Proteasome Interactome and Its Role in the Mechanisms of Brain Plasticity.

Proteasomes are highly conserved multienzyme complexes responsible for proteolytic degradation of the short-lived, regulatory, misfolded, and damaged proteins. They play an important role in the processes of brain plasticity, and decrease in their function is accompanied by the development of neurodegenerative pathology. Studies performed in different laboratories both on cultured mammalian and human cells and on preparations of the rat and rabbit brain cortex revealed a large number of proteasome-associated proteins. Since the identified proteins belong to certain metabolic pathways, multiple enrichment of the proteasome fraction with these proteins indicates their important role in proteasome functioning. Extrapolation of the experimental data, obtained on various biological objects, to the human brain suggests that the proteasome-associated proteins account for at least 28% of the human brain proteome. The proteasome interactome of the brain contains a large number of proteins involved in the assembly of these supramolecular complexes, regulation of their functioning, and intracellular localization, which could be changed under different conditions (for example, during oxidative stress) or in different phases of the cell cycle. In the context of molecular functions of the Gene Ontology (GO) Pathways, the proteins of the proteasome interactome mediate cross-talk between components of more than 30 metabolic pathways annotated in terms of GO. The main result of these interactions is binding of adenine and guanine nucleotides, crucial for realization of the nucleotide-dependent functions of the 26S and 20S proteasomes. Since the development of neurodegenerative pathology is often associated with regioselective decrease in the functional activity of proteasomes, a positive therapeutic effect would be obviously provided by the factors increasing proteasomal activity. In any case, pharmacological regulation of the brain proteasomes seems to be realized through the changes in composition and/or activity of the proteins associated with proteasomes (deubiquitinase, PKA, CaMKIIα, etc.).

DJ-1 Protein and Its Role in the Development of Parkinson's Disease: Studies on Experimental Models.

DJ-1, also known as Parkinson's disease protein 7, is a multifunctional protein ubiquitously expressed in cells and tissues. Interacting with proteins of various intracellular compartments, DJ-1 plays an important role in maintaining different cellular functions. Mutant DJ-1 forms containing amino acid substitutions (especially L166P), typical of Parkinson's disease, are characterized by impaired dimerization, stability, and folding. DJ-1 exhibits several types of catalytic activity; however, in the enzyme classification it exists as protein deglycase (EC 3.5.1.124). Apparently, in different cell compartments DJ-1 exhibits catalytic and non-catalytic functions, and their ratio still remains unknown. Oxidative stress promotes dissociation of cytoplasmic DJ-1 dimers into monomers, which are translocated to the nucleus, where this protein acts as a coactivator of various signaling pathways, preventing cell death. In mitochondria, DJ-1 is found in the synthasome, where it interacts with the ß ATP synthase subunit. Downregulation of the DJ-1 gene under conditions of experimental PD increases sensitivity of the cells to neurotoxins, and introduction of the recombinant DJ-1 protein attenuates manifestation of this pathology. The thirteen-membered fragment of the DJ-1 amino acid sequence attached to the heptapeptide of the TAT protein penetrating into the cells exhibited neuroprotective properties in various PD models both in cell cultures and after administration to animals. Low molecular weight DJ-1 ligands also demonstrate therapeutic potential, providing neuroprotective effects seen during their incubation with cells and administration to animals.

Proteomic profiling data of HEK293 proteins bound to human recombinant renalases-1 and -2.

Renalase (RNLS) is a recently discovered protein involved in blood pressure regulation. It exists both as an intracellular catalytically active flavoprotein (EC 1.6.3.5 dihydro-NAD(P):oxygen oxidoreductase) and an extracellular protein that demonstrates various cell protecting effects. Using a twenty-membered peptide corresponding to the residues 220-239 of the renalase sequence (RP-220) and the HK-2 cell line Wang et al. identified a renalase-binding protein, which was considered as a receptor for extracellular renalase crucial for MAPK signaling (Wang et al., 2015) [1]. In this study we have investigated profiles of renalase binding proteins in HEK293 cells by using affinity based proteomic profiling with full-length recombinant human RNLS-1 and human RNLS-2 as affinity ligands followed by analysis of bound proteins by liquid chromatography-mass spectrometry. Both renalases (RNLS-1 and RNLS-2) contain the RP-220 sequence (residues 220-239) but differ in their C-terminal region (residues 293-342 and 293-325, respectively). Profiling of HEK293 proteins resulted in identification of two different sets of proteins specifically bound to RNLS-1 and RNLS-2, respectively. We thus demonstrate that the C-terminal region is crucial for specific binding of renalase to its targets and/or receptors.

