Calcium-dependent conformational flexibility of a CUB domain controls activation of the complement serine protease C1r.

C1, the first component of the complement system, is a Ca(2+)-dependent heteropentamer complex of C1q and two modular serine proteases, C1r and C1s. Current functional models assume significant flexibility of the subcomponents. Noncatalytic modules in C1r have been proposed to provide the flexibility required for function. Using a recombinant CUB2-CCP1 domain pair and the individual CCP1 module, we showed that binding of Ca(2+) induces the folding of the CUB2 domain and stabilizes its structure. In the presence of Ca(2+), CUB2 shows a compact, folded structure, whereas in the absence of Ca(2+), it has a flexible, disordered conformation. CCP1 module is Ca(2+)-insensitive. Isothermal titration calorimetry revealed that CUB2 binds a single Ca(2+) with a relatively high K(D) (430 mum). In blood, the CUB2 domain of C1r is only partially (74%) saturated by Ca(2+), therefore the disordered, Ca(2+)-free form could provide the flexibility required for C1 activation. In accordance with this assumption, the effect of Ca(2+) on the autoactivation of native, isolated C1r zymogen was proved. In the case of infection-inflammation when the local Ca(2+) concentration decreases, this property of CUB2 domain could serve as subtle means to trigger the activation of the classical pathway of complement. The CUB2 domain of C1r is a novel example for globular protein domains with marginal stability, high conformational flexibility, and proteolytic sensitivity. The physical nature of the behavior of this domain is similar to that of intrinsically unstructured proteins, providing a further example of functionally relevant ligand-induced reorganization of a polypeptide chain

Proteolízis az idegrendszerben: a humán agyi tripszin szerkezetének és funkciójának vizsgálata = Proteolysis in central nervous system: investigations of the structure and function of human brain trypsin

Klónoztunk a humán tripszinogén 4 teljes kódoló szekvenciáját. Bakterális expressziós rendszerben kifejeztük a két alternatív iniciációval keletkező izoformáját. Expresszáltuk és enzimatikusan karakterizáltuk az a tripszin 4-et. Meghatároztuk az aktív enzim benzamidin komplexének térszerkezetét. Feltérképeztük a mRNS és a fehérje eloszlását a humán agy 17 különböző régiójában. Vizsgáltuk az agyban előforduló potenciális szubsztrátjait. Megállapítottuk, hogy számos, a citoszkeletális váz felépítésében és regulálásában szereplő fehérje és a mielin bázikus fehérje szubsztrátja lehet a tripszin 4-nek. Izoláltuk a tripszinogén 4-et emberi agyszövetből és meghatároztuk amino terminális szekvenciáját. Tranziens szövettenyészetben megfelelően tervezett expressziós vektorok segítségével megállapítottuk, hogy az amino terminális leucin beépülését nem-konvencionális CTG iniciátor kodon irányítja. Meghatároztuk a tripszin - metilubellireril guanidinobenzoáttal történő reakciójának elemi sebességi állandóit. Vizsgáltuk számos természetes és szintetikus inhibitor gátló képességét, és valószínű magyarázatot adtunk arra, miért lehetetlen kanonikus mechanizmus alapján gátolni a tripszin 4-et. | We have isolated the complete coding sequence of human trypsinogen 4. In bacterial expression system we expressed and purified the two putative isoforms of the enzyme. We isolated the active enzyme as well, and characterized its enzymatic properties. We determined the X-ray structure of the trypsin 4 ? benzamidine complex. By quantitative real time PCR and sandwich ELISA we determined the determined the distribution of the mRNA and protein in 17 different regions of the human brain. By sequencing the zymogen isolated from human brain we concluded that predominant form in the human brain possesses leucine amino terminus. Using human tissue cultures transiently transfected with appropriately designed expression vectors we proved that the incorporation of the amino terminal leucine is directed by a non-conventional CTG initiation codon. We determined the elementary kinetic constants of the trypsin 4 - methylumbelliferyl guanidine benzoate reaction. We studied the inhibitory potential of several natural and synthetic polypeptides, and we reasoned why is it impossible to inhibit trypsin 4 by a canonical inhibitor

Tanulással összefüggő előagyi rendszerek madarakban = Learning-associated forebrain systems in birds

