Molecular Regulation of Copper Homeostasis in the Male Gonad during the Process of Spermatogenesis

Owing to its redox properties, copper is a cofactor of enzymes that catalyze reactions in fundamental metabolic processes. However, copper–oxygen interaction, which is a source of toxic oxygen radicals generated by the Fenton reaction, makes copper a doubled-edged-sword in an oxygen environment. Among the microelements influencing male fertility, copper plays a special role because both copper deficiency and overload in the gonads worsen spermatozoa quality and disturb reproductive function in mammals. Male gametes are produced during spermatogenesis, a multi-step process that consumes large amounts of oxygen. Germ cells containing a high amount of unsaturated fatty acids in their membranes are particularly vulnerable to excess copper-mediated oxidative stress. In addition, an appropriate copper level is necessary to initiate meiosis in premeiotic germ cells. The balance between essential and toxic copper concentrations in germ cells at different stages of spermatogenesis and in Sertoli cells that support their development is handled by a network of copper importers, chaperones, recipient proteins, and exporters. Here, we describe coordinated regulation/functioning of copper-binding proteins expressed in germ and Sertoli cells with special emphasis on copper transporters, copper transporting ATPases, and SOD1, a copper-dependent antioxidant enzyme. These and other proteins assure copper bioavailability in germ cells and protection against copper toxicity.     

Frustrated Interfaces Facilitate Dynamic Interactions between Native Client Proteins and Holdase Chaperones

Molecular chaperones are crucial for cellular life to ensure that all proteins obtain their right fold and functionality. Many chaperones promiscuously bind a wide spectrum of client proteins, ranging from nascent to quasi-native and native proteins. Several recent studies have investigated, at atomic resolution, how chaperones interact with native proteins. Native proteins feature a wide variety of structural conformations, and therefore, a given chaperone cannot accomplish full surface complementarity to all of its client proteins. This limitation is circumvented by the recognition of frustrated regions on the client protein surface by the chaperone. In this interaction mode, the chaperone forms a multitude of transient local interactions with some segments of the client, whereas other parts are transiently not in favorable interactions. A permanent rearrangement of the client conformation on the chaperone occurs. Reconfiguration on the chaperone surface also gives the client a chance to fold into its correct, minimally frustrated conformation.     

Regulation by Different Types of Chaperones of Amyloid Transformation of Proteins Involved in the Development of Neurodegenerative Diseases

The review highlights various aspects of the influence of chaperones on amyloid proteins associated with the development of neurodegenerative diseases and includes studies conducted in our laboratory. Different sections of the article are devoted to the role of chaperones in the pathological transformation of alpha-synuclein and the prion protein. Information about the interaction of the chaperonins GroE and TRiC as well as polymer-based artificial chaperones with amyloidogenic proteins is summarized. Particular attention is paid to the effect of blocking chaperones by misfolded and amyloidogenic proteins. It was noted that the accumulation of functionally inactive chaperones blocked by misfolded proteins might cause the formation of amyloid aggregates and prevent the disassembly of fibrillar structures. Moreover, the blocking of chaperones by various forms of amyloid proteins might lead to pathological changes in the vital activity of cells due to the impaired folding of newly synthesized proteins and their subsequent processing. The final section of the article discusses both the little data on the role of gut microbiota in the propagation of synucleinopathies and prion diseases and the possible involvement of the bacterial chaperone GroE in these processes.     

Specificity of Lipoprotein Chaperones for the Characteristic Lipidated Structural Motifs of their Cognate Lipoproteins

Lipoprotein‐binding chaperones mediate intracellular transport of lipidated proteins and determine their proper localisation and functioning. Understanding of the exact structural parameters that determine recognition and transport by different chaperones is of major interest. We have synthesised several lipid‐modified peptides, representative of different lipoprotein classes, and have investigated their binding to the relevant chaperones PDEδ, UNC119a, UNC119b, and galectins‐1 and ‐3. Our results demonstrate that PDEδ recognises S‐isoprenylated C‐terminal peptidic structures but not N‐myristoylated peptides. In contrast, UNC119 proteins bind only mono‐N‐myristoylated, but do not recognise doubly lipidated and S‐isoprenylated peptides at the C terminus. For galectins‐1 and ‐3, neither binding to N‐acylated, nor to C‐terminally prenylated peptides could be determined. These results shed light on the specificity of the chaperone‐mediated cellular lipoprotein transport systems.     

