An eccentric calpain, CAPN3/p94/calpain-3

AbstractCalpains are Ca2+-regulated proteolytic enzymes that are involved in a variety of biological phenomena. Calpains process substrates by limited proteolysis to modulate various protein functions in the cell, and are thus called “modulator proteases.” CAPN3, previously called p94 or calpain-3, has unique features that are not found in any of the other 14 human calpains, or even in other proteases.For instance, CAPN3 undergoes extremely rapid and exhaustive autodegradation. CAPN3 is also the first (and so far, the only) intracellular enzyme found to depend on Na+ for its activation. CAPN3 has both proteolytic and non-proteolytic functions. It has the interesting distinction of being the only protease, other than a few virus proteases, with the ability to regain protease function after its autolytic dissociation; this occurs through a process known as intermolecular complementation (iMOC). Gene mutations causing CAPN3 defects are responsible for limb-girdle muscular dystrophy type 2A (LGMD2A).Unusual characteristics of CAPN3 have fascinated researchers, but have also hampered conventional biochemical analysis. In this review, we describe significant findings about CAPN3 from its discovery to the present, and suggest promising avenues for future CAPN3 research

Functional Analysis of Human Long Non-coding RNAs and Their Associations with Diseases

Within this study, we sought to leverage knowledge from well-characterized protein coding genes to characterize the lesser known long non-coding RNA (lncRNA) genes using computational methods to find functional annotations and disease associations. Functional genome annotation is an essential step to a systems-level view of the human genome. With this knowledge, we can gain a deeper understanding of how humans develop and function, and a better understanding of human disease. LncRNAs are transcripts greater than 200 nucleotides, which do not code for proteins. LncRNAs have been found to regulate development, tissue and cell differentiation, and organ formation. Their dysregulation has been linked to several diseases including autism spectrum disorder (ASD) and cancer. While a great deal of research has been dedicated to protein-coding genes, the relatively recently discovered lncRNA genes have yet to be characterized. LncRNA function is tied closely to when and where they are expressed. Co-expression network analysis offer a means of functional annotation of uncharacterized genes through a guilt by association approach. We have constructed two co-expression networks using known disease-associated protein-coding genes and lncRNA genes. Through clustering of the networks, gene set enrichment analysis, and centrality measures, we found enrichment for disease association and functions as well as identified high-confidence lncRNA disease gene targets. We present a novel approach to the identification of disease state associations by demonstrating genes that are associated with the same disease states share patterns that can be discerned from transcriptomes of healthy tissues. Using a machine learning algorithm, we built a model to classify ASD versus non-ASD genes using their expression profiles from healthy developing human brain tissues. Feature selection during the model-building process also identified critical temporospatial points for the determination of ASD genes. We constructed a webserver tool for the prioritization of genes for ASD association. The webserver tool has a database containing prioritization and co-expression information for nearly every gene in the human genome

Alfaviiruse mittestruktuurne proteaas ja tema liitvalgust substraat: täiuslikult korraldatud kooselu reeglid

