Reactivation of the chloroplast CF1-ATPase beta subunit by trace amounts of the CF1 alpha subunit suggests a chaperonin-like activity for CF1 alpha.

Incubation of tobacco and lettuce thylakoids with 2 M LiCl in the presence of MgATP removes the beta subunit from their CF1-ATPase (CF1 beta) together with varying amounts of the CF1 alpha subunit (CF1 alpha). These 2 M LiCl extracts, as with the one obtained from spinach thylakoids (Avital, S., and Gromet-Elhanan, Z. (1991) J. Biol. Chem. 266, 7067-7072), could form active hybrid ATPases when reconstituted into inactive beta-less Rhodospirillum rubrum chromatophores. Pure CF1 beta fractions that have been isolated from these extracts could not form such active hybrids by themselves, but could do so when supplemented with trace amounts (less than 5%) of CF1 alpha. A mitochondrial F1-ATPase alpha subunit was recently reported to be a heat-shock protein, having two amino acid sequences that show a highly conserved identity with sequences found in molecular chaperones (Luis, A. M., Alconada, A., and Cuezva, J. M. (1990) J. Biol. Chem. 265, 7713-7716). These sequences are also conserved in CF1 alpha isolated from various plants, but not in F1 beta subunits. The above described reactivation of CF1 beta by trace amounts of CF1 alpha could thus be due to a chaperonin-like function of CF1 alpha, which involves the correct, active folding of isolated pure CF1 beta.     

Brain Lactate Is an Obligatory Aerobic Energy Substrate for Functional Recovery After Hypoxia: Further In Vitro Validation

Abstract: This study used the rat hippocampal slice preparation and the monocarboxylate transporter inhibitor, α-cyano-4-hydroxycinnamate (4-CIN), to assess the obligatory role that lactate plays in fueling the recovery of synaptic function after hypoxia upon reoxygenation. At a concentration of 500 µ M , 4-CIN blocked lactate-supported synaptic function in hippocampal slices under normoxic conditions in 15 min. The inhibitor had no effect on glucose-supported synaptic function. Of control hippocampal slices exposed to 10-min hypoxia, 77.8 ± 6.8% recovered synaptic function after 30-min reoxygenation. Of slices supplemented with 500 µ M 4-CIN, only 15 ± 10.9% recovered synaptic function despite the large amount of lactate formed during the hypoxic period and the abundance of glucose present before, during, and after hypoxia. These results indicate that 4-CIN, when present during hypoxia and reoxygenation, blocks lactate transport from astrocytes, where the bulk of anaerobic lactate is formed, to neurons, where lactate is being utilized aerobically to support recovery of function after hypoxia. These results unequivocally validate that brain lactate is an obligatory aerobic energy substrate for posthypoxia recovery of function.     

The Crossroads of Synaptic Growth Signaling,Membrane Traffic and Neurological Disease: Insights from Drosophila

Neurons require target‐derived autocrine and paracrine growth factors to maintain proper identity, innervation, homeostasis and survival. Neuronal growth factor signaling is highly dependent on membrane traffic, both for the packaging and release of the growth factors themselves, and for regulation of intracellular signaling by their transmembrane receptors. Here, we review recent findings from the Drosophila larval neuromuscular junction (NMJ) that illustrate how specific steps of intracellular traffic and inter‐organelle interactions impinge on signaling, particularly in the bone morphogenic protein, Wingless and c‐Jun‐activated kinase pathways, regulating elaboration and stability of NMJ arbors, construction of synapses and synaptic transmission and homeostasis. These membrane trafficking and signaling pathways have been implicated in human motor neuron diseases including amyotrophic lateral sclerosis and hereditary spastic paraplegia, highlighting their importance for neuronal health and survival.      

Statistics is not enough: revisiting Ronald A. Fisher's critique (1936) of Mendel's experimental results (1866)

This paper is concerned with the role of rational belief change theory in the philosophical understanding of experimental error. Today, philosophers seek insight about error in the investigation of specific experiments, rather than in general theories. Nevertheless, rational belief change theory adds to our understanding of just such cases: R. A. Fisher's criticism of Mendel's experiments being a case in point. After an historical introduction, the main part of this paper investigates Fisher's paper from the point of view of rational belief change theory: what changes of belief about Mendel's experiment does Fisher go through and with what justification. It leads to surprising insights about what Fisher had done right and wrong, and, more generally, about the limits of statistical methods in detecting error.     