Neurotoxic Effects of Aß6-42 Peptides Mimicking Putative Products Formed by the Angiotensin Converting Enzyme.

Angiotensin converting enzyme (ACE) is involved in proteolytic processing of the amyloid-ß(Aß) peptide implicated in the development of Alzheimer's disease (AD) and known products of ACE-based processing of Aß42 are characterized by reduced aggregability and cytotoxicity. Recently it has been demonstrated that ACE can act as an arginine specific endopeptidase cleaving the N-terminal pentapeptide (Aß1-5) from synthetic Aß peptide analogues. In the context of proteolytic processing of full length Aß42, this suggests possible formation of Aß6-42 species. The aim of this study was to test a hypothesis that some N-terminally truncated Aß peptide(s) could retain aggregability and neurotoxic properties typical for Aß42. We have investigated aggregability of two amyloid-ß peptides, Aß6-42 and isoD7-Aß6-42, mimicking potential proteolytic products of Aß42 and isoD7-Aß42, and evaluated their effects on the repertoire of brain Aß binding proteins, and cytotoxicity towards neuroblastoma SH-SY5Y cells. Aggregability of isoD7-Aß6-42 and Aß6-42 was higher than that of full-length peptides Aß42 and isoD7-Aß42, while the repertoire of mouse brain Aß binding proteins dramatically decreased. Aß6-42 and isoD7-Aß6-42 exhibited higher neurotoxicity towards SH-SY5Y cells than Aß42 and isoD7-Aß42, respectively. They effectively stimulated production of ROS and NO, and also TNFα secretion by cells. Thus, our results suggest that ACE-dependent processing of full-length Aßs could result in formation of more pathogenic peptides.

MPTP Mouse Model of Preclinical and Clinical Parkinson's Disease as an Instrument for Translational Medicine.

Parkinson's disease (PD) is characterized by the appearance of motor symptoms many years after the onset of neurodegeneration, which explains low efficiency of therapy. Therefore, one of the priorities in neurology is to develop an early diagnosis and preventive treatment of PD, based on knowledge of molecular mechanisms of neurodegeneration and neuroplasticity in the nigrostriatal system. However, due to inability to diagnose PD at preclinical stage, research and development must be performed in animal models by comparing the nigrostriatal system in the models of asymptomatic and early symptomatic stages of PD. In this study, we showed that despite the progressive loss of neurons in the substantia nigra at the presymptomatic and symptomatic stage, almost no change was observed in the main functional characteristics of this brain region, including dopamine (DA) uptake and release, dopamine transporter (DAT) and vesicular monoamine transporter 2 (VMAT2) expression, and activity of MAO-A and MAO-B. In the striatum of presymptomatic mice, some parameters (DA release and uptake, MAO-A activity) remained compensatory unchanged or compensatory decreased (MAO-B gene expression and activity), while others-a reduction in DA levels in tissue and extracellular space and in VMAT2 and DAT expression-manifest the functional failure. In symptomatic mice, only a few parameters (spontaneous DA release and uptake, MAO-B gene expression and activity) remained at the same level as at presymptomatic stage, while most parameters (DA level in tissue and extracellular space, DA-stimulated release, VMAT2 and DAT contents), decreased, showing decompensation, which was enhanced by increasing MAO-A activity. Thus, this study provides a comprehensive assessment of the molecular mechanisms of neuroplasticity in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine models of preclinical and clinical stages of PD, which could potentially serve as a powerful tool for translational medicine.

Chemical modifications of amyloid-ß(1-42) have a significant impact on the repertoire of brain amyloid-ß(1-42) binding proteins.

