Regulation of REGγ cellular distribution and function by SUMO modification

Discovery of emerging REGγ-regulated proteins has accentuated the REGγ-proteasome as an important pathway in multiple biological processes, including cell growth, cell cycle regulation, and apoptosis. However, little is known about the regulation of the REGγ-proteasome pathway. Here we demonstrate that REGγ can be SUMOylated in vitro and in vivo by SUMO-1, SUMO-2, and SUMO-3. The SUMO-E3 protein inhibitor of activated STAT (PIAS)1 physically associates with REGγ and promotes SUMOylation of REGγ. SUMOylation of REGγ was found to occur at multiple sites, including K6, K14, and K12. Mutation analysis indicated that these SUMO sites simultaneously contributed to the SUMOylation status of REGγ in cells. Posttranslational modification of REGγ by SUMO conjugation was revealed to mediate cytosolic translocation of REGγ and to cause increased stability of this proteasome activator. SUMOylation-deficient REGγ displayed attenuated ability to degrade p21(Waf//Cip1) due to reduced affinity of the REGγ SUMOylation-defective mutant for p21. Taken together, we report a previously unrecognized mechanism regulating the activity of the proteasome activator REGγ. This regulatory mechanism may enable REGγ to function as a more potent factor in protein degradation with a broader substrate spectrum.     

Decorin and its galactosaminoglycan chain: extracellular regulator of cellular function?

A molecular network of extracellular matrix molecules determines the tissue architecture and accounts for mechanical properties like compressibility or stretch resistance. It is widely accepted that the elements of the cellular microenvironment are important regulators of the cellular behavior in vitro and in vivo. One large group comprising these molecules is the family of proteoglycans. Both, the core proteins and, in particular, the attached galactosaminoglycans, contribute to the regulation network as they bind a variety of signaling molecules, e.g. cytokines, chemokines, growth, and differentiation factors. We would like to emphasize specific patterns of epimerization and sulfation within the galactosaminoglycans chains, because these result in "motifs" that are responsible for the modulation of signal factor binding, release and activity. This property is crucial in physiological and pathological conditions, for example development and wound healing.     

DISC1 and the aggresome: a disruption to cellular function?

Disrupted in Schizophrenia 1 (DISC1) is a key susceptibility gene for major psychiatric disorders. DISC1 plays a role in key neuronal processes such as neuronal proliferation, migration, integration and function via DISC1's roles at the centrosome and synapse, and in the regulation of intracellular signaling pathways. Recently, the idea of protein aggregation as a disease mechanism for DISC1 has been suggested. In our recent paper we explore these DISC1 protein aggregates in cell lines and neurons and find they are recruited to the aggresome and cause disruption of DISC1 function in intracellular transport.     

The cellular and subcellular localization of neuroglobin and cytoglobin -- a clue to their function?

Neuroglobin and cytoglobin are recently discovered respiratory proteins of vertebrates with yet ill-defined physiological functions. Neuroglobin is widely expressed in neurons, but not glia, in the vertebrate central and peripheral nervous systems. Other major expression sites are the retina and endocrine tissues. This distribution is indicative of a function of neuroglobin in metabolically most active, oxygen-consuming cell types, but does not yet allow to safely distinguish between different cellular roles, such as oxygen homeostasis, scavenging of reactive oxygen species or sustaining energy metabolism. Cytoglobin is predominantly expressed in connective tissue fibroblasts and related cell types in the body organs. Its main function may therefore be related to the specific amounts of extracellular matrix. Cytoglobin may hypothetically be involved in the oxygen-consuming maturation of collagen proteins. Cytoglobin is also expressed in distinct cell types of brain and retina. Its distribution strikingly differs from neuroglobin, suggesting an independent, yet unknown function.     

Acquisition of tolerance to Δ-9-THC as measured by the response of a cellular function

The development of tolerance to delta-9-tetrahydrocannabinol (Δ-9-THC) was investigated by measuring respiration in brain tissue after acute or chronic administration. Mice were given either single or seven daily repeated intraperitoneal injections of 50 mg/Kg of delta-9-tetrahydrocannabinol (Δ-9-THC) or control vehicle. The final injection for all drug treated animals included radiolabeled ³H-Δ-9-THC. The mice were sacrificed at 1 hour, 2 hours, 4 hours, 24 hours, and 7 days after the final injection. Δ-9-THC depressed respiration, but after repeated injections was significantly less effective in this regard, indicating acquisition of tolerance to Δ-9-THC. Because the concentration of radiolabeled cannabinoids in brain tissue from each group is not appreciably different, a cellular as opposed to distributional mode of tolerance is suggested.     