A kutatóprogram a viselkedés motivációjának idegi alapjait vizsgálja multidiszciplináris megközelítéssel, madár idegrendszeri modell felhasználásával. A vizsgálatból extrapolálható eredmények felhasználhatók az emlős viszonyok leírására és értelmezésére is. A kutatócsoport korábbi nemzetközi tapasztalataira épülő program megvalósításával a következő területeken értünk el eredményeket: (1) A tanulással és motivációval összefüggő agyterületek (thalamus, septum, nucl. accumbens, ventralis tegmentalis area) funkciós morfológiai jellemzése. Ezen belül a jutalmazásban és addikcióban kulcsszerepet játszó nucleus accumbens pontos lokalizációja, szubdiviziói és kapcsolatrendszerének feltárása madarakon, valamint az amygdalo-accumbens pályarendszer elhárító tanulásban játszott szerepének igazolása; (2) Dopaminerg és dopaminoceptív neuronrendszerek szerepe háziszárnyas tanulási és motivációs folyamataiban. Dopamin receptor antagonisták hatása a memória retenciójára. Striato-tegmentalis pályarendszerek és szelektív projekciókkal rendelkező neuronjaik jellemzése. (3) A serkentő neurotranszmitterek és a dopamin striatalis kölcsönhatása, motivációban, döntéshozatalban és tanulásban játszott szerepe. Az aszpartát sajátos magatartásfüggő szerepének kimutatása a striatum és a törzsdúcok neurális hálózatában. (4) Cannabinoid receptorok megoszlása és a tanulási folyamatra gyakorolt hatása. | In the research program we investigated the neural foundations of motivation, using multidisciplinary approach and the avian nervous system as a model. The results extrapolated from the study may be adapted also for the description and interpretation of mammalian systems. Based upon international experience of the group, progress has been made primarily in the following areas: (1) Functional morphological characterisation of learning and behaviour associated brain regions (thalamus, septum, nucl. accumbens, ventral tegmental area). In particular, precise localization of the nucleus accumbens, its subdivisions and connectivity, and evidence for the role of amygdalo-accumbens pathway in avoidance learning. (2) The role of dopaminergic and dopaminoceptive neuronal systems in learning and motivation of domestic chicks. Effect of dopamine receptor antagonists on memory retention. Characterisation of striato-tegmental pathways and neurons with selective projections. (3) Striatal interaction between excitatory transmitter amino acids and dopamine, its role in motivation, decision-making and learning. Verification of specific behaviour-linked effect of the excitatory transmitter aspartate in the neural circuits of striatum and basal ganglia. (4) Tissue distribution of cannabinoid receptors and their effect on the learning process

The Effect of Sleep Deprivation and Subsequent Recovery Period on the Synaptic Proteome of Rat Cerebral Cortex

Sleep deprivation (SD) is commonplace in the modern way of life and has a substantial social, medical, and human cost. Sleep deprivation induces cognitive impairment such as loss of executive attention, working memory decline, poor emotion regulation, increased reaction times, and higher cognitive functions are particularly vulnerable to sleep loss. Furthermore, SD is associated with obesity, diabetes, cardiovascular diseases, cancer, and a vast majority of psychiatric and neurodegenerative disorders are accompanied by sleep disturbances. Despite the widespread scientific interest in the effect of sleep loss on synaptic function, there is a lack of investigation focusing on synaptic transmission on the proteome level. In the present study, we report the effects of SD and recovery period (RP) on the cortical synaptic proteome in rats. Synaptosomes were isolated after 8 h of SD performed by gentle handling and after 16 h of RP. The purity of synaptosome fraction was validated with western blot and electron microscopy, and the protein abundance alterations were analyzed by mass spectrometry. We observed that SD and RP have a wide impact on neurotransmitter-related proteins at both the presynaptic and postsynaptic membranes. The abundance of synaptic proteins has changed to a greater extent in consequence of SD than during RP: we identified 78 proteins with altered abundance after SD and 39 proteins after the course of RP. Levels of most of the altered proteins were upregulated during SD, while RP showed the opposite tendency, and three proteins (Gabbr1, Anks1b, and Decr1) showed abundance changes with opposite direction after SD and RP. The functional cluster analysis revealed that a majority of the altered proteins is related to signal transduction and regulation, synaptic transmission and synaptic assembly, protein and ion transport, and lipid and fatty acid metabolism, while the interaction network analysis revealed several connections between the significantly altered proteins and the molecular processes of synaptic plasticity or sleep. Our proteomic data implies suppression of SNARE-mediated synaptic vesicle exocytosis and impaired endocytic processes after sleep deprivation. Both SD and RP altered GABA neurotransmission and affected protein synthesis, several regulatory processes and signaling pathways, energy homeostatic processes, and metabolic pathways. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12035-021-02699-x

Chronic Cerebral Hypoperfusion Induced Synaptic Proteome Changes in the rat Cerebral Cortex