Molecular Chaperones and Thyroid Cancer

Thyroid cancers are the most common of the endocrine system malignancies and progress must be made in the areas of differential diagnosis and treatment to improve patient management. Advances in the understanding of carcinogenic mechanisms have occurred in various fronts, including studies of the chaperone system (CS). Components of the CS are found to be quantitatively increased or decreased, and some correlations have been established between the quantitative changes and tumor type, prognosis, and response to treatment. These correlations provide the basis for identifying distinctive patterns useful in differential diagnosis and for planning experiments aiming at elucidating the role of the CS in tumorigenesis. Here, we discuss studies of the CS components in various thyroid cancers (TC). The chaperones belonging to the families of the small heat-shock proteins Hsp70 and Hsp90 and the chaperonin of Group I, Hsp60, have been quantified mostly by immunohistochemistry and Western blot in tumor and normal control tissues and in extracellular vesicles. Distinctive differences were revealed between the various thyroid tumor types. The most frequent finding was an increase in the chaperones, which can be attributed to the augmented need for chaperones the tumor cells have because of their accelerated metabolism, growth, and division rate. Thus, chaperones help the tumor cell rather than protect the patient, exemplifying chaperonopathies by mistake or collaborationism. This highlights the need for research on chaperonotherapy, namely the development of means to eliminate/inhibit pathogenic chaperones.     

Structural and Kinetic Views of Molecular Chaperones in Multidomain Protein Folding

Despite recent developments in protein structure prediction, the process of the structure formation, folding, remains poorly understood. Notably, folding of multidomain proteins, which involves multiple steps of segmental folding, is one of the biggest questions in protein science. Multidomain protein folding often requires the assistance of molecular chaperones. Molecular chaperones promote or delay the folding of the client protein, but the detailed mechanisms are still unclear. This review summarizes the findings of biophysical and structural studies on the mechanism of multidomain protein folding mediated by molecular chaperones and explains how molecular chaperones recognize the client proteins and alter their folding properties. Furthermore, we introduce several recent studies that describe the concept of kinetics–activity relationships to explain the mechanism of functional diversity of molecular chaperones.     

Chaperoning roles of macromolecules interacting with proteins in vivo

The principles obtained from studies on molecular chaperones have provided explanations for the assisted protein folding in vivo. However, the majority of proteins can fold without the assistance of the known molecular chaperones, and little attention has been paid to the potential chaperoning roles of other macromolecules. During protein biogenesis and folding, newly synthesized polypeptide chains interact with a variety of macromolecules, including ribosomes, RNAs, cytoskeleton, lipid bilayer, proteolytic system, etc. In general, the hydrophobic interactions between molecular chaperones and their substrates have been widely believed to be mainly responsible for the substrate stabilization against aggregation. Emerging evidence now indicates that other features of macromolecules such as their surface charges, probably resulting in electrostatic repulsions, and steric hindrance, could play a key role in the stabilization of their linked proteins against aggregation. Such stabilizing mechanisms are expected to give new insights into our understanding of the chaperoning functions for de novo protein folding. In this review, we will discuss the possible chaperoning roles of these macromolecules in de novo folding, based on their charge and steric features.     

