Diseño y evaluación computacional de LYTACS para la degradación selectiva de MMP-2 / Computational design and evaluation of LYTACS for the selective degradation of MMP-2

Los LYTACs (LYsosome TArgeting Chimeras) son una novedosa estrategia farmacológica basada en la degradación dirigida de proteínas extracelulares y transmembrana. Su mecanismo de acción se basa en la utilización de un receptor de membrana para internalizar a una proteína diana y promover su degradación lisosomal. Hasta la fecha, su desarrollo se ha basado en el uso de anticuerpos para la unión a la proteína diana, lo cual presenta ciertas desventajas desde el punto de vista farmacocinético y sintético. El objetivo de este trabajo es diseñar un LYTAC capaz de inducir la degradación selectiva de MMP-2 (LYTAC-MMP2), una metaloproteasa de la matriz que se encuentra sobreexpresada en diversos tipos de cáncer. LYTAC-MMP2 está formado por un ligando del receptor de manosa-6-fosfato independiente de cationes (CI- MPR) y un inhibidor selectivo de MMP2 previamente descrito. Se han empleado métodos computacionales de modelado por homología, docking y dinámica molecular para estudiar el receptor CI-MPR y su mecanismo de internalización, así como para la comparación del comportamiento dinámico libre en agua de un ligando de CI-MPR descrito en la bibliografía y el LYTAC-MMP2.(AU)

LYTACs (LYsosome TArgeting Chimeras) are a novel pharmacological strategy based on the targeted protein degradation of extracellular and transmembrane proteins. Their mechanism of action is based on the use of a membrane receptor to internalize a target protein and mediate its lysosomal degradation. To date, its development has been focused on the use of antibodies for target binding, which has certain disadvantages from the pharmacokinetic and synthetic point of view. The aim of this work is to design a LYTAC capable of inducing the selective degradation of MMP-2 (LYTAC-MMP2), a matrix metalloprotease that is overexpressed in many types of cancer. LYTAC-MMP2 consists of a cation-independent mannose-6-phosphate receptor (CI-MPR) ligand and a selective MMP-2 inhibitor developed by our research group. Computational methods of homology modelling, docking and molecular dynamics have been used to study the CI-MPR receptor and its internalization mechanism, as well as for the comparison of the dynamic behaviour in water of a CI-MPR ligand described in the literature and LYTAC-MMP2.(AU)

Enfermedad CLN8 congénita de lipofuscinosis neuronal ceroidea: un nuevo fenotipo / Congenital CLN8 disease of neuronal ceroid lipofuscinosis: a novel phenotype

Introducción. La enfermedad CLN8 es uno de los 13 tipos genéticos reconocidos de lipofuscinosis neuronal ceroidea, un grupo de trastornos neurodegenerativos de acumulación lisosómica, los más frecuentes en la infancia. La causan mutaciones en la proteína transmembrana CLN8 de 286 aminoácidos, cuya función se desconoce. Las variantes patológicas en el gen CLN8 se asociaron con dos fenotipos diferentes: la variante infantil tardía en individuos de diversos países alrededor del mundo, y la epilepsia progresiva con retraso mental, que aparece en pacientes finlandeses y turcos. Caso clínico. Niña que mostró retraso psicomotor y demencia desde el nacimiento, convulsiones tonicoclónicas, mioclonía, ataxia con atrofia cerebelosa y muerte temprana a los 12 años. La microscopia electrónica de la piel mostró una mezcla de citosomas con patrones de depósitos osmiofílicos granulares, curvilíneos y de «huella digital», y mitocondrias hipertrofiadas. Se encontraron dos variantes patológicas de ADN en el gen CLN8 (exón 2 c.1A>G; p.?/ exón 3 c.792C>G; p.Asn264Lys), lo que confirmó un genotipo heterocigoto compuesto. Conclusión. Éste es el caso índice en América Latina para el nuevo fenotipo congénito de la enfermedad CLN8. La sospecha de esta patología debería sustentarse genéticamente en casos de síndrome neurodegenerativo con retraso psicomotor desde el nacimiento, dificultad del habla y convulsiones. El curso clínico incluye ataxia, atrofia cerebelosa y muerte temprana