The amyloid-ß peptide(1-42) (Aß) is a key player in the development and progression of Alzheimer's disease (AD). Although much attention is paid to its role in formation of extracellular amyloid plaques and protein aggregates as well as to corresponding mechanisms of their toxicity, good evidence exists that intracellular Aß can accumulate intraneuronally and interact with intracellular target proteins. However, intracellular Aß binding proteins as well as conditions favoring their interactions with Aß are poorly characterized. In this study we have investigated the effect of two known pathogenic Aß modifications, isomerization of Asp7 and phosphorylation of Ser8, on the proteomic profiles of mouse brain Aß binding proteins. Phosphorylation of Ser8 and especially isomerization of Asp7 significantly extended the repertoire of mouse brain Aß binding proteins. However, there were 61 proteins, common for three types of the affinity ligands. They obviously represent potential targets for direct interaction with all Aß species. Taking into consideration spontaneous mode of Asp7 isomerization and reports on initial accumulation of phosphorylated Aß species inside neurons it is reasonable to suggest that these modifications of intracellular Aß peptides causing the significant increase in the repertoire of Aß binding proteins represent a primary pathogenic effect that precedes formation of extracellular pathogenic oligomerization/aggregation of Aß peptides well described in the literature.

Human urinary renalase lacks the N-terminal signal peptide crucial for accommodation of its FAD cofactor.

Renalase is a recently discovered secretory protein involved in the regulation of blood pressure. Cells synthesize all known isoforms of human renalase (1 and 2) as flavoproteins. Accommodation of FAD in the renalase protein requires the presence of its N-terminal peptide. However, in secretory proteins, such peptides are usually cleaved during their export from the cell. In the present study, we have isolated human renalase from urinary samples of healthy volunteers and human recombinant renalases 1 and 2 expressed in Escherichia coli cells. In these proteins, we investigated the presence of the renalase N-terminal peptide and the FAD cofactor and performed computer-aided molecular analysis of the renalase crystal structure to evaluate possible consequences of removal of the N-terminal peptide. In contrast to human recombinant renalase isoforms 1 and 2 containing non-covalently bound FAD and clearly detectable N-terminal peptide, renalase purified from human urine lacks both the N-terminal signal peptide and FAD. The computer-aided analysis indicates that the removal of this peptide results in inability of the truncated renalase to bind the FAD cofactor. Thus, our results indicate that human renalase secreted in urine lacks its N-terminal peptide, and therefore catalytic activities of urinary renalase reported in the literature cannot be attributed to FAD-dependent mechanisms. We suggest that FAD-dependent catalytic functions are intrinsic properties of intracellular renalases, whereas extracellular renalases act in FAD- and possibly catalytic-independent manner.

The effects of endogenous non-peptide molecule isatin and hydrogen peroxide on proteomic profiling of rat brain amyloid-ß binding proteins: relevance to Alzheimer's disease?

The amyloid-ß peptide is considered as a key player in the development and progression of Alzheimer's disease (AD). Although good evidence exists that amyloid-ß accumulates inside cells, intracellular brain amyloid-binding proteins remain poorly characterized. Proteomic profiling of rat brain homogenates, performed in this study, resulted in identification of 89 individual intracellular amyloid-binding proteins, and approximately 25% of them were proteins that we had previously identified as specifically binding to isatin, an endogenous neuroprotector molecule. A significant proportion of the amyloid-binding proteins (more than 30%) are differentially expressed or altered/oxidatively modified in AD patients. Incubation of brain homogenates with 70 µM hydrogen peroxide significantly influenced the profile of amyloid-ß binding proteins and 0.1 mM isatin decreased the number of identified amyloid-ß binding proteins both in control and hydrogen peroxide treated brain homogenates. The effects of hydrogen peroxide and isatin have been confirmed in optical biosensor experiments with purified glyceraldehyde-3-phosphate dehydrogenase, one of the known crucial amyloid-ß binding proteins (also identified in this study). Data obtained suggest that isatin protects crucial intracellular protein targets against amyloid binding, and possibly favors intracellular degradation of this protein via preventing formation of amyloid-ß oligomers described in the literature for some isatin derivatives.