Compartmentalization and regulation of iron metabolism proteins protect male germ cells from iron overload

The universal importance of iron, its high toxicity, and complex chemistry present a challenge to biological systems in general and to protected compartments in particular. The high mitotic rate and avid mitochondriogenesis of developing male germ cells imply high iron requirements. Yet access to germ cells is tightly regulated by the blood-testis barrier that protects the meiotic and postmeiotic germ cells. To elucidate how iron is supplied to developing male germ cells, we analyzed iron deposition and iron transport proteins in testes of mice with iron overload and with genetic ablation of the iron regulators Hfe and iron regulatory protein 2. Iron accumulated mainly around seminiferous tubules, and only small amounts localized within the seminiferous tubules. The localization and regulation of proteins involved in iron import, storage, and export such as transferrin, transferrin receptor, the divalent metal transporter-1, cytosolic ferritin, and ferroportin strongly support a model of a largely autonomous iron cycle within seminiferous tubules. We show evidence that ferritin secretion from Sertoli cells may play an important role in iron acquisition of primary spermatocytes. During spermatogenic development iron is carried along from primary spermatocytes to spermatids, and from spermatids iron is recycled to the apical compartment of Sertoli cells, which traffic it back to a new generation of spermatocytes. Losses are replenished by the peripheral circulation. Such an internal iron cycle essentially detaches the iron homeostasis within the seminiferous tubule from the periphery and protects developing germ cells from iron fluctuations. This model explains how compartmentalization can optimize cellular and systemic nutrient homeostasis.     

A micellar model system for the role of zeaxanthin in the non-photochemical quenching process of photosynthesis—chlorophyll fluorescence quenching by the xanthophylls

To get an insight to the mechanism of the zeaxanthin-dependent non-photochemical quenching in photosystem II of photosynthesis, we probed the interaction of some xanthophylls with excited chlorophyll-a by trapping both pigments in micelles of triton X-100. Optimal distribution of pigments among micelles was obtained by proper control of the micelle concentration, using formamide in the reaction mixture, which varies the micellar aggregation number over three orders of magnitude. The optimal reaction mixture was obtained around 40% (v/v) formamide in 0.2-0.4% (v/v) triton X-100 in water. Zeaxanthin in the micellar solution exhibited initially absorption and circular dichroism spectral features corresponding to a J-type aggregate. The spectrum was transformed over time (half-time values vary—an average characteristic figure is roughly 20 min) to give features representing an H-type aggregate. The isosbestic point in the series of spectral curves favors the supposition of a rather simple reaction between two pure J and H-types dimeric species. Violaxanthin exhibited immediately stable spectral features corresponding to a mixture of J-type and more predominately H-type dimers. Lutein, neoxanthin and β-carotene did not show any aggregated spectral forms in micelles. The spectral features in micelles were compared to spectra in aqueous acetone, where the assignment to various aggregated types was established previously. The specific tendency of zeaxanthin to form the J-type dimer (or aggregate) could be important for its function in photosynthesis. The abilities of five carotenoids (zeaxanthin, violaxanthin, lutein, neoxanthin and β-carotene) to quench chlorophyll-a fluorescence were compared. Zeaxanthin, in its two micellar dimeric forms, and β-carotene were comparable good quenchers of chlorophyll-a fluorescence. Violaxanthin was a much weaker quencher, if at all. Lutein and neoxanthin rather enhanced the fluorescence. The implications to non-photochemical quenching process in photosynthesis are discussed.     

The MitCHAP-60 Disease Is Due to Entropic Destabilization of the Human Mitochondrial Hsp60 Oligomer

