The ability of the cellular prion protein (PrP(C)) to bind copper in vivo points to a physiological role for PrP(C) in copper transport. Six copper binding sites have been identified in the nonstructured N-terminal region of human PrP(C). Among these sites, the His111 site is unique in that it contains a MKHM motif that would confer interesting Cu(I) and Cu(II) binding properties. We have evaluated Cu(I) coordination to the PrP(106-115) fragment of the human PrP protein, using NMR and X-ray absorption spectroscopies and electronic structure calculations. We find that Met109 and Met112 play an important role in anchoring this metal ion. Cu(I) coordination to His111 is pH-dependent: at pH >8, 2N1O1S species are formed with one Met ligand; in the range of pH 5-8, both methionine (Met) residues bind to Cu(I), forming a 1N1O2S species, where N is from His111 and O is from a backbone carbonyl or a water molecule; at pH <5, only the two Met residues remain coordinated. Thus, even upon drastic changes in the chemical environment, such as those occurring during endocytosis of PrP(C) (decreased pH and a reducing potential), the two Met residues in the MKHM motif enable PrP(C) to maintain the bound Cu(I) ions, consistent with a copper transport function for this protein. We also find that the physiologically relevant Cu(I)-1N1O2S species activates dioxygen via an inner-sphere mechanism, likely involving the formation of a copper(II) superoxide complex. In this process, the Met residues are partially oxidized to sulfoxide; this ability to scavenge superoxide may play a role in the proposed antioxidant properties of PrP(C). This study provides further insight into the Cu(I) coordination properties of His111 in human PrP(C) and the molecular mechanism of oxygen activation by this site.

Asunto(s)

Cobre/metabolismo , Fragmentos de Péptidos/metabolismo , Proteínas Priónicas/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Cinética , Modelos Teóricos , Resonancia Magnética Nuclear Biomolecular , Oxidación-Reducción , Proteínas Priónicas/química , Unión Proteica , Espectroscopía de Absorción de Rayos X

RESUMEN

Cataract is the leading cause of blindness in the world. It results from aggregation of eye lens proteins into high-molecular-weight complexes, causing light scattering and lens opacity. Copper and zinc concentrations in cataractous lens are increased significantly relative to a healthy lens, and a variety of experimental and epidemiological studies implicate metals as potential etiological agents for cataract. The natively monomeric, ß-sheet rich human γD (HγD) crystallin is one of the more abundant proteins in the core of the lens. It is also one of the most thermodynamically stable proteins in the human body. Surprisingly, we found that both Cu(II) and Zn(II) ions induced rapid, nonamyloid aggregation of HγD, forming high-molecular-weight light-scattering aggregates. Unlike Zn(II), Cu(II) also substantially decreased the thermal stability of HγD and promoted the formation of disulfide-bridged dimers, suggesting distinct aggregation mechanisms. In both cases, however, metal-induced aggregation depended strongly on temperature and was suppressed by the human lens chaperone αB-crystallin (HαB), implicating partially folded intermediates in the aggregation process. Consistently, distinct site-specific interactions of Cu(II) and Zn(II) ions with the protein and conformational changes in specific hinge regions were identified by nuclear magnetic resonance. This study provides insights into the mechanisms of metal-induced aggregation of one of the more stable proteins in the human body, and it reveals a novel and unexplored bioinorganic facet of cataract disease.

Asunto(s)

Cobre/farmacología , Cristalinas/metabolismo , Agregación Patológica de Proteínas/inducido químicamente , Zinc/farmacología , Cobre/química , Electroforesis en Gel de Poliacrilamida , Humanos , Iones , Espectroscopía de Resonancia Magnética , Microscopía Electrónica de Transmisión , Modelos Moleculares , Pliegue de Proteína , Estabilidad Proteica , Temperatura , Zinc/química