Tubulin–Na+ ,K +-ATPase interaction: Involvement in enzymatic regulation and cellular function

A new function for tubulin was described by our laboratory: acetylated tubulin forms a complex with Na⁺,K ⁺-ATPase (NKA) and inhibits its activity. This process was shown to be a regulatory factor of physiological importance in cultured cells, human erythrocytes, and several rat tissues. Formation of the acetylated tubulin–NKA complex is reversible. We demonstrated that in cultured cells, high concentrations of glucose induce translocation of acetylated tubulin from cytoplasm to plasma membrane with a consequent inhibition of NKA activity. This effect is reversed by adding glutamate, which is coctransported to the cell with Na ⁺. Another posttranslational modification of tubulin, detyrosinated tubulin, is also involved in the regulation of NKA activity: it enhances the NKA inhibition induced by acetylated tubulin. Manipulation of the content of these modifications of tubulin could work as a new strategy to maintain homeostasis of Na ⁺ and K ⁺, and to regulate a variety of functions in which NKA is involved, such as osmotic fragility and deformability of human erythrocytes. The results summarized in this review show that the interaction between tubulin and NKA plays an important role in cellular physiology, both in the regulation of Na ⁺/K ⁺ homeostasis and in the rheological properties of the cells, which is mechanically different from other roles reported up to now.     

Randomness in microtubule dynamics: an error that requires correction or an inherent plasticity required for normal cellular function?

Microtubules (MTs) play roles in regulating the mechanical structure and dynamics of cells. While MTs appear to be highly ordered structures, recent data suggest some randomness in their structure and dynamics. Part of this inherent randomness is attributed to errors and correction mechanisms are being investigated to overcome these ‘mistakes.’ However, this randomness may also be part of the normal intracellular function of MTs. It is possible that random events in MT structure and dynamics may contribute to their normal function and may even be part of an improved efficacy mechanism. An alternative view, wherein MT and kinetochore errors are part of required cell plasticity, is also discussed. These data may further support the concept of randomness in biological pathways as part of self‐organization or accurate and enhanced function.     

Lipophilic methylene blue analogues enhance mitochondrial function and increase frataxin levels in a cellular model of Friedreich’s ataxia

Friedreich’s ataxia (FRDA) is an autosomal recessive neurodegenerative disorder resulting from reduced expression of the protein frataxin (FXN). Although its function is not fully understood, frataxin appears to help assemble iron sulfur clusters; these are critical for the function of many proteins, including those needed for mitochondrial energy production. Finding ways to increase FXN levels has been a major therapeutic strategy for this disease. Previously, we described a novel series of methylene violet analogues and their structural optimization as potential therapeutic agents for neurodegenerative and mitochondrial disorders. Presently, a series of methylene blue analogues has been synthesized and characterized for their in vitro biochemical and biological properties in cultured Friedreich’s ataxia lymphocytes. Favorable methylene blue analogues were shown to increase frataxin levels and mitochondrial biogenesis, and to improve aconitase activity. The analogues were found to be good ROS scavengers, and able to protect cultured FRDA lymphocytes from oxidative stress resulting from inhibition of complex I and from glutathione depletion. The analogues also preserved mitochondrial membrane potential and augmented ATP production. Our results suggest that analogue 5, emerging from the initial structure of the parent compound methylene blue (MB), represents a promising lead structure and lacks the cytotoxicity associated with the parent compound MB.     

Vesicular stomatitis virus mRNA and inhibition of translation of cellular mRNA--is there a P function in vesicular stomatitis virus?