The 60-kDa heat shock protein (mHsp60) is a vital cellular complex that mediates the folding of many of the mitochondrial proteins. Its function is executed in cooperation with the co-chaperonin, mHsp10, and requires ATP. Recently, the discovery of a new mHsp60-associated neurodegenerative disorder, MitCHAP-60 disease, has been reported. The disease is caused by a point mutation at position 3 (D3G) of the mature mitochondrial Hsp60 protein, which renders it unable to complement the deletion of the homologous bacterial protein in Escherichia coli (Magen, D., Georgopoulos, C., Bross, P., Ang, D., Segev, Y., Goldsher, D., Nemirovski, A., Shahar, E., Ravid, S., Luder, A., Heno, B., Gershoni-Baruch, R., Skorecki, K., and Mandel, H. (2008) Am. J. Hum. Genet. 83, 30–42). The molecular basis of the MitCHAP-60 disease is still unknown. In this study, we present an in vitro structural and functional analysis of the purified wild-type human mHsp60 and the MitCHAP-60 mutant. We show that the D3G mutation leads to destabilization of the mHsp60 oligomer and causes its disassembly at low protein concentrations. We also show that the mutant protein has impaired protein folding and ATPase activities. An additional mutant that lacks the first three amino acids (N-del), including Asp-3, is similarly impaired in refolding activity. Surprisingly, however, this mutant exhibits profound stabilization of its oligomeric structure. These results suggest that the D3G mutation leads to entropic destabilization of the mHsp60 oligomer, which severely impairs its chaperone function, thereby causing the disease.Type I chaperonins are essential molecular chaperones of the Hsp60 family found in eubacteria, mitochondria, and chloroplasts (1). They are key players in mediating the correct folding of newly translated, translocated, and stress-denatured proteins. The folding function of chaperonins is executed by the coordinated action of two oligomeric proteins, Hsp60 (also named cpn60 and in bacteria GroEL) and its co-chaperonin Hsp10 (also named cpn10 and in bacteria GroES). The GroEL molecule is composed of 14 subunits that form a barrel-like structure that consists of two back-to-back stacked heptameric rings with a large cavity at each end termed the “Anfinsen cage.” GroES is a heptameric ring formed by ∼10-kDa subunits. The co-chaperonin binds to the chaperonin in the presence of ATP and Mg²⁺ via a short, unstructured, but highly conserved region known as the mobile loop (2, 3). Due to the stability of the GroEL/ES oligomers and the ease with which they can be purified from bacteria, they have become the primary targets for study in the field of chaperonins. As a result, almost all of our knowledge concerning the structure and mechanism of chaperonins, both Hsp60 and Hsp10, is based largely on data from experiments on these E. coli proteins. The chaperonin reaction cycle starts when a non-folded protein substrate binds to the surface of the cavity of one of the GroEL rings. ATP-dependent GroES binding to that (cis) ring causes a dramatic conformational change that leads to a doubling of the cavity size and a switch in the cavity surface from being hydrophobic to hydrophilic. These conformational changes trigger the release of the encapsulated substrate protein into the cavity, where it can fold in a protected environment (4–9). Subsequent binding of ATP and GroES to the opposite (trans) ring promotes the release of GroES, ADP, and protein substrate from the cis ring into bulk solution.The homologous mitochondrial chaperonin system was shown to be responsible for refolding proteins imported into the mitochondria (10, 11). The mitochondrial Hsp60 (mHsp60)³ is similar to GroEL in that it is made up of heptameric rings (12–14) that can refold denatured substrates in vitro with the assistance of a co-chaperonin and ATP (15). However, the mHsp60 oligomer is less stable than the bacterial homolog, and it exhibits unique nucleotide binding properties and specificity for co-chaperonin (13, 14, 16).In addition to their essential function in mediating protein folding, the mammalian mitochondrial chaperonins were also suggested to be involved in extramitochondrial activities. A number of reports have suggested that mHsp60 can stimulate human leukocytes and vascular endothelial cells to produce proinflammatory cytokines (17). Furthermore, it has been reported that mHsp60 has proapoptotic and antiapoptotic roles, depending on its cellular localization (18, 19). Finally, mHsp60 and mHsp10 were found to change their expression pattern in tumor cells (20, 21).The importance of mHsp60 for human cell function has been demonstrated through the autosomal dominant hereditary spastic paraplegia SPG13, a neurodegenerative disorder associated with two independent mutations in the gene encoding mHsp60 (22, 23). Recently, a large kindred including 23 patients suffering from MitCHAP-60 disease, an autosomal recessive neurodegenerative disorder, has been identified (24). Magnetic resonance imaging of the brains of the patients showed diffuse hypomyelination and leukodystrophy, in which myelin is not formed properly. The disease-causing mutation was identified to be a homozygous missense mutation in the human HSPD1 gene encoding the mHsp60 protein (24), namely D3G in the mature protein. Initial studies showed that, in contrast to wild-type mHsp60, the mutant, together with mHsp10, was not able to fully complement a deletion of the bacterial homologues, GroEL and GroES, in E. coli (24). The mechanism by which the D3G mutation may compromise the function of mHsp60 has not been reported. In this study, we suggest that the D3G mutation impedes the function of mHsp60 by entropic destabilization of the oligomeric structure of the molecule.     

Discovery of Novel Proteasome Inhibitors Using a High-Content Cell-Based Screening System

The regulated degradation of damaged or misfolded proteins, as well as down-regulation of key signaling proteins, within eukaryotic and bacterial cells is catalyzed primarily by large, ATP-dependent multimeric proteolytic complexes, termed proteasomes. Inhibition of proteasomal activity affects a wide variety of physiological and pathological processes, and was found to be particularly effective for cancer therapy. We report here on the development of a novel high throughput assay for proteasome inhibition using a unique, highly sensitive live-cell screening, based on the cytoplasm-to-nucleus translocation of a fluorescent proteasome inhibition reporter (PIR) protein, consisting of nuclear localization signal-deficient p53 derivative. We further show here that mdm2, a key negative regulator of p53 plays a key role in the accumulation of PIR in the nucleus upon proteasome inhibition. Using this assay, we have screened the NCI Diversity Set library, containing 1,992 low molecular weight synthetic compounds, and identified four proteasome inhibitors. The special features of the current screen, compared to those of other approaches are discussed.