Construction of the coding sequence of the transcription variant 2 of the human Renalase gene and its expression in the prokaryotic system.

Renalase is a recently discovered protein, involved in regulation of blood pressure in humans and animals. Although several splice variants of human renalase mRNA transcripts have been recognized, only one protein product, hRenalase1, has been found so far. In this study, we have used polymerase chain reaction (PCR)-based amplification of individual exons of the renalase gene and their joining for construction of full-length hRenalase2 coding sequence followed by expression of hRenalase2 as a polyHis recombinant protein in Escherichia coli cells. To date this is the first report on synthesis and purification of hRenalase2. Applicability of this approach was verified by constructing hRenalase1 coding sequence, its sequencing and expression in E. coli cells. hRenalase1 was used for generation of polyclonal antiserum in sheep. Western blot analysis has shown that polyclonal anti-renalase1 antibodies effectively interact with the hRenalase2 protein. The latter suggests that some functions and expression patterns of hRenalase1 documented by antibody-based data may be attributed to the presence of hRenalase2. The realized approach may be also used for construction of coding sequences of various (especially weakly expressible) genes, their transcript variants, etc.

Strain differences in profiles of dopaminergic neurotransmission in the prefrontal cortex of the BALB/C vs. C57Bl/6 mice: consequences of stress and afobazole.

We found that in mice the basal activity of monoamine oxidase B (MAO-B) in the medial prefrontal cortex (mPFC) is lower in BALB/C than in C57Bl/6J mice, whereas activity of MAO-A is similar between strains. BALB/C mice, in comparison to C57Bl/6N mice, have higher basal content of dopamine in the mPFC, in both microdialysates and tissue content. Novelty stress (open field test) elicits a further increase in the microdialysate levels of dopamine in BALB/C, but not in C57Bl/6N mice; a subsequent accumulation of extracellular 3,4-dioxyphenylacetic acid (DOPAC) reaffirms the difference in catabolic capacity of monoaminergic systems between the strains. We demonstrated that in stress-susceptible BALB/C mice the novel anxiolytic afobazole, 5mg/kg, selectively mitigates trait anxiety; however it does not change the behavioral response in stress-resilient C57Bl/6N mice. Afobazole inhibits MAO-A in in vitro; it also lowers the microdialysate DOPAC levels in both strains (which testifies to its MAO-A inhibiting activity in vivo) and slightly suppresses dopamine release when elevated. Therefore, it is likely that the drug may mediate its anxiolytic activity via modulation of volume dopaminergic transmission at level of the mPFC.

Use of biotinylated ubiquitin for analysis of rat brain mitochondrial proteome and interactome.

Applicability of in vitro biotinylated ubiquitin for evaluation of endogenous ubiquitin conjugation and analysis of ubiquitin-associated protein-protein interactions has been investigated. Incubation of rat brain mitochondria with biotinylated ubiquitin followed by affinity chromatography on avidin-agarose, intensive washing, tryptic digestion of proteins bound to the affinity sorbent and their mass spectrometry analysis resulted in reliable identification of 50 proteins belonging to mitochondrial and extramitochondrial compartments. Since all these proteins were bound to avidin-agarose only after preincubation of the mitochondrial fraction with biotinylated ubiquitin, they could therefore be referred to as specifically bound proteins. A search for specific ubiquitination signature masses revealed several extramitochondrial and intramitochondrial ubiquitinated proteins representing about 20% of total number of proteins bound to avidin-agarose. The interactome analysis suggests that the identified non-ubiquitinated proteins obviously form tight complexes either with ubiquitinated proteins or with their partners and/or mitochondrial membrane components. Results of the present study demonstrate that the use of biotinylated ubiquitin may be considered as the method of choice for in vitro evaluation of endogenous ubiquitin-conjugating machinery in particular subcellular organelles and changes in ubiquitin/organelle associated interactomes. This may be useful for evaluation of changes in interactomes induced by protein ubiquitination under norm and various brain pathologies.