Infection of animal cells by vesicular stomatitis virus (VSV) results in inhibition of translation of cellular mRNA. We showed previously that, in BHK cells infected by the Glasgow isolate of VSV Indiana, this is due to competition during the initiation step of protein synthesis of viral and cellular mRNA for a constant, limiting number of ribosomes. We show here that infection of the same cells with the San Juan isolate of VSV resulted in a more rapid shutoff of host protein synthesis and that this was paralleled by a more rapid accumulation of viral mRNA. Extending our conclusion that shutoff is due to mRNA competition, we show further that the average size of polysomes translating viral and cellular mRNA was threefold smaller in cells infected by VSV San Juan than by VSV Glasgow, which, in turn, was about one-half that of uninfected cells. In all cases, cellular and viral mRNA's which encoded the same-sized polypeptides were found on the same-sized polysomes, a result indicating that the efficiency of translation of both types of mRNA's is about the same in the infected cell. Also, there was no preferential sequestration of viral or cellular mRNA's in ribonucleoprotein particles. Additional correlations between the levels of viral mRNA's and the inhibition of protein synthesis came from studies of three other wild-type VSV strains and also from studies with Vero and L cells. In particular, the rate of shutoff of L-cell protein synthesis after infection by any VSV isolate was slower than that in BHK cells, and this was correlated with a slower rate of accumulation of viral mRNA. VSV temperature-sensitive mutants which synthesized, at the nonper-missive temperature, no VSV mRNA failed to inhibit synthesis of cellular proteins. Stanners and co-workers (C. P. Stanners, A. M. Francoeur, and T. Lam, Cell 11:273-281, 1977) claimed that VSV mutant R1 inhibited synthesis of L cell protein synthesis less rapidly than did its parent wild-type strain HR. They concluded that this effect was due to a mutation in an unspecified VSV protein, “P.” We found, in both L and BHK cells, that R1 infection resulted in a slightly slower inhibition of cellular mRNA translation than did HR infection and that this was correlated with a slightly reduced accumulation of VSV mRNA. The level of VSV mRNA, rather than any specific VSV protein, appeared to be the key factor in determining the rate of shutoff of host protein synthesis.     

Calcium ion as cellular messenger

<正>The prominent role Ca2+ion plays as a major small biological messenger is fascinating.There are many physiologically important ions,such as Na+,K+,H+,Cl?,Ca2+,Mg2+,Fe3+,and PO43?that participate in cell signaling.Among them,monovalent ions primarily contribute to rapid electrical signaling,while multivalent ions generally act as a co-factor for chemical reactions by associating with the host molecule through electrostatic or covalent interactions.Ca2+plays both roles.Its transmembrane influx supports the plateau phase of cardiac action potential and helps set the speed of pacemaker potential.Inside the cell,Ca2+switches     

α-Cleavage of cellular prion protein

The cellular prion protein (PrP^C) is subjected to various processing under physiological and pathological conditions, of which the α-cleavage within the central hydrophobic domain not only disrupts a region critical for both PrP toxicity and PrP^C to PrP^Sc conversion but also produces the N1 fragment that is neuroprotective and the C1 fragment that enhances the pro-apoptotic effect of staurosporine in one report and inhibits prion in another. The proteases responsible for the α-cleavage of PrP^C are controversial. The effect of ADAM10, ADAM17, and ADAM9 on N1 secretion clearly indicates their involvement in the α-cleavage of PrP^C, but there has been no report of direct PrP^C α-cleavage activity with any of the three ADAMs in a purified protein form. We demonstrated that, in muscle cells, ADAM8 is the primary protease for the α-cleavage of PrP^C, but another unidentified protease(s) must also play a minor role. We also found that PrP^C regulates ADAM8 expression, suggesting that a close examination on the relationships between PrP^C and its processing enzymes may reveal novel roles and underlying mechanisms for PrP^C in non-prion diseases such as asthma and cancer.     