Conformational Analysis of Misfolded Protein Aggregation by FRET and Live-Cell Imaging Techniques

Cellular homeostasis is maintained by several types of protein machinery, including molecular chaperones and proteolysis systems. Dysregulation of the proteome disrupts homeostasis in cells, tissues, and the organism as a whole, and has been hypothesized to cause neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS) and Huntington’s disease (HD). A hallmark of neurodegenerative disorders is formation of ubiquitin-positive inclusion bodies in neurons, suggesting that the aggregation process of misfolded proteins changes during disease progression. Hence, high-throughput determination of soluble oligomers during the aggregation process, as well as the conformation of sequestered proteins in inclusion bodies, is essential for elucidation of physiological regulation mechanism and drug discovery in this field. To elucidate the interaction, accumulation, and conformation of aggregation-prone proteins, in situ spectroscopic imaging techniques, such as Förster/fluorescence resonance energy transfer (FRET), fluorescence correlation spectroscopy (FCS), and bimolecular fluorescence complementation (BiFC) have been employed. Here, we summarize recent reports in which these techniques were applied to the analysis of aggregation-prone proteins (in particular their dimerization, interactions, and conformational changes), and describe several fluorescent indicators used for real-time observation of physiological states related to proteostasis.     

Expression of Huntingtin and TDP-43 Derivatives in Fission Yeast Can Cause Both Beneficial and Toxic Effects

Many neurodegenerative disorders display protein aggregation as a hallmark, Huntingtin and TDP-43 aggregates being characteristic of Huntington disease and amyotrophic lateral sclerosis, respectively. However, whether these aggregates cause the diseases, are secondary by-products, or even have protective effects, is a matter of debate. Mutations in both human proteins can modulate the structure, number and type of aggregates, as well as their toxicity. To study the role of protein aggregates in cellular fitness, we have expressed in a highly tractable unicellular model different variants of Huntingtin and TDP-43. They each display specific patterns of aggregation and toxicity, even though in both cases proteins have to be very highly expressed to affect cell fitness. The aggregation properties of Huntingtin, but not of TDP-43, are affected by chaperones such as Hsp104 and the Hsp40 couple Mas5, suggesting that the TDP-43, but not Huntingtin, derivatives have intrinsic aggregation propensity. Importantly, expression of the aggregating form of Huntingtin causes a significant extension of fission yeast lifespan, probably as a consequence of kidnapping chaperones required for maintaining stress responses off. Our study demonstrates that in general these prion-like proteins do not cause toxicity under normal conditions, and in fact they can protect cells through indirect mechanisms which up-regulate cellular defense pathways.     

Identification of interaction site of pseudoazurin with its redox partner, copper-containing nitrite reductase from Alcaligenes faecalis S-6

Pseudoazurin, a low molecular weight protein containing a singletype I copper, functions as an electron donor to a copper-containingnitrite reductase (NIR) in a denitrifying bacterium Alcaligenesfaecalis S-6. To elucidate the proteinprotein interaction betweenthese two copper-containing proteins, each of nine out of 13lysine residues on the surface of pseudoazurin were independentlyreplaced by alanine or aspartate, and the effects of the mutationson the interaction with NIR, as well as the physicochemicalproperties of pseudoazurin, were analyzed. All of the mutatedpseudoazurins showed optical spectra and oxidation-reductionpotentials almost identical to those of wildtype pseudoazurin,suggesting that none of the replacements of these lysine residuesaffected the environment around the type I copper site. Kineticanalysis of electron transfer between mutated pseudoazurinsand NIR reveals that the lysine mutations have very little effecton the rate of electron transfer to NIR, but substitution atresidues 10, 38, 57 and 77, all close to the copper site, substantiallydecreases the affinity of pseudoazurin for NIR. This suggeststhat pseudoazurin interacts with NIR through the region closeto the type I copper site. The refined X-ray structures of Lys38Aspand Lys10Asp/Lys38Asp show that the molecular structure hasindeed changed little. A new space group is observed for theLys109Ala mutant crystal. Crystal packing interactions changefor the Lys10Asp/Lys38Asp mutant but remain the same for Lys38Aspand Lys59Ala mutants.     

