Tubulin chaperone E binds microtubules and proteasomes and protects against misfolded protein stress

Mutation of tubulin chaperone E (TBCE) underlies hypoparathyroidism, retardation, and dysmorphism (HRD) syndrome with defective microtubule (MT) cytoskeleton. TBCE/yeast Pac2 comprises CAP-Gly, LRR (leucine-rich region), and UbL (ubiquitin-like) domains. TBCE folds α-tubulin and promotes α/β dimerization. We show that Pac2 functions in MT dynamics: the CAP-Gly domain binds α-tubulin and MTs, and functions in suppression of benomyl sensitivity of pac2Δ mutants. Pac2 binds proteasomes: the LRR binds Rpn1, and the UbL binds Rpn10; the latter interaction mediates Pac2 turnover. The UbL also binds the Skp1-Cdc53-F-box (SCF) ubiquitin ligase complex; these competing interactions for the UbL may impact on MT dynamics. pac2Δ mutants are sensitive to misfolded protein stress. This is suppressed by ectopic PAC2 with both the CAP-Gly and UbL domains being essential. We propose a novel role for Pac2 in the misfolded protein stress response based on its ability to interact with both the MT cytoskeleton and the proteasomes.     

Characterization of the role of metallothionein-3 in an animal model of Alzheimer’s disease

Among the dementias, Alzheimer’s disease (AD) is the most commonly diagnosed, but there are still no effective drugs available for its treatment. It has been suggested that metallothionein-3 (MT-3) could be somehow involved in the etiology of AD, and in fact very promising results have been found in in vitro studies, but the role of MT-3 in vivo needs further analysis. In this study, we analyzed the role of MT-3 in a mouse model of AD, Tg2576 mice, which overexpress human Amyloid Precursor Protein (hAPP) with the Swedish mutation. MT-3 deficiency partially rescued the APP-induced mortality of females, and mildly affected APP-induced changes in behavior assessed in the hole-board and plus-maze tests in a gender-dependent manner. Amyloid plaque burden and/or hAPP expression were decreased in the cortex and hippocampus of MT-3-deficient females. Interestingly, exogenously administered Zn₇MT-3 increased soluble Aβ40 and Aβ42 and amyloid plaques and gliosis, particularly in the cortex, and changed several behavioral traits (increased deambulation and exploration and decreased anxiety). These results highlight that the control of the endogenous production and/or action of MT-3 could represent a powerful therapeutic target in AD.     

IFT54 regulates IFT20 stability but is not essential for tubulin transport during ciliogenesis

Intraflagellar transport (IFT) is required for ciliogenesis by ferrying ciliary components using IFT complexes as cargo adaptors. IFT54 is a component of the IFT-B complex and is also associated with cytoplasmic microtubules (MTs). Loss of IFT54 impairs cilia assembly as well as cytoplasmic MT dynamics. The N-terminal calponin homology (CH) domain of IFT54 interacts with tubulins/MTs and has been proposed to transport tubulin during ciliogenesis, whereas the C-terminal coiled-coil (CC) domain binds IFT20. However, the precise function of these domains in vivo is not well understood. We showed that in Chlamydomonas, loss of IFT54 completely blocks ciliogenesis but does not affect spindle formation and proper cell cycle progression, even though IFT54 interacts with mitotic MTs. Interestingly, IFT54 lacking the CH domain allows proper flagellar assembly. The CH domain is required for the association of IFT54 with the axoneme but not with mitotic MTs, and also regulates the flagellar import of IFT54 but not IFT81 and IFT46. The C-terminal CC domain is essential for IFT54 to bind IFT20, and for its recruitment to the basal body and incorporation into IFT complexes. Complete loss of IFT54 or the CC domain destabilizes IFT20. ift54 mutant cells expressing the CC domain alone rescue the stability of IFT20 and form stunted flagella with accumulation of both IFT-A component IFT43 and IFT-B component IFT46, indicating that IFT54 also functions in IFT turn-around at the flagellar tip.     

