Glutamate Transporter Homolog-Based Model Predicts Anion-{pi} Interaction is the Mechanism for the Voltage-Dependent Response of Prestin [Neurobiology]

August 18th, 2015 by

Prestin is the motor protein of cochlear outer hair cells. Its unique capability to perform direct, rapid and reciprocal electromechanical conversion depends on membrane potential and interaction with intracellular anions. How prestin senses the voltage change and interacts with anions is still unknown. Our three-dimensional model of prestin, using molecular dynamics simulations, predicts that prestin contains eight transmembrane spanning segments, two helical re-entry loops and that tyrosyl residues are the structural specialization of the molecule for the unique function of prestin. Using site-directed mutagenesis and electrophysiological techniques, we confirmed that residues Y367, Y486, Y501 and Y508 contribute to anion binding, interacting with intracellular anions through novel anion-π interactions. Such weak interactions, sensitive to voltage and mechanical stimulation, confer prestin with a unique capability to perform electromechanical and mechanoelectric conversions with exquisite sensitivity. This novel mechanism is completely different from all known mechanisms seen in ion channels, transporters and motor proteins.
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Alternative activation mechanisms of Protein Kinase B trigger distinct downstream signaling responses [Signal Transduction]

August 18th, 2015 by

Protein kinase B (PKB/Akt) is an important mediator of signals that control various cellular processes including cell survival, growth, proliferation and metabolism. PKB promotes these processes by phosphorylating many cellular targets, which trigger distinct downstream signaling events. However, how PKB is able to selectively target its substrates to induce specific cellular functions remains elusive. Here we perform a systematic study to dissect mechanisms that regulate intrinsic kinase activity versus mechanisms that specifically regulate activity towards specific substrates. We demonstrate that activation loop phosphorylation and the C-terminal hydrophobic motif (HM) are essential for high PKB activity in general. On the other hand we identify membrane targeting, which for decades has been regarded as an essential step in PKB activation, as a mechanism mainly affecting substrate selectivity. Further, we show that PKB activity in cells can be triggered independently of PI3K by initial HM phosphorylation, presumably through a mechanism analogous to other AGC kinases. Importantly, different modes of PKB activation result in phosphorylation of distinct downstream targets. Our data indicate that specific mechanisms have evolved for signaling nodes, like PKB, to select between various downstream events. Targeting such mechanisms selectively could facilitate the development of therapeutics that might limit toxic side effects.

MicroRNA-542-3p Suppresses Tumor Cell Invasion via Targeting AKT Pathway in Human Astrocytoma [Signal Transduction]

August 18th, 2015 by

The molecular mechanism underlying constitutive activation of AKT signaling, which plays essential roles in astrocytoma progression, is not fully characterized. Increasing studies have reported that microRNAs are involved in the malignant behavior of astrocytoma cells via directly targeting multiple oncogenes or tumor suppressors. Here, we found that miR-542-3p expression was decreased in glioblastoma cell lines and astrocytoma tissues, and reduced levels of miR-542-3p expression correlated with high histopathological grades and poor prognosis of astrocytoma patients. Exogenous miR-542-3p suppressed glioblastoma cell invasion through not only targeting AKT1 itself, but also directly downregulating its two important upstream regulators, namely, ILK and PIK3R1. Notably, overexpressing miR-542-3p decreased AKT1 phosphorylation and, directly and indirectly, repressed nuclear translocation and transactivation activity of β-catenin to exert its anti-invasive effect. Furthermore, miR-542-3p expression level negatively correlated with AKT activity, as well as levels of ILK and PIK3R1 in human astrocytoma specimens. These findings suggest that miR-542-3p acts as a negative regulator in astrocytoma progression, and miR-542-3p downregulation contributes to aberrant activation of the AKT signaling, leaving open the possibility that miR-542-3p may be a potential therapeutic target for high-grade astrocytoma.

Dephosphorylation during Bleach and Regeneration of Visual Pigment in Carp Rod and Cone Membranes [Cell Biology]

August 18th, 2015 by Yamaoka, H., Tachibanaki, S., Kawamura, S.