Functional cell-surface display of a lipase-specific chaperone

Lipases are important enzymes in biotechnology. Extracellular bacterial lipases from Pseudomonads and related species require the assistance of specific chaperones, designated "Lif" proteins (lipase specific foldases). Lifs, a unique family of steric chaperones, are anchored to the periplasmic side of the inner membrane where they convert lipases into their active conformation. We have previously shown that the autotransporter protein EstA from P. aeruginosa can be used to direct a variety of proteins to the cell surface of Escherichia coli. Here we demonstrate for the first time the functional cell-surface display of the Lif chaperone and FACS (fluorescence-activated cell sorting)-based analysis of bacterial cells that carried foldase-lipase complexes. The model Lif protein, LipH from P. aeruginosa, was displayed at the surface of E. coli cells. Surface exposed LipH was functional and efficiently refolded chemically denatured lipase. The foldase autodisplay system reported here can be used for a variety of applications including the ultrahigh-throughput screening of large libraries of foldase variants generated by directed evolution.     

Cellular Chaperone Function of Intrinsically Disordered Dehydrin ERD14

Disordered plant chaperones play key roles in helping plants survive in harsh conditions, and they are indispensable for seeds to remain viable. Aside from well-known and thoroughly characterized globular chaperone proteins, there are a number of intrinsically disordered proteins (IDPs) that can also serve as highly effective protecting agents in the cells. One of the largest groups of disordered chaperones is the group of dehydrins, proteins that are expressed at high levels under different abiotic stress conditions, such as drought, high temperature, or osmotic stress. Dehydrins are characterized by the presence of different conserved sequence motifs that also serve as the basis for their categorization. Despite their accepted importance, the exact role and relevance of the conserved regions have not yet been formally addressed. Here, we explored the involvement of each conserved segment in the protective function of the intrinsically disordered stress protein (IDSP) A. thaliana’s Early Response to Dehydration (ERD14). We show that segments that are directly involved in partner binding, and others that are not, are equally necessary for proper function and that cellular protection emerges from the balanced interplay of different regions of ERD14.     

Heat Shock Protein 90 as Therapeutic Target for CVDs and Heart Ageing

Cardiovascular diseases (CVDs) are the leading cause of death globally, representing approximately 32% of all deaths worldwide. Molecular chaperones are involved in heart protection against stresses and age-mediated accumulation of toxic misfolded proteins by regulation of the protein synthesis/degradation balance and refolding of misfolded proteins, thus supporting the high metabolic demand of the heart cells. Heat shock protein 90 (HSP90) is one of the main cardioprotective chaperones, represented by cytosolic HSP90a and HSP90b, mitochondrial TRAP1 and ER-localised Grp94 isoforms. Currently, the main way to study the functional role of HSPs is the application of HSP inhibitors, which could have a different way of action. In this review, we discussed the recently investigated role of HSP90 proteins in cardioprotection, atherosclerosis, CVDs development and the involvements of HSP90 clients in the activation of different molecular pathways and signalling mechanisms, related to heart ageing.     

Solvent extraction of copper in a reciprocating plate column

Equilibrium data for the distribution of copper between an aqueous solution and a 20% solution of di-(2 ethyl hexyl) phosphoric acid in kerosene has been obtained by shake-flask experiments. The capacity of the organic phase for copper is greatly improved when the ammonium salt of D2EHPA is used. The performance of a 5 cm diameter Karr reciprocating plate extraction column for continuous counter-current extraction of copper by ammoniated D2EHPA has been measured. The results, expressed as heights of a transfer unit, have been compared with data for a non-metallic system (kerosene/acetic acid/water) in the same column. The lowest H.T.U. values measured were 43 cm for copper transfer and 23 cm for acetic acid transfer, with the difference being attributed mainly to the lower molecular diffusivity in the copper/D2EHPA system.     

Structural studies of electrochemically activated glassy carbon electrode: Effects of chloride anion on the redox responses of copper deposition

The redox behaviors of copper species at electrochemically activated glassy carbon electrodes have been investigated in aqueous solutions containing chloride anions. Experimental results showed that the voltammetric responses of copper species were influenced by the electrochemical activation means employed. Abnormal copper stripping was observed at electrodes obtained by cyclic polarization. Cyclic polarization would cause changes in the interwoven graphitic crystalline surface of glassy carbon, producing electrode interface with low distribution density of electron transfer sites for the early nucleation of metallic copper in the presence of chloride anion. Potentiostatic activation would generate oxygen-containing functionalities and maintain the basic surface structure of graphitic crystalline with high distribution density of electron transfer sites.     

