Interaction of small heat shock proteins with BAG3.

BAG3 is a universal adapter protein involved in various cellular processes, including the regulation of apoptosis, chaperone-assisted selective autophagy, and heat shock protein function. The interaction between small heat shock proteins (sHsps) and their α-crystallin domains (Acds) with full-length BAG3 protein and its IPV domain was analyzed using size-exclusion chromatography, native gel electrophoresis, and chemical cross-linking. HspB7 and the 3D mutant of HspB1 (which mimics phosphorylation) showed no interaction, HspB6 weakly interacted, and HspB8 strongly interacted with full-length BAG3.

Role of Filamin C in Muscle Cells.

Filamin C (FLNC) is a member of a high-molecular weight protein family, which bind actin filaments in the cytoskeleton of various cells. In human genome FLNC is encoded by the gene located on chromosome 7 and is expressed predominantly in striated skeletal and cardiac muscle cells. Filamin C is involved in organization and stabilization of thin actin filaments three-dimensional network in sarcomeres, and is supposed to play a role of mechanosensor transferring mechanical signals to different protein targets.

Universal Adapter Protein Bag3 and Small Heat Shock Proteins.

Bag3 (Bcl-2-associated athanogene 3) protein contains a number of functional domains and interacts with a wide range of different partner proteins, including small heat shock proteins (sHsps) and heat shock protein Hsp70. The ternary Bag3-sHsp-and Hsp70 complex binds denatured proteins and transports them to phagosomes, thus playing a key role in the chaperone-assisted selective autophagy (CASA). This complex also participates in the control of formation and disassembly of stress granules (granulostasis) and cytoskeleton regulation.

Interaction of the C-terminal immunoglobulin-like domains (Ig 22-24) of filamin C with human small heat shock proteins.

Small heat shock proteins are the well-known regulators of the cytoskeleton integrity, yet their complexes with actin-binding proteins are underexplored. Filamin C, a dimeric 560 kDa protein, abundant in cardiac and skeletal muscles, crosslinks actin filaments and contributes to Z-disc formation and membrane-cytoskeleton attachment. Here, we analyzed the interaction of a human filamin C fragment containing immunoglobulin-like domains 22-24 (FLNC) with five small heat shock proteins (HspB1, HspB5, HspB6, HspB7, HspB8) and their α-crystallin domains.

Investigation of NO Role in Neural Tissue in Brain and Spinal Cord Injury.

Nitric oxide (NO) production in injured and intact brain regions was compared by EPR spectroscopy in a model of brain and spinal cord injury in Wistar rats. The precentral gyrus of the brain was injured, followed by the spinal cord at the level of the first lumbar vertebra. Seven days after brain injury, a reduction in NO content of 84% in injured brain regions and 66% in intact brain regions was found.

α-Crystallin Domains of Five Human Small Heat Shock Proteins (sHsps) Differ in Dimer Stabilities and Ability to Incorporate Themselves into Oligomers of Full-Length sHsps.

The α-crystallin domain (ACD) is the hallmark of a diverse family of small heat shock proteins (sHsps). We investigated some of the ACD properties of five human sHsps as well as their interactions with different full-length sHsps. According to size-exclusion chromatography, at high concentrations, the ACDs of HspB1 (B1ACD), HspB5 (B5ACD) and HspB6 (B6ACD) formed dimers of different stabilities, which, upon dilution, dissociated to monomers to different degrees.

Role of Small Heat Shock Proteins in the Remodeling of Actin Microfilaments.

Small heat shock proteins (sHsps) play an important role in the maintenance of proteome stability and, particularly, in stabilization of the cytoskeleton and cell contractile apparatus. Cell exposure to different types of stress is accompanied by the translocation of sHsps onto actin filaments; therefore, it is commonly believed that the sHsps are true actin-binding proteins. Investigations of last years have shown that this assumption is incorrect.

Is the small heat shock protein HSPB7 (cvHsp) a genuine actin-binding protein?

It is postulated that the small heat shock proteins directly interact with actin, affect formation and stabilize actin filaments. To verify this suggestion, we have analyzed interaction of recombinant human small heat shock protein HspB7 with skeletal muscle actin. In blot overlay HspB7 binds both G- and F-actin.

Quaternary Structure and Hetero-Oligomerization of Recombinant Human Small Heat Shock Protein HspB7 (cvHsp).