On absorption of light by vertebrate visual pigment, the chromophore, 11-cis retinal, is isomerized to all-trans retinal to activate the phototransduction cascade, which leads to a hyperpolarizing light response. Activated pigment is inactivated by phosphorylation on the protein moiety, opsin. Isomerized all-trans retinal is ultimately released from opsin, and the pigment is regenerated by binding to 11-cis retinal. In this pigment regeneration cycle, the phosphates incorporated should be removed in order that the pigment regains the capability of activating the phototransduction cascade. However, it is not clear yet how pigment dephosphorylation takes place in the regeneration cycle. First in this study, we tried to estimate the dephosphorylation activity in living carp rods and cones, and found that the activity, which is present mainly in the cytoplasm in both rods and cones, is 3-times higher in cones than in rods. Second, we examined at which stage the dephosphorylation takes place; before or after the release of all-trans retinal, during pigment regeneration or after pigment regeneration. For this purpose, we prepared three types of phosphorylated substrates in purified carp rod and cone membranes: phosphorylated bleaching intermediate, phosphorylated opsin, and phosphorylated-and-regenerated pigment. We also examined the effect of pigment regeneration on the dephosphorylation. The results showed that the dephosphorylation does not show substrate preference in the regeneration cycle, and suggested that the dephosphorylation takes place constantly. The results also suggest that, under bright light, some of the regenerated visual pigment remain phosphorylated to reduce the light sensitivity in cones.

Substrate Specificity and Possible Heterologous Targets of Phytaspase, a Plant Cell Death Protease [Plant Biology]

August 17th, 2015 by

Plants lack aspartate-specific cell death proteases homologous to animal caspases. Instead, a subtilisin-like serine-dependent plant protease named phytaspase shown to be involved in the accomplishment of programmed death of plant cells is able to hydrolyze a number of peptide-based caspase substrates. Here, we determined the substrate specificity of rice (Oryza sativa) phytaspase by using the positional scanning substrate combinatorial library approach. Phytaspase was shown to display an absolute specificity of hydrolysis after an aspartic acid residue. The preceding amino acid residues however significantly influence the efficiency of hydrolysis. Efficient phytaspase substrates demonstrated a remarkable preference for an aromatic amino acid residue in the P3 position. The deduced optimum phytaspase recognition motif has the sequence IWLD and is strikingly hydrophobic. The established pattern was confirmed through synthesis and kinetic analysis of cleavage of a set of optimized peptide substrates. An amino acid motif similar to the phytaspase cleavage site is shared by the human gastrointestinal peptide hormones gastrin and cholecystokinin. In agreement with the established enzyme specificity, phytaspase was shown to hydrolyze gastrin-1 and cholecystokinin at the predicted sites in vitro, thus destroying the active moieties of the hormones.

Membrane Curvature-Sensing and -Inducing Activity of Islet Amyloid Polypeptide and Its Implications for Membrane Disruption [Membrane Biology]

August 17th, 2015 by

Islet amyloid polypeptide (IAPP) is a 37-amino-acid amyloid protein intimately associated with pancreatic islet β-cell dysfunction and death in type II diabetes. In this study, we combine spectroscopic methods and microscopy to investigate α-helical IAPP-membrane interactions. Using light scattering and fluorescence microscopy we observe that larger vesicles become smaller upon treatment with human or rat IAPP. Electron microscopy shows the formation of various highly curved structures such as tubules or smaller vesicles in a membrane-remodeling process, and spectrofluorometric detection of vesicle leakage shows disruption of membrane integrity. This effect is stronger for hIAPP than for the less toxic rIAPP. From CD spectra in the presence of different-sized vesicles, we also uncover the membrane curvature-sensing ability of IAPP and find that it transitions from inducing to sensing membrane curvature when lipid negative charge is decreased. Our in vivo EM images of immunogold-labeled rIAPP and hIAPP show both forms to localize to mitochondrial cristae, which contain not only locally curved membranes but also phosphatidylethanolamine and cardiolipin, lipids with high spontaneous negative curvature. Disruption of membrane integrity by induction of membrane curvature could apply more broadly to other amyloid proteins and be responsible for membrane damage observed in other amyloid diseases as well.
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Localization of Proteins to the 1,2-Propanediol Utilization Microcompartment by Non-native Signal Sequences Is Mediated by a Common Hydrophobic Motif [Microbiology]

August 17th, 2015 by

Various bacteria localize metabolic pathways to proteinaceous organelles known as bacterial microcompartments (MCPs), enabling the metabolism of carbon sources to enhance survival and pathogenicity in the gut. There is considerable interest in exploiting bacterial MCPs for metabolic engineering applications, but little is known about the interactions between MCP signal sequences and the protein shells of different MCP systems. We found that the N-terminal sequences from the ethanolamine utilization (Eut) and glycyl radical-generating protein (Grp) MCPs are able to target reporter proteins to the 1,2-propanediol utilization (Pdu) MCP, mediated by a conserved hydrophobic residue motif. Recapitulation of this motif by the addition of a single amino acid confers targeting function on an N-terminal sequence from the ethanol utilization (Etu) MCP system that previously did not act as a Pdu signal sequence. Moreover, the Pdu-localized signal sequences compete with native Pdu targeting sequences for encapsulation in the Pdu MCP. Salmonella enterica natively possesses both the Pdu and Eut operons, and our results suggest that Eut proteins might be localized to the Pdu MCP in vivo. We further demonstrate that S. enterica LT2 retains the ability to grow on 1,2-propanediol as the sole carbon source when a Pdu enzyme is replaced with its Eut homolog. While the relevance of this finding to the native system remains to be explored, we show that the Pdu-localized signal sequences described herein allow control over the ratio of heterologous proteins encapsulated within Pdu MCPs.
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Disruption of nucleotide homeostasis by the antiproliferative drug AICAR (5-aminoimidazole-4-carboxamide-1-{beta}-D-ribofuranoside monophosphate) [Metabolism]