RESUMEN

The increasing prevalence of conformational diseases, including Alzheimer's disease, type 2 Diabetes Mellitus and Cancer, poses a global challenge at many different levels. It has devastating effects on the sufferers as well as a tremendous economic impact on families and the health system. In this work, we apply a cross-functional approach that combines ideas, concepts and technologies from several disciplines in order to study, in silico and in vitro, the role of a novel chemical chaperones family (NCHCHF) in processes of protein aggregation in conformational diseases. Given that Serum Albumin (SA) is the most abundant protein in the blood of mammals, and Bovine Serum Albumin (BSA) is an off-the-shelf protein available in most labs around the world, we compared the ligandability of BSA:NCHCHF with the interaction sites in the Human Islet Amyloid Polypeptide (hIAPP):NCHCHF, and in the amyloid pharmacophore fragments (Aß17-42 and Aß16-21):NCHCHF. We posit that the merging of this interaction sites is a meta-structure of pharmacophore which allows the development of chaperones that can prevent protein aggregation at various states from: stabilizing the native state to destabilizing oligomeric state and protofilament. Furthermore to stabilize fibrillar structures, thus decreasing the amount of toxic oligomers in solution, as is the case with the NCHCHF. The paper demonstrates how a set of NCHCHF can be used for studying and potentially treating the various physiopathological stages of a conformational disease. For instance, when dealing with an acute phase of cytotoxicity, what is needed is the recruitment of cytotoxic oligomers, thus chaperone F, which accelerates fiber formation, would be very useful; whereas in a chronic stage it is better to have chaperones A, B, C, and D, which stabilize the native and fibril structures halting self-catalysis and the creation of cytotoxic oligomers as a consequence of fiber formation. Furthermore, all the chaperones are able to protect and recondition the cerebellar granule cells (CGC) from the cytotoxicity produced by the hIAPP20-29 fragment or by a low potassium medium, regardless of their capacity for accelerating or inhibiting in vitro formation of fibers. In vivo animal experiments are required to study the impact of chemical chaperones in cognitive and metabolic syndromes.

Asunto(s)

Proteínas Amiloidogénicas/metabolismo , Chaperonas Moleculares/metabolismo , Péptidos beta-Amiloides/efectos de los fármacos , Péptidos beta-Amiloides/metabolismo , Proteínas Amiloidogénicas/efectos de los fármacos , Animales , Sitios de Unión , Dicroismo Circular , Simulación por Computador , Descubrimiento de Drogas/métodos , Humanos , Técnicas In Vitro , Microscopía Electrónica de Transmisión , Chaperonas Moleculares/farmacología , Simulación del Acoplamiento Molecular , Fragmentos de Péptidos/efectos de los fármacos , Fragmentos de Péptidos/metabolismo , Agregación Patológica de Proteínas/tratamiento farmacológico , Albúmina Sérica/metabolismo , Albúmina Sérica/farmacología , Albúmina Sérica Bovina/metabolismo , Albúmina Sérica Bovina/farmacología

RESUMEN

Light chain amyloidosis (AL) is a deadly disease characterized by the deposition of monoclonal immunoglobulin light chains as insoluble amyloid fibrils in different organs and tissues. Germ line λ VI has been closely related to this condition; moreover, the R24G mutation is present in 25% of the proteins of this germ line in AL patients. In this work, five small molecules were tested as inhibitors of the formation of amyloid fibrils from the 6aJL2-R24G protein. We have found by thioflavin T fluorescence and transmission electron microscopy that EGCG inhibits 6aJL2-R24G fibrillogenesis. Furthermore, using nuclear magnetic resonance spectroscopy, dynamic light scattering, and isothermal titration calorimetry, we have determined that the inhibition is due to binding to the protein in its native state, interacting mainly with aromatic residues.

Asunto(s)

Amiloide/antagonistas & inhibidores , Amiloide/genética , Amiloidosis/tratamiento farmacológico , Amiloidosis/genética , Catequina/análogos & derivados , Cadenas Ligeras de Inmunoglobulina/efectos de los fármacos , Cadenas Ligeras de Inmunoglobulina/genética , Mutación Missense , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Amiloide/biosíntesis , Amiloidosis/metabolismo , Catequina/farmacología , Humanos , Cadenas Ligeras de Inmunoglobulina/biosíntesis , Técnicas In Vitro , Melatonina/farmacología , Microscopía Electrónica de Transmisión , Modelos Moleculares , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Unión Proteica/efectos de los fármacos , Multimerización de Proteína/efectos de los fármacos , Quercetina/farmacología , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/efectos de los fármacos , Proteínas Recombinantes/genética , Rifampin/farmacología , Tetraciclina/farmacología