Three Cdk1 sites in the kinesin-5 Cin8 catalytic domain coordinate motor localization and activity during anaphase

The bipolar kinesin-5 motors perform essential functions in mitotic spindle dynamics. We previously demonstrated that phosphorylation of at least one of the Cdk1 sites in the catalytic domain of the Saccharomyces cerevisiae kinesin-5 Cin8 (S277, T285, S493) regulates its localization to the anaphase spindle. The contribution of these three sites to phospho-regulation of Cin8, as well as the timing of such contributions, remains unknown. Here, we examined the function and spindle localization of phospho-deficient (serine/threonine to alanine) and phospho-mimic (serine/threonine to aspartic acid) Cin8 mutants. In vitro, the three Cdk1 sites undergo phosphorylation by Clb2-Cdk1. In cells, phosphorylation of Cin8 affects two aspects of its localization to the anaphase spindle, translocation from the spindle-pole bodies (SPBs) region to spindle microtubules (MTs) and the midzone, and detachment from the mitotic spindle. We found that phosphorylation of S277 is essential for the translocation of Cin8 from SPBs to spindle MTs and the subsequent detachment from the spindle. Phosphorylation of T285 mainly affects the detachment of Cin8 from spindle MTs during anaphase, while phosphorylation at S493 affects both the translocation of Cin8 from SPBs to the spindle and detachment from the spindle. Only S493 phosphorylation affected the anaphase spindle elongation rate. We conclude that each phosphorylation site plays a unique role in regulating Cin8 functions and postulate a model in which the timing and extent of phosphorylation of the three sites orchestrates the anaphase function of Cin8.     

Regulation of end-binding protein EB1 in the control of microtubule dynamics

The regulation of microtubule dynamics is critical to ensure essential cell functions, such as proper segregation of chromosomes during mitosis or cell polarity and migration. End-binding protein 1 (EB1) is a plus-end-tracking protein (+TIP) that accumulates at growing microtubule ends and plays a pivotal role in the regulation of microtubule dynamics. EB1 autonomously binds an extended tubulin-GTP/GDP-Pi structure at growing microtubule ends and acts as a molecular scaffold that recruits a large number of regulatory +TIPs through interaction with CAP-Gly or SxIP motifs. While extensive studies have focused on the structure of EB1-interacting site at microtubule ends and its role as a molecular platform, the mechanisms involved in the negative regulation of EB1 have only started to emerge and remain poorly understood. In this review, we summarize recent studies showing that EB1 association with MT ends is regulated by post-translational modifications and affected by microtubule-targeting agents. We also present recent findings that structural MAPs, that have no tip-tracking activity, physically interact with EB1 to prevent its accumulation at microtubule plus ends. These observations point out a novel concept of “endogenous EB1 antagonists” and emphasize the importance of finely regulating EB1 function at growing microtubule ends.     

Bidirectional motility of kinesin-5 motor proteins: structural determinants,cumulative functions and physiological roles

Mitotic kinesin-5 bipolar motor proteins perform essential functions in mitotic spindle dynamics by crosslinking and sliding antiparallel microtubules (MTs) apart within the mitotic spindle. Two recent studies have indicated that single molecules of Cin8, the Saccharomyces cerevisiae kinesin-5 homolog, are minus end-directed when moving on single MTs, yet switch directionality under certain experimental conditions (Gerson-Gurwitz et al., EMBO J 30:4942–4954, 2011; Roostalu et al., Science 332:94–99, 2011). This finding was unexpected since the Cin8 catalytic motor domain is located at the N-terminus of the protein, and such kinesins have been previously thought to be exclusively plus end-directed. In addition, the essential intracellular functions of kinesin-5 motors in separating spindle poles during mitosis can only be accomplished by plus end-directed motility during antiparallel sliding of the spindle MTs. Thus, the mechanism and possible physiological role of the minus end-directed motility of kinesin-5 motors remain unclear. Experimental and theoretical studies from several laboratories in recent years have identified additional kinesin-5 motors that are bidirectional, revealed structural determinants that regulate directionality, examined the possible mechanisms involved and have proposed physiological roles for the minus end-directed motility of kinesin-5 motors. Here, we summarize our current understanding of the remarkable ability of certain kinesin-5 motors to switch directionality when moving along MTs.     