Introduction. CLN8 disease is one of the thirteen recognized genetic types of neuronal ceroid lipofuscinosis, a group of neurodegenerative lysosomal storage disorders, most frequent in childhood. A putative 286 amino acids transmembrane CLN8 protein with unknown function is affected. Pathological variants in the CLN8 gene were associated with two different phenotypes: variant late-infantile in individuals from many countries worldwide, and epilepsy progressive with mental retardation, appearing in Finnish and Turkish subjects. Case report. The girl showed psychomotor delay and dementia since birth, tonic-clonic seizures, myoclonus, ataxia with cerebellar atrophy, and early death at 12 years old. Electron microscopy of the skin showed mixed GROD, curvilinear, fingerprint cytosomes and mitochondrial hypertrophy. Two pathological DNA variants in the CLN8 gene (exon 2 c.1A>G; p.?/ exon 3 c.792C>G; p.Asn264Lys) were found confirming a compound heterozygous genotype. Conclusion. This case is the Latin American index for a new congenital phenotype of the CLN8 disease. The congenital phenotype has to be added to the clinical spectrum of the CLN8 disease. The suspicion of CLN8 disease should be genetically sustained in challenging cases of a neurodegenerative syndrome with psychomotor delay since birth, speech difficulty and seizures. The course includes ataxia, cerebellar atrophy, and early death

Reducing mitochondrial bound hexokinase II mediates transition from non-injurious into injurious ischemia/reperfusion of the intact heart

Ischemia/reperfusion (I/R) of the heart becomes injurious when duration of the ischemic insult exceeds a certain threshold (approximately ≥20 min). Mitochondrial bound hexokinase II (mtHKII) protects against I/R injury, with the amount ofmtHKII correlating with injury. Here, we examine whether mtHKII can induce the transition from non-injurious to injurious I/R, by detaching HKII from mitochondria during a non-injurious I/R interva l . Additionally, we examine possible underlying mechanisms (increased reactive oxygen species (ROS), increased oxygen consumption (MVO2) and decreased cardiac energetics) associated with this transition. Langendorff perfused rat hearts were treated for 20 min with saline, TAT-only or 200 nM TAT-HKII, a peptide that translocates HKII from mitochondria. Then, hearts were exposed to non-injurious 15-min ischemia, followed by 30-min reperfusion. I/R injury was determined by necrosis (LDH release) and cardiac mechanical recovery. ROS were measured by DHE fluorescence. Changes in cardiac respiratory activity (cardiac MVO2 and efficiency and mitochondrial oxygen tension (mitoPO2) using protoporphyrin IX) and cardiac energetics (ATP, PCr, ΔGATP) were determined following peptide treatment.When exposed to 15-min ischemia, control hearts had no necrosis and 85% recovery of function. Conversely, TAT-HKII treatment resulted in significant LDH release and reduced cardiac recovery (25%), indicating injurious I/R. This was associated with increased ROS during ischemia and reperfusion. TAT-HKII treatment reducedMVO2 and improved energetics (increased PCr) before ischemia, without affecting MVO2/RPP ratio or mitoPO2. In conclusion, a reduction in mtHKII turns non-injurious I/R into injurious I/R. Loss of mtHKII was associated with increased ROS during ischemia and reperfusion, but not with increased MVO2 or decreased cardiac energetics before damage occurs

Phosphorylation and subcellular localization of Na+/H+ exchanger isoform 3 (NHE3) are associated with altered gallbladder absorptive function after formation of cholesterol gallstones

Na+/H+ exchanger isoform 3 (NHE3) dysfunction is thought to contribute to the altered gallbladder absorption that occurs in cholesterol gallstone disease, but the mechanism is unknown. The current study was undertaken to examine the expression, phosphorylation, and subcellular localization of NHE3 in gallbladder epithelium cells (GBECs) of male C57BL/6 mice on a control or lithogenic diet. Thirty-six 8-week-old male C57BL/6 mice were randomly assigned to receive a high cholesterol diet or a regular diet for 8 weeks. Gallstone formation was recorded. Gallbladder bile cholesterol, phospholipid, and total bile acids were examined. RT-PCR was used to measure NHE3 mRNA expression. NHE3 protein expression and subcellular localization were examined by Western blotting and immunofluorescence microscopy, respectively. Gallstones were formed in all mice fed the lithogenic diet. Despite higher NHE3 mRNA expression in gallbladders of the mice on the lithogenic diet than in those on the control diet, there was no significant difference in expression of total NHE3 protein. However, a higher level of NHE3 phosphorylated at serine-552 (P-NHE3) was seen on the lithogenic diet. In immunofluorescence studies, NHE3 protein was expressed both on the apical membrane and in the cytoplasm of mouse GBEC. This pattern of subcellular distribution of NHE3 strongly corroborates an exchanger trafficking mechanism in NHE3 activity regulation in mouse GBEC. We conclude that increased phosphorylation of NHE3 following gallstone formation leads to turnover of the exchanger, resulting in decreased gallbladder concentrating function (AU)

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The role of sirtuns in cellular homeostasis