Chronic cerebral hypoperfusion (CCH) evokes mild cognitive impairment (MCI) and contributes to the progression of vascular dementia and Alzheimer’s disease (AD). How CCH induces these neurodegenerative processes that may spread along the synaptic network and whether they are detectable at the synaptic proteome level of the cerebral cortex remains to be established. In the present study, we report the synaptic protein changes in the cerebral cortex after stepwise bilateral common carotid artery occlusion (BCCAO) induced CCH in the rat. The occlusions were confirmed with magnetic resonance angiography 5 weeks after the surgery. Synaptosome fractions were prepared using sucrose gradient centrifugation from cerebral cortex dissected 7 weeks after the occlusion. The synaptic protein differences between the sham operated and CCH groups were analyzed with label-free nanoUHPLC-MS/MS. We identified 46 proteins showing altered abundance due to CCH. In particular, synaptic protein and lipid metabolism, as well as GABA shunt-related proteins showed increased while neurotransmission and synaptic assembly-related proteins showed decreased protein level changes in CCH rats. Protein network analysis of CCH-induced protein alterations suggested the importance of increased synaptic apolipoprotein E (APOE) level as a consequence of CCH. Therefore, the change in APOE level was confirmed with Western blotting. The identified synaptic protein changes would precede the onset of dementia-like symptoms in the CCH model, suggesting their importance in the development of vascular dementia. © 2017 Springer Science+Business Media New Yor

Maternal alterations in the proteome of the medial prefrontal cortex in rat

Proteomic differences between rat dams and control mothers deprived of their pups immediately after delivery were investigated in the medial prefrontal cortex (mPFC).A 2-D DIGE minimal dye technique combined with LC-MS/MS identified 32 different proteins that showed significant changes in expression in the mPFC, of which, 25 were upregulated and 7 were downregulated in dams. The identity of one significantly increased protein, the small heat-shock protein alpha-crystallin B chain (Cryab), was confirmed via Western blot analysis. Alpha-crystallin B chain was distributed in scattered cells in the mPFC, as demonstrated by immunohistochemistry. Furthermore, it was found to be localized in parvalbumin-containing neurons using double labeling. The elevation of its mRNA level in rat dams was also demonstrated via RT-PCR.The functional classification of the altered proteins was conducted using the UniProt and Gene Ontology protein databases. The identified proteins predominantly participate in synaptic transport and plasticity, neuron development, oxidative stress and apoptosis, and cytoskeleton organization. A common regulator and target analysis of these proteins determined using the Elsevier Pathway Studio Platform suggests that protein level changes associated with pup nursing are driven by growth factors and cytokines, while the MAP kinase pathway was identified as a common target. A high proportion of the proteins that were found to be altered in the mPFC are associated with depression. Biological significance: The behavior and emotional state of females change robustly when they become mothers. The brain, which governs these changes, may also undergo molecular alterations in mothers. As no proteomics approaches have been applied regarding maternal changes in the brain, we addressed this issue in the mPFC as this brain area is the uppermost cortical center of maternal control and the associated mood changes. The high number of protein-level alterations found between mothers taking care of their litter and those without pups indicates that pup nursing is associated with cortical protein-level changes. Alterations in proteins participating in synaptic transport, plasticity and neuron development suggest neuroplastic changes in the maternal brain. In turn, the relatively high number of altered proteins in the mPFC associated with depression suggests that the physiological effects of the protein-level alterations in the maternal mPFC could promote the incidence of postpartum depression. Cryab, a protein confirmed to be increased during maternal behaviors, was selectively found in parvalbumin cells, which, as fast-spiking interneurons, are associated with depression. The function of Cryab should be further investigated to establish whether it can be used to identify drug targets for future drug development. © 2016 Elsevier B.V

Cell Surface Protein mRNAs Show Differential Transcription in Pyramidal and Fast-Spiking Cells as Revealed by Single-Cell Sequencing

The prefrontal cortex (PFC) plays a key role in higher order cognitive functions and psychiatric disorders such as autism, schizophrenia, and depression. In the PFC, the two major classes of neurons are the glutamatergic pyramidal (Pyr) cells and the GABAergic interneurons such as fast-spiking (FS) cells. Despite extensive electrophysiological, morphological, and pharmacological studies of the PFC, the therapeutically utilized drug targets are restricted to dopaminergic, glutamatergic, and GABAergic receptors. To expand the pharmacological possibilities as well as to better understand the cellular and network effects of clinically used drugs, it is important to identify cell-type-selective, druggable cell surface proteins and to link developed drug candidates to Pyr or FS cell targets. To identify the mRNAs of such cell-specific/enriched proteins, we performed ultra-deep single-cell mRNA sequencing (19 685 transcripts in total) on electrophysiologically characterized intact PFC neurons harvested from acute brain slices of mice. Several selectively expressed transcripts were identified with some of the genes that have already been associated with cellular mechanisms of psychiatric diseases, which we can now assign to Pyr (e.g., Kcnn2, Gria3) or FS (e.g., Kcnk2, Kcnmb1) cells. The earlier classification of PFC neurons was also confirmed at mRNA level, and additional markers have been provided