Väitekirja elektrooniline versioon ei sisalda publikatsioone.Alfaviirused (sugukond Togaviridae) on artriiti ja entsefaliiti põhjustavad RNA genoomsed viirused. Nende paljunemise strateegia aluseks on viiruse replikaasi valkude süntees ühe nn. mittestruktuurse eelvalgu P1234 kujul ning selle ajaliselt reguleeritud lõikamine valmis valkudeks nsP2 proteaasi abil. Käesoleva väitekirja aluseks olevad uurimistööd viisid nsP2 substraat-spetsiifilisust tagavate mehhanismide väljaselgitamiseni; muu hulgas kirjeldati uudset proteolüütiliste lõikamiste regulatsioonimehhanismi, mis põhineb liitvalgu erinevate regioonide vahelisel „suhtlemisel“ viiruse replikatsiooni kompleksi moodustamise käigus. Sellest saab järeldada, et P1234 lõikamise ajaline regulatsioon sõltub otseselt replikatsioonikompleksi konfiguratsioonidest, millised omakorda on määratud selle komponentide vaheliste interaktsioonide poolt. Seega tõuseb viiruse nsP2 proteaas esile kui keerulise signaalvõrgustiku keskne element, mille roll viirus infektsiooni regulatsioonis seisneb replikatsiooniga kaasnevate sündmuste „jälgimises“ ja nendele reageerimises. Viimane põhineb sellel, et kui viiruse paljunemine jõuab kindla vahe-etapini, siis kaasneb sellega lõikamiskohtade ja/või muude oluliste struktuuride „esitlemine“ proteaasile, mis reageerib toimunud muudatustele lokaalse signaalülekande, mis lõppkokkuvõttes viib replikaasi kompleksi struktuuri järjestikulistele muudatustele, käivitamisega. Kokkuvõttes, tõid läbiviidud uurimised välja asjaolu, et lisaks varem teada olnud lõikamisjärjestuste äratundmisele, omab ka makromolekulaarsete struktuuride moodustamine viiruse valkude poolt olulist (ja mitmel juhul isegi määravat) rolli viiruse proteaasi töö reguleerimisel. Veel enam, eeldati, et seesugune mitmetahuline regulatsioon võib olla paljukomponentsete proteolüütiliste süsteemide üldine omadus. Kirjeldatud avastused ja nende lahtimõtestamine omavad olulist rolli uurimistöödele, mille eesmärgiks on alfaviiruste paljunemist takistavate lähenemiste väljatöötamine. Nii võib saadud tulemuste põhjal järeldada, et lisaks proteaasi aktiivsuse otsesele mõjutamisele võib viiruse replikatsiooni takistada ka mõjutades proteolüüsi regulatsiooni tagavaid molekulide vahelised seoseid.Alphaviruses from the Togaviridae family are RNA viruses that may cause arthritic syndroms and encephalitis. The alphavirus replication strategy relies on the production of replicase proteins initially in the form of non-structural (ns) polyprotein precursor P1234, which during the course of replication becomes proteolytically processed by the virus-encoded nsP2 protease in a temporally regulated manner. The studies that constitute the basis of this thesis led to identification of the requirements for substrate specificity of nsP2 protease and revealed novel mechanism for the regulation of processing based on the specific communication between distant parts of the viral polyprotein brought together during assembly of replication complex. It was concluded that the order of alphaviral ns-polyprotein processing is mostly dependent on the configuration of the replication complex imposed by intermolecular interactions meant to guarantee timely cleavages. The alphaviral protease therefore emerges as an integral part of the sophisticated signaling mechanism, in which the regulatory task of the protease consists of monitoring the succession and completion of the events of viral infection. Once the respective replication status-induced conformational changes within replicase allow the presentation of the scissile bond and/or other essential determinants of substrate recognition like exosites, the local protease signaling is initiated, which apparently leads to further reconfiguration of the viral replication complex. Combined, the studies unveiled the decisive role played by the macromolecular assembly-dependent component of substrate recognition in addition to the sequence-dependent component, the combination of which may be expected to constitute the basis of regulation in multi-site proteolytic systems in general. Described findings and their interpretations are expected to provide with essential grounds and directions for further studies on the restriction of alphaviral replication through affecting the center of viral proteolytic activity or via intervention with its regulation by targeting intramolecular interactions

Papel de las calpaínas en la remodelación tisular de la glándula mamaria tras el ciclo embarazo/lactancia