Sirtuins are evolutionarily conserved nicotinamide adenine dinucleotide (NAD+)-dependent lysine deacylases or ADP-ribosyltransferases. These cellular enzymes are metabolic sensors sensitive to NAD+ levels that maintain physiological homeostasis in the animal and plant cells (AU)

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Chlamydia pneumoniae CPj0783 interaction with Huntingtin-protein14

Chlamydia pneumoniae is a Gram-negative, obligate intracellular pathogen that causes community-acquired respiratory infections. After C. pneumoniae invades host cells, it disturbs the vesicle transport system to escape host lysosomal or autophagosomal degradation. By using a yeast mis-sorting assay, we found 10 C. pneumoniae candidate genes involved in aberrant vesicular trafficking in host cells. One of the candidate genes, CPj0783, was recognized by antibodies from C. pneumoniae-infected patients. The expression of CPj0783 was detected at mid to late-cycle time points and increased during the inclusion maturation. Two-hybrid screening in yeast cells revealed that CPj0783 interacted with Huntingtin-interacting protein 14 (HIP14). The specific interaction between CPj0783 and HIP14 could be demonstrated by an in vivo co-immunoprecipitation assay and an in vitro GST pull-down assay. It was also demonstrated that HIP14 was localized in the Golgi apparatus and colocalized with CPj0783. HIP14 has a palmitoyl transferase activity that is involved in the palmitoylation-dependent vesicular trafficking of several acylated proteins. These findings suggest that CPj0783 might cause abnormal vesicle-mediated transport by interacting with HIP14 (AU)

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Hemodiafiltración en línea secuencial (HDF-OL-S): una nueva opción terapéutica / No disponible

La hemodiafiltración en línea proporciona una alta eficacia depurativa de moléculas de mediano y gran peso molecular. Existe consenso sobre la necesidad de conseguir al menos 20 L de ultrafiltración en postdilución y tasas de reducción de B2-microglobulina mayores del 70%. Desafortunadamente muchos pacientes tienen un acceso vascular inadecuado siendo muy difícil lograr esos volúmenes de ultrafiltración sin complicaciones clínicas. El objetivo de este trabajo fue conseguir un volumen de ultrafiltración equivalente a 20 L en postdilucional, mediante la técnica «Secuencial» (HDF-OL-S) que comienza siendo postdilucional y cuando la PTM alcanza los 250 mmHg se trasforma en predilucional. Se realizó una sesión de hemodiálisis de alto flujo (HD-HF), una de hemodiafiltración postdilucional (HDF-OL-P) y otra sesión en modo secuencial a 16 pacientes durante 3 semanas consecutivas, en la sesión de mitad de semana. Se compararon los rendimientos de eliminación de pequeñas y medianas moléculas entre las diferentes técnicas. Se midió la presión prefiltro (PPF) mediante manómetro predializador. No encontramos diferencias en el Kt/V, tasa de reducción de urea y de creatinina entre las3 técnicas. La tasa de reducción de B2-microglobulina y mioglobina fue significativamente mayor tanto en HDFOL-P como en HDF-OL-S con respecto a la HD-HF, no habiendo diferencias entre ambas técnicas de HDF. Existió una correlación directa entre PTM y PPF a lo largo de la sesión en todas las técnicas. La PPF horaria se correlacionaba mejor que PTM con los niveles basales de albúmina sérica, hematocrito y porcentaje de hemoconcentración al final de la diálisis. La HDF-OL-S es una técnica de hemodiálisis con los mismos beneficios de la postdilucional que permite lograr volúmenes de ultrafiltración dentro de los objetivos planteados. Creemos podría ser útil en pacientes con flujos sanguíneos limitados para lo cual habría que diseñar nuevos estudios. La PPF aporta información complementaria a la PTM (AU)