August 17th, 2015 by

5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside monophosphate (AICAR) is a natural metabolite with potent anti-proliferative and low-energy mimetic properties. At high concentration, AICAR is toxic for yeast and mammalian cells but the molecular basis of this toxicity is poorly understood. Here, we report the identification of yeast purine salvage pathway mutants which are synthetic lethal with AICAR accumulation. Genetic suppression revealed that this synthetic lethality is in part due to low expression of adenine phosphoribosyl transferase under high AICAR conditions. In addition, metabolite profiling points to the AICAR/nucleotide triphosphate (NTP) balance as crucial for optimal utilization of glucose as a carbon source. Indeed, we found that AICAR toxicity in yeast and human cells is alleviated when glucose is replaced by an alternative carbon source. Together, our metabolic analyses unveil the AICAR/NTP balance as a major factor of AICAR antiproliferative effects.
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Cyclic-di-GMP regulates production of sortase substrates of Clostridium difficile and their surface exposure through ZmpI protease-mediated cleavage [Gene Regulation]

August 17th, 2015 by

In Gram-positive pathogens, surface proteins may be covalently anchored to the bacterial peptidoglycan by sortase, a cysteine transpeptidase enzyme. In contrast to other Gram-positive bacteria, only one single sortase enzyme, SrtB, is conserved between strains of Clostridium difficile. Sortase-mediated peptidase activity has been reported in vitro and seven potential substrates have been identified. Here we demonstrate the functionality of sortase in C. difficile. We identify two sortase-anchored proteins, the putative adhesins CD2831 and CD3246, and determine the cell wall anchor structure of CD2831. The C-terminal PPKTG sorting motif of CD2831 is cleaved between the threonine and glycine residues and the carboxyl group of threonine is amide-linked to the side chain amino group of diaminopimelic acid within the peptidoglycan peptide stem. We show that CD2831 protein levels are elevated in presence of high intracellular cyclic di-GMP (c-di-GMP) concentrations, in agreement with the control of CD2831 expression by a c-di-GMP-dependent type II riboswitch. Low c-di-GMP levels induce the release of CD2831, and presumably CD3246, from the surface of cells. This regulation is mediated by proteolytic cleavage of CD2831 and CD3246 by the zinc metalloprotease ZmpI, whose expression is controlled by a type II c-di-GMP riboswitch. These data reveal a novel regulatory mechanism for expression of two sortase substrates by the secondary messenger c-di-GMP, on which surface anchoring is dependent.
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Regulation of Rubisco Activase: Product Inhibition, Cooperativity, and Magnesium Activation [Plant Biology]

August 17th, 2015 by

In many photosynthetic organisms, tight-binding Rubisco inhibitors are released by the motor protein Rubisco activase (Rca). In higher plants, Rca plays a pivotal role in regulating CO2 fixation. Here, the ATPase activity of 0.005 mM tobacco Rca was monitored under steady-state conditions, and global curve fitting was utilized to extract kinetic constants. The kcat was best fit by 22.3 +/- 4.9 min-1, the Km for ATP by 0.104 +/- 0.024 mM, and the Ki for ADP by 0.037 +/- 0.007 mM. Without ADP, the Hill coefficient for ATP hydrolysis was extracted to be 1.0 +/- 0.1, indicating non-cooperative behavior of homo-oligomeric Rca assemblies. However, the addition of ADP was shown to introduce positive cooperativity between two or more subunits (Hill coefficient 1.9 +/- 0.2), allowing for regulation via the prevailing ATP/ADP ratio. ADP-mediated activation was not observed, whereas larger amounts led to competitive product inhibition of hydrolytic activity. The catalytic efficiency increased 8.4-fold upon cooperative binding of a second magnesium ion (Hill coefficient 2.5 +/- 0.5), suggesting at least three conformational states (ATP-bound, ADP-bound, empty) within assemblies containing an average of about six subunits. The addition of excess Rubisco (144:1, L8S8:Rca) and crowding agents did not modify catalytic rates. However, high magnesium provided for thermal Rca stabilization. We propose that magnesium mediates the formation of closed hexameric toroids capable of high turnover rates and amenable to allosteric regulation. We suggest that in vivo, the Rca hydrolytic activity is tuned by fluctuating [Mg2+] in response to changes in available light.