En el presente proyecto de tesis se ha contribuido a elucidar el papel de las calpaínas en la muerte celular programada y la remodelación tisular de la glándula mamaria tras el ciclo embarazo/lactancia. La involución de la glándula mamaria se caracteriza por la muerte celular programada (MCP) del epitelio secretor y por la proliferación e invasión de los adipocitos, con el fin de conseguir el estado glandular pre-gestacional. Estudios previos demuestran que las calpaínas, proteasas calcio-dependientes, están activas a lo largo de esta regresión post-lactancia. Estas enzimas, participan en la escisión de numerosas proteínas ubicadas la mayoría en membranas celulares, al poseer las calpaínas un dominio de unión a fosfolípidos que facilita su activación. El incremento del calcio intracelular que acontece durante el destete como consecuencia de la falta de succión por parte de las crías, también incrementa su actividad. Durante la involución de la glándula mamaria, la translocación lisosomal, mitocondrial y nuclear de las calpaínas está involucrada en la desestabilización progresiva de estos orgánulos durante la muerte celular programada de las células epiteliales secretoras. La liberación del contenido lisosomal al citosol constituye el primer paso en la ejecución de esta muerte celular programada. Al translocarse al lisosoma, las calpaínas participan en la degradación de proteínas asociadas a la membrana lisosomal como LAMP2a y VATB2, identificadas por primera vez como dianas lisosomales de estas proteasas. La translocación mitocondrial de las calpaínas induce una desestabilización de la membrana mitocondrial, con la consiguiente liberación de citocromo c al citosol. En el núcleo de células epiteliales, las calpaínas degradan nucleoporinas ubicadas en posiciones periféricas del complejo del poro nuclear. De hecho, nucleoporinas localizadas en partes centrales del poro, no son degradadas por su inaccesible localización. Sin embargo, también se ha detectado la presencia de calpaína-1 en núcleos de células adipocíticas a las 48 horas de destete. En estas células, se observa que dicha proteasa presenta una función diferente a la presente en células epiteliales. Experimentos realizados in vivo e in vitro demuestran que la calpaina-1 escinde el extremo amino de la Histona H3 en células estromales de ratones destetados. Ensayos de ChIP señalan la presencia de calpaína-1 en los promotores de genes involucrados en la diferenciación y adquisición del fenotipo de adipocito, como CEBPalfa y leptina, sugiriendo la idea de una participación de la calpaína-1 en la diferenciación y proliferación de los adipocitos, a través de la remodelación de la estructura de la cromatina. Nuestras observaciones revelan que, durante la involución de la glándula mamaria, la calpaína-1 estaría involucrada en la retirada masiva del epitelio secretor, además de presentar un posible papel en la adipogénesis. La desregulación de la actividad de estas proteasas se ha correlacionado con un gran número de enfermedades, como distrofias musculares, neurodegeneración y cáncer, entre otras. De hecho, un reciente estudio determinó que un incremento de la actividad de calpaína-2 desemboca en un peor pronóstico y menor supervivencia específica de pacientes con tumores de mama triple negativos. De ahí que, conocer en profundidad el mecanismo de acción, las dianas proteolíticas, y en general, el funcionamiento de las calpaínas en el contexto de la glándula mamaria, pueda contribuir a avanzar en el conocimiento de estas proteasas con potencial implicación en el cáncer de mama.Mammary gland involution is characterized by programmed cell death of the secretory epithelium, followed by differentiation/proliferation of adipocytes, in order to leave the gland in its pre-gestational state. Prior studies demonstrate that calpains, calcium-dependent proteases, increase their activity during weaning. We observed that lysosomal, mitochondrial, and nuclear calpain translocation is involved in the subsequent organelle membrane destabilization, leading to epithelial cell death. Calpains translocate to lysosomal membranes, where they proteolyze lysosome-associated membrane proteins such as VATB2 and LAMP2a, identified for the first time as calpain targets within the lysosomal membrane. Mitochondrial calpain translocation is involved in the destabilization of mitochondrial membrane, triggering cytochrome c release. Calpains are also able to translocate to nuclear membrane of epithelial cells, degrading nucleoporins localized in peripheral positions of the nuclear pore complex. However, CAPN1 has also been found in the nuclei of adipose cells. This protease is involved in the differentiation process of adipocytes during mammary gland post-lactational remodeling, playing a different role that the one observed in epithelial cells. Histone H3, was first identified as a nuclear target of CAPN1 in adipocytes. The proteolysis occurs in the aminoterminal domain of the protein, around lysine in position 9 and serine in position 10. The removal of this aminoterminal domain favours the activation of adipogenic-related genes through chromatin remodeling. Herein, we have reviewed the major role that calpains play in the involution of the mammary gland after the pregnancy/lactation cycle. CAPN’s targets within the lysosomal, mitochondrial and nuclear membranes, induce membrane leakiness and favors cell death. Whilst in adipose cells, CAPN1 is involved in the modulation of pre-adipocytes differentiation to mature adipocytes through chromatin remodeling