Background: On line haemodiafiltration provides the greatest clearance for low and high-molecular weight uremic toxins, which is associated with a lower risk of mortality in our patients. Nowadays, there’s increasing evidence about the need of achieving at least 20 litters ultrafiltration in postdilution mode and70% reduction of beta-2-microglobulin (B2M), however it requires a vascular access’s high blood flow. Unfortunately, we do not succeed in these objectives because of our patients being older, diabetic and with poor vascular access; in this situation highblood flows are more difficult to get at the expense of lower postdilution exchange volumes. The aim of this study was to assess the efficiency of OL-S-HDF to obtain an equivalent ultrafiltration volume as 20 L in OL-postdilution-HDF (OL-P-HDF). OL-SHD Finitially begins in postdilution mode changing to predilution once the transmembrane pressure (TMP) reached 250 mmHg. Methods: We performed one high-flux HD session (HF-HD), one OL-P-HDF session and one OL-S-HDF session in each of the 16adult patients who participated during 3 consecutive weeks. We compared the clearance rates of low and middle molecules such asurea, creatinine, B2M, myoglobulin and levels of albumin and haematocrit between the 3 different techniques. We measured the pre-filter pressure (PFP) by a manometer set before the dialyzer. Results: The main characteristics of the sessions are described in table Nº1. There wasn¿t significant difference in Kt/V, urea and creatinine removal between the three techniques. B2M and myoglobulin¿s clearance rates were significantly higher in both hemodiafiltration modes than in HF-HD (p = 0.000), however we didn¿t find differences between OL-P-HDF and OL-S-HDF. There was a direct correlation between PFP and TMP along the sessions in every technique (p < 0.05). We found that PFP was better than TMP to correlate with pre-dialysis levels of albumin and haematocrit and also with the haemoconcentration percentage at the end of the sessions. Conclusions: This study confirms that OL-S-HDF is as good as OL-P-HDF and it could be a useful technique to treat patients with suboptimal access¿s blood flow to get to achieve ultrafiltration volumes within the objectives. PFP could offer extra information than TMP (AU)

Toll-like receptor signaling: Involvement in graft transplantation

Los receptores tipo Toll (TLR) son componentes fundamentales del sistema inmune innato. La activación de TLRs conduce a la expresión de genes inflamatorios y requiere de diferentes proteínas adaptadoras,así como de una regulación muy fina a través de proteínasinhibidoras que permiten el control y la finalización de la respuesta inflamatoria. Los TLRs detectan patógenos exógenos gracias al reconocimiento de estructuras moleculares asociadas a los patógenos (pathogen-associated molecular patterns, PAMPs). Además, nuevos datos indican que los TLRs pueden ser activados por señales endógenas liberadas durante procesos de isquemia-reperfusión, y daño tisular. Esta activación puede tener relevancia en los fenómenos que determinan la supervivencia o el rechazo de los órganos transplantados.En esta revisión se discutirán los últimos avances en las rutas de señalización de TLRs, así como la posible implicación de los TLRs en el transplante de órganos (AU)

Toll like receptors (TLRs) are key components of the innate immune system. TLRs signaling leads to the expression of inflammatory genes, and relies on the usage of different intracellular adaptor proteinsand a tight modulation by negative regulators to attenuate inflammation. TLRs are key sensors of foreign pathogens through the recognition of pathogen-associated molecular patterns (PAMPs). In addition, emerging evidence shows that TLRs can be activated by endogenoussignals released in the settings of ischemiareperfusion, and tissue damage, which may potentially contribute to the survival or rejection of transplanted organs. This review describes the recent advances in TLR signaling and addresses the potential involvement of TLRs in organ transplantation (AU)

Intracellular transport of a heterologous membrane protein, the human transferrin receptor, in Saccharomyces cerevisiae / Transporte intracelular de una proteína de membrana heteróloga, el receptor de transferrina humano, en Saccharomyces cerevisiae

We have analyzed the intracellular behavior of the human transferrin receptor (TfR) in Saccharomyces cerevisiae. The major part of the heterologously expressed TfR, which has previously been used as a model for heterologous expression of membrane proteins in yeast, is localized in the endoplasmic reticulum (ER) membranes; a minor fraction is present in the plasma membrane (PM). The stability of the TfR depends on vacuolar proteases, implying that it is degraded in the vacuolar compartment. Degradation is further dependent on favorable transport conditions to this compartment. The main bottleneck of transport seems to be the transition from the ER to the PM. The chaperone Cne1p, which is involved in quality control in the ER, plays a role in regulating the amount of heterologous TfR, as deletion of CNE1 leads to significant accumulation of the protein. This is the first demonstration of the involvement of CNE1 in regulating the level of heterologous membrane proteins (AU)

Hemos analizado el comportamiento intracelular del receptor de la transferrina humano (TfR) en Saccharomyces cerevisiae. La mayor parte del TfR expresado heterólogamente, usado previamente como modelo de expresión heteróloga de proteínas de membrana en levaduras, se localiza en las membranas del retículo endoplásmico (RE); una fracción menor se encuentra en la membrana plasmática. La estabilidad del TfR depende de proteasas vacuolares, lo cual implica que es degradado en el compartimento vacuolar. La degradación depende asimismo de condiciones favorables de transporte a este compartimento. El factor limitante del transporte parece ser la transición desde el RE a la membrana plasmática. La chaperona Cne1p, implicada en el control de calidad en el RE, juega un papel importante en la regulación de los niveles de TfR heterólogo, ya que la deleción de CNE1 provoca una acumulación significativa de la proteína. Se trata de la primera demostración de la implicación de CNE1 en la regulación de los niveles de proteínas de membrana heterólogas (AU)