Restraint stress induced changes in rat liver and serum metallothionein and in Zn metabolism

Summary 24 h of a psychogenic stress (restraint) caused a strong increase of liver metallothionein (MT) levels. 3 h of stress were sufficient to induce an increase in liver MT, measured 21 h later, but the increase was much lower than in continuously restrained rats. Stress induction of liver MT was not due to food deprivation, since rats deprived for 24 h showed lower MT levels than stressed ones. Zn on MT presented the same qualitative but not quantitative pattern of response as MT protein. Liver cytosolic Zn was increased by restraint in spite of their being no decrease in serum Zn. Any treatment altered serum MT. Liver and serum MT were not correlated. The present results demonstrate that basically psychogenic stresses increased liver but not serum MT levels. No positive evidence for a relationship between corticosterone secretion and MT induction was found.     

Restraint stress induced changes in rat liver and serum metallothionein and in Zn metabolism

24 h of a psychogenic stress (restraint) caused a strong increase of liver metallothionein (MT) levels. 3 h of stress were sufficient to induce an increase in liver MT, measured 21 h later, but the increase was much lower than in continuously restrained rats. Stress induction of liver MT was not due to food deprivation, since rats deprived for 24 h showed lower MT levels than stressed ones. Zn on MT presented the same qualitative but not quantitative pattern of response as MT protein. Liver cytosolic Zn was increased by restraint in spite of their being no decrease in serum Zn. Any treatment altered serum MT. Liver and serum MT were not correlated. The present results demonstrate that basically psychogenic stresses increased liver but not serum MT levels. No positive evidence for a relationship between corticosterone secretion and MT induction was found.     

Understanding microtubule dynamics for improved cancer therapy

Microtubules (MTs), key components of the cytoskeleton, are dynamic polymers of tubulin that form a well-organized network of polarized tube filaments. MT dynamics are highly regulated both spacially and temporally by several MT-related proteins, themselves regulated by several kinases and phosphatases via signaling cascades, and also by coordinated interactions with actin cytoskeleton and adhesion sites. Regulation of MT dynamics is crucial for mitosis, cell migration, cell signaling and trafficking. MT-targeted drugs (MTDs), which constitute a major anticancer drug family with antimitotic and antiangiogenic properties, inhibit tumor progression mainly by altering MT dynamics in both cancer and endothelial cells. Identification of proteins regulating the MT network will lead to a better understanding of tumor progression regulators and will be helpful in improving cancer therapy. Received 22 July 2005; received after revision 8 September 2005; accepted 12 September 2005     

Isolation,characterization, and determination of human liver (copper/zinc) metallothionein

Summary A copper-containing protein was purified from the liver of a patient with primary biliary cirrhosis by a combination of gel filtration and anion exchange chromatography. This copper-protein had UV absorption and emission spectra, an amino acid composition, and a molecular mass which were characteristic for metallothionein (MT).From 8 livers (3 control, 1 fetal and 4 primary biliary cirrhosis) MT was extracted with non-reducing buffer and centrifuged, and the pellets were re-extracted with a 1% 2-mercaptoethanol-containing buffer. The non-reducing buffer extracted a predominantly copper-containing MT from the livers of patients with primary biliary cirrhosis and a predominantly zinc-containing MT from control lives and the fetal liver. Only from the fetal liver was a copper/zinc containing MT solubilized during the re-extraction with 2-mercaptoethanol-containing buffer. These results indicate that human MT is a unique metalloprotein with age and disease-dependent characteristics.     