In this study, a reliable and simple method of untagged recombinant human HspB7 preparation was developed. Recombinant HspB7 is presented in two oligomeric forms with an apparent molecular weight of 36 kDa (probably dimers) and oligomers with an apparent molecular weight of more than 600 kDa. By using hydrophobic and size-exclusion chromatography, we succeeded in preparation of HspB7 dimers.

Cardio-Vascular Heat Shock Protein (cvHsp, HspB7), an Unusual Representative of Small Heat Shock Protein Family.

HspB7 is one of ten human small heat shock proteins. This protein is expressed only in insulin-dependent tissues (heart, skeletal muscle, and fat tissue), and expression of HspB7 is regulated by many different factors. Single nucleotide polymorphism is characteristic for the HspB7 gene and this polymorphism correlates with cardio-vascular diseases and obesity.

Effect of cataract-associated mutations in the N-terminal domain of αB-crystallin (HspB5).

Physico-chemical properties of three cataract-associated missense mutants of αB-crystallin (HspB5) (R11H, P20S, R56W) were analyzed. The oligomers formed by the R11H mutant were smaller, whereas the oligomers of the P20S and R56W mutants were larger than those of the wild-type protein. The P20S mutant possessed lower thermal stability than the wild-type HspB5 or two other HspB5 mutants.

Changes in Membrane and Threshold Potentials of Command Neurons in Terrestrial Snail during Development of a Conditioned Situational Defensive Reflex.

Changes of the electrical characteristics of command neurons of defensive behavior caused by the development of a conditioned situational defensive reflex were studied experimentally under in vitro conditions on preparations of the nervous system of snails. After learning, the membrane and threshold potentials of command neurons LPa3 and RPa3 significantly decreased and excitability of the studied neurons increased.

Mutations in HspB1 and hereditary neuropathies.

Charcot-Marie-Tooth (CMT) disease is major hereditary neuropathy. CMT has been linked to mutations in a range of proteins, including the small heat shock protein HspB1. Here we review the properties of several HspB1 mutants associated with CMT.

Effects of Thryptophan Hydroxylase Blockade by P-Chlorophenylalanine on Contextual Memory Reconsolidation after Training of Different Intensity.

The processes of memory formation and its storage are extremely dynamic. Therefore, the determination of the nature and temporal evolution of the changes that underlie the molecular mechanisms of retrieval and cause reconsolidation of memory is the key to understanding memory formation. Retrieval induces the plasticity, which may result in reconsolidation of the original memory and needs critical molecular events to stabilize the memory or its extinction.

Small Heat Shock Proteins and Human Neurodegenerative Diseases.

The review discusses the role of small heat shock proteins (sHsps) in human neurodegenerative disorders, such as Charcot-Marie-Tooth disease (CMT), Parkinson's and Alzheimer's diseases, and different forms of tauopathies. The effects of CMT-associated mutations in two small heat shock proteins (HspB1 and HspB8) on the protein stability, oligomeric structure, and chaperone-like activity are described. Mutations in HspB1 shift the equilibrium between different protein oligomeric forms, leading to the alterations in its chaperone-like activity and interaction with protein partners, which can induce damage of the cytoskeleton and neuronal death.

αB-Crystallin Phosphorylation: Advances and Problems.

The review is dedicated to phosphorylation of αB-crystallin (HspB5), one of ubiquitously expressed small heat shock proteins. We describe the structure and properties of αB-crystallin and protein kinases involved in its phosphorylation in different cells and tissues, advantages and drawbacks of pseudophosphorylation mutants in elucidation of the mechanism of αB-crystallin functioning, effects of phosphorylation on the quaternary structure and intracellular location of αB-crystallin, interactions of αB-crystallin with different elements of the cytoskeleton, and effect of phosphorylation on the chaperone-like activity of αB-crystallin. We also discuss the validity of experimental data obtained by overexpression of pseudophosphorylation mutants for understanding the effect of phosphorylation on physiologically important properties of αB-crystallin, as well as the question why multiple attempts to phosphorylate αB-crystallin in vitro have been unsuccessful so far.

Impairing of Serotonin Synthesis by P-Chlorphenylanine Prevents the Forgetting of Contextual Memory After Reminder and the Protein Synthesis Inhibition.

The injection of p-chlorophenylalanine, specific blocker of 5-HT synthesis 3 days before reminder with anisomycin administration prevented forgetting. It is known that the reminder cause reactivation of the long-term memory and it leads to reconsolidation of memory. We showed earlier that the disruption of the reconsolidation of contextual memory in terrestrial snail was caused by anisomycin, the inhibitor of protein syntheses (Gainutdinova et al.