RESUMEN

Type 2 diabetes (T2D) is one of the most common chronic diseases, affecting over 300 million people worldwide. One of the hallmarks of T2D is the presence of amyloid deposits of human islet amyloid polypeptide (IAPP) in the islets of Langerhans of pancreatic ß-cells. Recent reports indicate that Cu(II) can inhibit the aggregation of human IAPP, although the mechanism for this inhibitory effect is not clear. In this study, different spectroscopic techniques and model fragments of IAPP were employed to shed light on the structural basis for the interaction of Cu(II) with human IAPP. Our results show that Cu(II) anchors to His18 and the subsequent amide groups toward the C-terminal, forming a complex with an equatorial coordination mode 3N1O at physiological pH. Cu(II) binding to truncated IAPP at the His18 region is the key event for its inhibitory effect in amyloid aggregation. Electron paramagnetic resonance studies indicate that the monomeric Cu(II)-IAPP(15-22) complex differs significantly from Cu(II) bound to mature IAPP(15-22) fibers, suggesting that copper binding to monomeric IAPP(15-22) competes with the conformation changes needed to form ß-sheet structures, thus delaying fibril formation. A general mechanism is proposed for the inhibitory effect of copper and other imidazole-binding metal ions in IAPP amyloid formation, providing further insights into the bioinorganic chemistry of T2D.

Asunto(s)

Cobre/farmacología , Diabetes Mellitus Tipo 2/metabolismo , Polipéptido Amiloide de los Islotes Pancreáticos/química , Polipéptido Amiloide de los Islotes Pancreáticos/metabolismo , Agregado de Proteínas/efectos de los fármacos , Cobre/química , Humanos , Polipéptido Amiloide de los Islotes Pancreáticos/aislamiento & purificación , Estructura Molecular , Agregación Patológica de Proteínas/metabolismo

RESUMEN

Metal ions play a wide range of important functional roles in biology, and they often serve as cofactors in enzymes. Some of the metal ions that are essential for life are strongly associated with proteins, forming obligate metalloproteins, while others may bind to proteins with relatively low affinity. The spectroscopic tools presented in this chapter are suitable to study metal ion-protein interactions. Metal sites in proteins are usually low symmetry centers that differentially absorb left and right circularly polarized light. The combination of electronic absorption and circular dichroism (CD) in the UV-visible region allows the characterization of electronic transitions associated with the metal-protein complex, yielding information on the geometry and nature of the metal-ligand interactions. For paramagnetic metal centers in proteins, electron paramagnetic resonance (EPR) is a powerful tool that provides information on the chemical environment around the unpaired electron(s), as it relates to the electronic structure and geometry of the metal-protein complex. EPR can also probe interactions between the electron spin and nuclear spins in the vicinity, yielding valuable information on some metal-ligand interactions. This chapter describes each spectroscopic technique and it provides the necessary information to design and implement the study of metal ion-protein interactions by electronic absorption, CD, and EPR.

Asunto(s)

Complejos de Coordinación/química , Electrones , Metales Pesados/química , Proteínas/química , Sitios de Unión , Dicroismo Circular , Espectroscopía de Resonancia por Spin del Electrón , Cinética , Unión Proteica , Espectrofotometría , Temperatura , Termodinámica