Isolation, characterization, and determination of human liver (copper/zinc) metallothionein

A copper-containing protein was purified from the liver of a patient with primary biliary cirrhosis by a combination of gel filtration and anion exchange chromatography. This copper-protein had UV absorption and emission spectra, an amino acid composition, and a molecular mass which were characteristic for metallothionein (MT). From 8 livers (3 control, 1 fetal and 4 primary biliary cirrhosis) MT was extracted with non-reducing buffer and centrifuged, and pellets were re-extracted with a 1% 2-mercaptoethanol-containing buffer. The non-reducing buffer extracted a predominantly copper-containing MT from the livers of patients with primary biliary cirrhosis and a predominantly zinc-containing MT from control livers and the fetal liver. Only from the fetal liver was a copper/zinc containing MT solubilized during the re-extraction with 2-mercaptoethanol-containing buffer. These results indicate that human MT is a unique metalloprotein with age and disease-dependent characteristics.     

Recombinant synthesis of mouse Zn3-β and Zn4-α metallothionein 1 domains and characterization of their cadmium(II) binding capacity

Genetic engineering, coupled with spectro scopic analyses, has enabled the metal binding proper ties of the α and β subunits of mouse metallothionein 1 (MT) to be characterized. A heterologous expression system in E.coli has led to high yields of their pure zinc-complexed forms. The cadmium(II) binding properties of recombinant Zn₄-αMT and Zn₃-βMT have been studied by electronic absorption and circular dichroism. The former binds Cd(II) identically to α fragments obtained from mammalian organs, showing that the recombinant polypeptide behaves like the na tive protein. Titration of Zn₃-βMT with CdCl₂ results in the formation of Cd₃-βMT. The addition of excess Cd(II) leads to Cd₄-βMT which, with the extra loading of Cd(II), unravels to give rise isodichroically to Cd₉-βMT. The effect of cadmium-displaced Zn(II) ions and excess Cd(II) above the full metal occupancy of three has been studied using Chelex-100. The Cd₃-βMT species is stable in the presence of this strong metal-chelating agent. Received 20 May 1997; received after revision 7 July 1997; accepted 9 July 1997     

Redox modulation of the NMDA receptor

Redox modulation has been recognized to be an important mechanism of regulation for the N-methyl-D-aspartate (NMDA) receptor. Sulfhydryl reducing agents enhance, whereas oxidizing agents decrease, NMDA-evoked currents. Multiple cysteine residues located in different NMDA receptor subunits have been identified as molecular determinants underlying redox modulation. The NMDA receptor is also regulated by nitric oxide (NO)-related species directly, not involving cyclic GMP, but the molecular mechanism of this action has heretofore not been entirely clear. The confusion arose at least partly due to the fact that various redox forms of NO (NO+, NO*, NO-, each having an additional electron compared with the previous) have distinct mechanisms of action. Recently, a critical cysteine residue (Cys 399) on the NR2A subunit has been shown to react under physiological conditions with NO by S-nitrosylation (transfer of the NO+ to cysteine thiol) or by reaction with NO- (nitroxyl anion) to underlie this form of modulation.     

Regulation of long-distance transport of mitochondria along microtubules

Mitochondria are cellular organelles of crucial importance, playing roles in cellular life and death. In certain cell types, such as neurons, mitochondria must travel long distances so as to meet metabolic demands of the cell. Mitochondrial movement is essentially microtubule (MT) based and is executed by two main motor proteins, Dynein and Kinesin. The organization of the cellular MT network and the identity of motors dictate mitochondrial transport. Tight coupling between MTs, motors, and the mitochondria is needed for the organelle precise localization. Two adaptor proteins are involved directly in mitochondria-motor coupling, namely Milton known also as TRAK, which is the motor adaptor, and Miro, which is the mitochondrial protein. Here, we discuss the active mitochondria transport process, as well as motor–mitochondria coupling in the context of MT organization in different cell types. We focus on mitochondrial trafficking in different cell types, specifically neurons, migrating cells, and polarized epithelial cells.     