Characterization of human small heat shock protein HSPB1 α-crystallin domain localized mutants associated with hereditary motor neuron diseases.

Congenital mutations in human small heat shock protein HSPB1 (HSP27) have been linked to Charcot-Marie-Tooth disease, a commonly occurring peripheral neuropathy. Understanding the molecular mechanism of such mutations is indispensable towards developing future therapies for this currently incurable disorder. Here we describe the physico-chemical properties of the autosomal dominant HSPB1 mutants R127W, S135F and R136W.

Responses of Withdrawal Interneurons to Serotonin Applications in Naïve and Learned Snails Are Different.

Long-term changes in membrane potential after associative training were described previously in identified premotor interneurons for withdrawal of the terrestrial snail . Serotonin was shown to be a major transmitter involved in triggering the long-term changes in mollusks. In the present study we compared the changes in electrophysiological characteristics of identifiable premotor interneurons for withdrawal in response to bath applications of serotonin (5-HT) or serotonin precursor 5-hydroxytryptophan (5-HTP) in preparations from naïve, neurotoxin-injected or associatively trained snails.

Effect of methylglyoxal modification on the structure and properties of human small heat shock protein HspB6 (Hsp20).

Human small heat shock protein HspB6 (Hsp20) was modified by metabolic α-dicarbonyl compound methylglyoxal (MGO). At low MGO/HspB6 molar ratio, Arg13, Arg14, Arg27, and Arg102 were the primary sites of MGO modification. At high MGO/HspB6 ratio, practically, all Arg and Lys residues of HspB6 were modified.

Effects of NO Donors and Inhibitors of NO Synthase and Guanylate Cyclase on the Acquisition of a Conditioned Defense Food Aversion Response in Edible Snails.

Experiments on edible snails revealed that NO synthase blockade with a nonspecific neuronal NO synthase inhibitor L-NAME before defense food aversion conditioning is followed by a decrease in the rate of learning. Exogenous NO donors, sodium nitroprusside and dinitrosyl iron complex, were shown to improve learning. Chronic administration of a specific soluble guanylate cyclase inhibitor ODQ also increased the rate of learning.

Small Heat Shock Proteins and Distal Hereditary Neuropathies.

Classification of small heat shock proteins (sHsp) is presented and processes regulated by sHsp are described. Symptoms of hereditary distal neuropathy are described and the genes whose mutations are associated with development of this congenital disease are listed. The literature data and our own results concerning physicochemical properties of HspB1 mutants associated with Charcot-Marie-Tooth disease are analyzed.

Modulation of defensive reflex conditioning in snails by serotonin.

Highlights Daily injection of serotonin before a training session accelerated defensive reflex conditioning in snails.Daily injection of 5-hydroxytryptophan before a training session in snails with a deficiency of serotonin induced by the "neurotoxic" analog of serotonin 5,7-dihydroxytryptamine, restored the ability of snails to learn.After injection of the "neurotoxic" analogs of serotonin 5,6- and 5,7-dihydroxytryptamine as well as serotonin, depolarization of the membrane and decrease of the threshold potential of premotor interneurons was observed.

Characterization of Mutants of Human Small Heat Shock Protein HspB1 Carrying Replacements in the N-Terminal Domain and Associated with Hereditary Motor Neuron Diseases.

Physico-chemical properties of the mutations G34R, P39L and E41K in the N-terminal domain of human heat shock protein B1 (HspB1), which have been associated with hereditary motor neuron neuropathy, were analyzed. Heat-induced aggregation of all mutants started at lower temperatures than for the wild type protein. All mutations decreased susceptibility of the N- and C-terminal parts of HspB1 to chymotrypsinolysis.

Attempt to optimize some properties of fluorescent chimeras of human small heat shock protein HspB1 by modifying linker length and nature.

Chimerical proteins consisting of enhanced yellow fluorescent protein (EYFP) connected by linkers of different length and nature to the N-terminal end of small heat shock protein HspB1 were obtained and characterized. To obtain fluorescent chimeras with properties similar to those of unmodified small heat shock protein, we used either 12-residue-long linkers of different nature (highly flexible Gly-Ser linker (L1), rigid α-helical linker (L2), or rigid Pro-Ala linker (L3)) or highly flexible Gly-Ser linker consisting of 12, 18, or 21 residues. The wild-type HspB1 formed large stable oligomers consisting of more than 20 subunits.