RESUMEN

The prion protein coordinates copper with high affinity in the regions encompassing residues 92-99 (GGGTHSQW) and 106-115 (KTNMKHMAGA). Cu(II) binding to these sites involves the coordination of the His96/His111 imidazole ring and backbone deprotonated amides that precede the His residue. Such a coordination arrangement involves the formation of hexa- and penta-membered cycles that provide further stabilization of the metal-peptide complex. The purpose of the present study is to introduce a methylene group in the peptide backbone, to evaluate the impact of increasing the size of these cycles in Cu(II) binding. Thus, a ß-alanine residue was inserted at different positions preceding the His residue in these prion fragments, and their Cu(II) coordination properties were assessed by UV-Visible absorption, circular dichroism, and electron paramagnetic resonance. Spectroscopic data show that the insertion of a methylene group leads to a completely different Cu(II) coordination that involves the His96/His111 imidazole ring and nitrogen or oxygen atoms provided by the peptide backbone towards the C-terminal. This study clearly shows that two main factors determine the nature of Cu(II)-peptide complexes involving an anchoring His residue and deprotonated amides from the backbone chain: i) the stabilization of Cu(II)-peptide complexes due to the formation of cyclic structures (i.e. chelate effect) and ii) the nature of the residues associated to the deprotonated amide groups that participate in metal ion coordination.

Asunto(s)

Complejos de Coordinación/química , Cobre/química , Péptidos/química , Priones/química , beta-Alanina/química , Cationes Bivalentes , Dicroismo Circular , Espectroscopía de Resonancia por Spin del Electrón , Histidina/química , Humanos , Péptidos/síntesis química , Unión Proteica , Dominios y Motivos de Interacción de Proteínas

RESUMEN

The prion protein (PrP(C)) binds Cu(II) in its N-terminal region, and it is associated to a group of neurodegenerative diseases termed transmissible spongiform encephalopaties (TSEs). The isoform PrP(Sc), derived from the normal PrP(C), is the pathogenic agent of TSEs. Using spectroscopic techniques (UV-vis absorption, circular dichroism, and electron paramagnetic resonance) and electronic structure calculations, we obtained a structural description for the different pH-dependent binding modes of Cu(II) to the PrP(92-96) fragment. We have also evaluated the possibility of water molecule ligation to the His96-bound copper ion. Geometry-optimized structural models that reproduce the spectroscopic features of these complexes are presented. Two Cu(II) binding modes are relevant at physiological pH: 4N and 3NO equatorial coordination modes; these are best described by models with no participation of water molecules in the coordination sphere of the metal ion. In contrast, the 2N2O and N3O coordination modes that are formed at lower pH involve the coordination of an axial water molecule. This study underscores the importance of including explicit water molecules when modeling copper binding sites in PrP(C).

Asunto(s)

Cobre/metabolismo , Proteínas PrPC/metabolismo , Sitios de Unión , Dicroismo Circular , Cobre/química , Espectroscopía de Resonancia por Spin del Electrón , Humanos , Concentración de Iones de Hidrógeno , Proteínas PrPC/química , Unión Proteica , Agua/química

RESUMEN

The prion protein (PrP(C)) is implicated in the spongiform encephalopathies in mammals, and it is known to bind Cu(II) at the N-terminal region. The region around His111 has been proposed to be key for the conversion of normal PrP(C) to its infectious isoform PrP(Sc). The principal aim of this study is to understand the role of protons and methionine residues 109 and 112 in the coordination of Cu(II) to the peptide fragment 106-115 of human PrP, using different spectroscopic techniques (UV-vis absorption, circular dichroism, and electron paramagnetic resonance) in combination with detailed electronic structure calculations. Our study has identified a proton equilibrium with a pK(a) of 7.5 associated with the Cu(II)-PrP(106-115) complex, which is ascribed to the deprotonation of the Met109 amide group, and it converts the site from a 3NO to a 4N equatorial coordination mode. These findings have important implications as they imply that the coordination environment of this Cu binding site at physiological pH is a mixture of two species. This study also establishes that Met109 and Met112 do not participate as equatorial ligands for Cu, and that Met112 is not an essential ligand, while Met109 plays a more important role as a weak axial ligand, particularly for the 3NO coordination mode. A role for Met109 as a highly conserved residue that is important to regulate the protonation state and redox activity of this Cu binding site, which in turn would be important for the aggregation and amyloidogenic properties of the protein, is proposed.

Asunto(s)

Cobre/metabolismo , Histidina/metabolismo , Metionina/metabolismo , Priones/metabolismo , Dicroismo Circular , Espectroscopía de Resonancia por Spin del Electrón , Humanos , Priones/química , Protones , Espectrofotometría Ultravioleta