Metallothionein induction by cadmium and zinc in rat secretory organs

Metallothionein (MT) levels were determined in four secretory organs of the rat following administration of zinc (Zn) and cadmium (Cd). The concentrations of MT in the lacrimal, parotid and adrenal glands of untreated rats were in the range of 2.2-4.9 micrograms/g wet weight tissue while in the pancreas it was shown to be 15.2 micrograms/g. Injection of zinc at total doses of 16, 32 and 80 mg/kg resulted in a 1.8-, 3.2- and 5.9-fold increase in lacrimal MT content, respectively, while a 10.2- and 13.1-fold elevation was observed following treatment with 4 and 8 mg/kg of Cd, respectively. Similar findings were found in the adrenal gland. The parotid MT was elevated 5.9 and 17 times following Zn treatment at doses of 16 and 80 mg/kg respectively, whereas 4 mg/kg of Cd increased MT 14.4 times in this gland. Pancreatic MT was elevated by 39- and 40-fold after injection of Zn at doses of 16 and 32 mg/kg respectively, whereas 4 and 8 mg/kg of Cd caused a 9.8- and 17.9-fold induction, respectively. These results may indicate that secretory organs participate in metabolism of heavy metals in the mammalian body.     

The role of ras and other low molecular weight guanine nucleotide (GTP)-binding proteins during hematopoietic cell differentiation

Recent progress in the understanding of signal transduction and gene regulation in hematopoietic cells has shown that many intracellular signalling pathways are modulated by low molecular weight guanine nucleotide (GTP)-binding proteins (LMWGs). LMWGs act as molecular switches for regulating a wide range of signal-transduction pathways in virtually all cells. In hematopoietic cells, LMWGs have been shown to participate in essential functions such as growth control, differentiation, cytoskeletal organization, cytokine and chemoattractant-induced signalling events, reduced nicotinamide adenine dinucleotide phosphate oxidase activity, intracellular vesicle transport and secretion. In human leukemias, myelodysplastic syndromes and myeloproliferative disorders, Ras activation occurs by point mutations, overexpression or by alteration of NF-1 Ras-GTPase activating protein (GAP). These are postinitiation events in leukemia but may modulate growth-factor-dependent and independent leukemic growth. Two animal models of mutated N-ras expression resulting in myelodysplastic and myeloproliferative features are discussed. The role of Ras in organ development is discussed in the context of transgenic knockout mice. More LMWG functions will certainly be identified as we gain a better understanding of regulatory pathways modulating myeloid signal transduction. This review will summarize our current understanding of this rapidly advancing area of research.     

Metallothionein expression in the central nervous system of multiple sclerosis patients

Multiple sclerosis (MS) is a major chronic demyelinating and inflammatory disease of the central nervous system (CNS) in which oxidative stress likely plays a pathogenic role in the development of myelin and neuronal damage. Metallothioneins (MTs) are antioxidant proteins induced in the CNS by tissue injury, stress and some neurodegenerative diseases, which have been postulated to play a neuroprotective role. In fact, MT-I+II-deficient mice are more susceptible to developing experimental autoimmune encephalomyelitis (EAE), and treatment of Lewis rats with Zn-MT-II reduces EAE severity. We show here that, as in EAE, MT-I+II proteins were expressed in brain lesions of MS patients. Cells expressing MT-I+II were mainly astrocytes and activated monocytes/macrophages. Interestingly, the levels of MT-I+II were slightly increased in the inactive MS lesions in comparison with the active lesions, suggesting that MTs may be important in disease remission.     

Microbial cycling of volatile organic sulfur compounds

Microbial cycling of volatile organic sulfur compounds (VOSCs), especially dimethyl sulfide (DMS) and methanethiol (MT), is intensively studied because these compounds play an important role in the processes of global warming, acid precipitation, and the global sulfur cycle. VOSC concentrations in freshwater sediments are low due to the balance between the formation and degradation of these compounds. These reactions occur for the greater part at the oxic/anoxic interphase of sediment and water column. In contrast to marine ecosystems, where dimethylsulfoniopropionate is the main precursor of MT and DMS, in freshwater ecosystems, VOSCs are formed mainly by methylation of sulfide and to a lesser extent from the degradation of S-containing amino acids. One of the major routes for DMS and MT formation through sulfide methylation is anaerobic O-demethylation of methoxylated aromatic compounds. Inhibition studies have revealed that the major part of the endogenously produced MT and DMS is degraded anaerobically by methanogens. The major bacterial groups involved in formation and consumption of VOSCs are described.