泡沫铜孔隙率和孔密度对流动与沸腾换热特性的影响

以去离子水作为工质，设计并搭建了以泡沫铜为研究对象的单相和两相换热实验系统。对于单相流动换热，当Re数较小时，孔隙率80%、孔密度90PPI的泡沫铜样品换热性能最好；当Re数较大时，孔隙率80%、孔密度45PPI的泡沫铜样品换热性能最好。泡沫铜最大换热系数为空通道的6倍，但同时需付出更大的泵功损耗为代价。对于两相流沸腾换热，低孔隙率样品70%~80%能有效地降低壁面过热度和强化沸腾换热性能。孔隙率对沸腾换热性能起决定性作用，孔隙率越低，沸腾换热系数越大；孔密度对沸腾换热性能起次要作用。90PPI泡沫铜样品，因其成核址密度高和毛细力较大，有助于提升泡沫铜的沸腾换热性能。     

基于MWCNTs/PA复合材料铜表面处理的传热性能

从换热器防腐和强化传热的角度出发,对铜板表面进行不同的涂膜处理：聚丙烯酸酯（PA）单双面涂膜、MWCNTs/PA复合材料单双面涂膜和无涂层铜板。通过对流换热实验,研究不同处理条件下的传热系数。结果表明,经PA处理过后的铜板传热系数明显降低。与无涂层铜板相比,单、双面PA处理的铜板传热系数分别下降21.91%和40.00%。经MWCNTs/PA复合材料处理过后的铜板传热系数得到了提高。与无涂层铜板相比,单、双面MWCNTs/PA处理的铜板的传热系数分别提高了16.74%、27.49%。     

单相自然循环回路的强化对流传热特性

通过实验对比分析了带有单相自然循环回路元件的铜板与光滑铜板相隔冷热空气逆流换热的换热效果。 结果表明,相同泵耗功情况下,加装单相自然循环回路元件的换热板的换热量是光滑铜板的1.1~1.3倍。在此基础上,本文通过数值模拟进一步对比分析了加装单相自然循环回路元件与相同形状、尺寸的铜翅片换热效果,针对影响自然循环回路传热性能的主要因素,如循环回路高度、横向与轴向倾斜角度、冷热源温差等,进行了数值模拟研究。结果表明：只有当温差超过等效温差点后,自然循环回路元件的换热效果才强于相同形状和尺寸的铜翅片;随着几何尺寸减少以及倾斜角度的增加,自然循环元件与铜翅片的等效温差点随之升高。     

Electrochemical behaviour of copper/LDPE composites in the simulated uterine solution

Two metal/insulator composites, copper/low-density polyethylene (LDPE) microcomposites and copper/LDPE nanocomposites, were prepared in a uniform metal mass fraction. The corrosion characteristics of the two composites in the simulated uterine solution have been studied by cyclic voltammetry, potentiodynamic polarization measurement and electrochemical impedance spectroscopy. Results obtained during this study show that there are obvious differences on the corrosive rates and mechanisms between the two materials. The nanocomposites have stable corrosive reaction, hardly passivation, low corrosion rate and high transformation ratio of copper to soluble cupric ions compared with microcomposites, which indicate that the nanocomposites are perfect to be the novel material for intrauterine device. The corrosion behaviour of the microcomposites was controlled by both the processes of the pervasion of Cu²⁺ and the transfer of the charge, while that of the nanocomposites was only controlled by the transfer of the charge. This paper also compared the corrosion behaviours of the composites and bulk copper in the simulated uterine solution, there were more insoluble subproducts in the corrosion process of bulk copper than the composites. Further, the electrochemical method in this study is proved to be useful to characterize the corrosion behaviour of high electrical resistivity materials.