��-Cleavage of cellular prion protein

The cellular prion protein (PrP^C) is subjected to various processing under physiological and pathological conditions, of which the α-cleavage within the central hydrophobic domain not only disrupts a region critical for both PrP toxicity and PrP^C to PrP^Sc conversion but also produces the N1 fragment that is neuroprotective and the C1 fragment that enhances the pro-apoptotic effect of staurosporine in one report and inhibits prion in another. The proteases responsible for the α-cleavage of PrP^C are controversial. The effect of ADAM10, ADAM17, and ADAM9 on N1 secretion clearly indicates their involvement in the α-cleavage of PrP^C, but there has been no report of direct PrP^C α-cleavage activity with any of the three ADAMs in a purified protein form. We demonstrated that, in muscle cells, ADAM8 is the primary protease for the α-cleavage of PrP^C, but another unidentified protease(s) must also play a minor role. We also found that PrP^C regulates ADAM8 expression, suggesting that a close examination on the relationships between PrP^C and its processing enzymes may reveal novel roles and underlying mechanisms for PrP^C in non-prion diseases such as asthma and cancer.     

Mechanism of cellular uptake of long-chain fatty acids: Do we need cellular proteins?

Molecular and Cellular Biochemistry - Defining the mechanism(s) of long-chain fatty acid movement through membranes is vital to understanding whether or not entry of fatty acids into cells can be...     

Anti-infectives: can cellular screening deliver?

In an era of emerging and reemerging infectious diseases, and increasing multidrug resistance, the need to identify novel therapy is imperative. Unfortunately, the recent shift of the drug discovery paradigm from cellular screening to target-based approaches has not delivered the anticipated benefits. A recent renaissance of the traditional cell-based approach, on the other hand, has yielded several clinical candidates. Three successful examples are illustrated in this review, namely spiroindolone, thiazolidinone, and diarylquinoline for the treatment of malaria, hepatitis C virus, and tuberculosis, respectively. We describe in detail their identification, mechanism of action (MoA), and common features in the chemical structures. The challenges of the cell-based approach for anti-infective drug discovery are also discussed. We propose a shift from standard libraries to synthetic natural-product-like compound collections to improve the success of phenotypic lead finding and to facilitate the validation of hits.     

How does cellular senescence prevent cancer?

It is widely believed that cellular senescence is a tumor suppressor mechanism; however, it has not been understood why it is advantageous for organisms to retain mutant cells is a postmitotic state rather than simply eliminating them by apoptosis. It has recently been proposed that the primary role of cellular senescence is in mitotic compartments of fixed size in which spatial considerations dictate that a deleted cell is replaced by a neighboring cell. In these situations, rather than eliminating the neoplastic clone, deletion of mutant cells can paradoxically lead to their increased turnover. If mutant cells become senescent, then the compartment is instead progressively filled by senescent cells until the mutant clone is eliminated. Since most of the genetic alterations responsible for malignancy arise in stem cells, this mechanism may have particular relevance to the stem cell niche. In this article the implications of this hypothesis are examined in detail and related to experimental results. It is further proposed here that blockage of stem cell niches by senescent stem cells may account for some of the functional alterations observed in stem cell compartments at old age. Clearly, the existence of senescent stem cells is central to the proposed hypothesis, and although there is preliminary evidence for this assertion it has yet to be proven in vivo. An experimental strategy involving double labeling of stem cells with a nucleotide label is described that can address this question.     

Is hsp70 the cellular thermometer?

Cells respond to an increase in temperature by inducing the synthesis of the heat shock proteins, which are a small set of evolutionarily conserved proteins. We review the evidence leading us to suggest that the free pool of one of these proteins, hsp70, serves as a cellular thermometer that regulates the expression of all heat shock proteins.     

Why does ethanol induce cellular heat-shock response?

At the time of induction of the periplasmic protein alkaline phosphatase (AP) in Escherichia coli, the presence of ethanol (10% v/v) in the growth medium did not allow the induced AP to be translocated out to the periplasm. The nontransported AP was stored in the cytoplasm as the unfolded precursor form (AP with its amino-terminal signal sequence), which had no enzymatic activity. The presence of 10% v/v ethanol in the growth medium also induced the heat-shock response in E. coli, which was evident from the enhanced syntheses of several heat-shock proteins (HSPs) over their cellular basal levels. These results, in conjunction with our earlier findings on the occurrence of heat-shock response in an AP-signal sequence mutant of E. coli due to the export deficiency of AP precursor, suggest that the membrane protein precursors, stored in the cytoplasm due to the ethanol-mediated inhibition of translocation, behaved to the cells as abnormal proteins, which ultimately triggered the signal for the induction of heat-shock response in E. coli.