Long-term Aggresome Accumulation Leads to DNA Damage, p53-dependent Cell-cycle Arrest and Steric Interference in Mitosis [Protein Synthesis and Degradation]

September 25th, 2015 by Lu, M., Boschetti, C., Tunnacliffe, A.

Juxtanuclear aggresomes form in cells when levels of aggregation-prone proteins exceed the capacity of the proteasome to degrade them. It is widely believed that aggresomes have a protective function, sequestering potentially damaging aggregates until these can be removed by autophagy. However, most in-cell studies have been carried out over a few days at most, and there is little information on the long-term effects of aggresomes. To examine these long-term effects, we created inducible, single-copy cell lines that expressed aggregation-prone polyQ proteins over several months. We present evidence that, as perinuclear aggresomes accumulate, they are associated with abnormal nuclear morphology and DNA double-strand breaks (DSBs), resulting in cell-cycle arrest via the P-p53(Ser15)-dependent pathway. Further analysis reveals that aggresomes can have a detrimental effect on mitosis by steric interference with chromosome alignment, centrosome positioning and spindle formation. The incidence of apoptosis also increased in aggresome-containing cells. These severe defects developed gradually following juxtanuclear aggresome formation and were not associated with small cytoplasmic aggregates alone. Thus, our findings demonstrate that, in dividing cells, aggresomes are detrimental over the long term, rather than protective. This suggests a novel mechanism for polyQ-associated developmental and cell biological abnormalities, particularly those with early onset and non-neuronal pathologies.
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The Natural Product N-palmitoyl-L-leucine Selectively Inhibits Late Assembly of Human Spliceosomes [RNA]

September 25th, 2015 by

The spliceosome is a dynamic complex of five structural RNA and dozens of proteins, which assemble together to remove introns from nascent eukaryotic gene transcripts in a process called splicing. Small molecules that target different components of the spliceosome represent valuable research tools to investigate this complicated macromolecular machine. However, the current collection of spliceosome inhibitors is very limited. To expand the toolkit we used a high-throughput in vitro splicing assay to screen a collection of pre-fractions of natural compounds derived from marine bacteria for splicing inhibition. Further fractionation of initial hits generated individual peaks of splicing inhibitors that interfere with different stages of spliceosome assembly. With additional characterization of individual peaks we identified N-palmitoyl-L-leucine as a new splicing inhibitor that blocks a late stage of spliceosome assembly. Structure activity relationship analysis of the compound revealed that length of carbon chain is important for activity in splicing, as well as for effects on the cytological profile of cells in culture. Together these results demonstrate that our combination of in vitro splicing analysis with complex natural product libraries is a powerful strategy for identifying new small molecule tools with which to probe different aspects of spliceosome assembly and function.

Membrane Potential Controls the Efficacy of Catecholamine-induced {beta}1-Adrenoceptor Activity [Molecular Biophysics]

September 25th, 2015 by Birk, A., Rinne, A., B&uumlnemann, M.

G protein-coupled receptors (GPCRs) are membrane located proteins and, therefore, are exposed to changes in membrane potential (VM) in excitable tissues. These changes have been shown to alter receptor activation of certain Gi-and Gq-coupled GPCRs. By means of a combination of whole-cell patch-clamp and Foerster resonance energy transfer (FRET) in single cells, we demonstrate that the activation of the Gs-coupled β1-adrenoceptor (β1-AR) by the catecholamines isoprenaline (Iso) and adrenaline (Adr) is regulated by VM. This voltage-dependence is also transmitted to G protein and arrestin 3 signaling. Voltage-dependence of β2-AR activation, however, was weak compared to β1-AR voltage-dependence. Drug efficacy is a major target of β1-AR voltage-dependence as depolarization attenuated receptor activation even under saturating concentrations of agonists with significantly faster kinetics than the inactivation upon agonist withdrawal. Also the efficacy of the endogenous full agonist adrenaline was reduced by depolarization. This is a unique finding since reports of natural full agonists at other voltage-dependent GPCRs only show alterations in affinity during depolarization. Based on a Boltzmann function fit to the relationship of VM and receptor-arrestin 3 interaction we determined the voltage-dependence with highest sensitivity in the physiological range of membrane potential. Our data suggests that under physiological conditions voltage regulates the activity of agonist-occupied β1-adrenoceptors on a very fast time scale.

Role of a Hydrophobic Pocket in Polyamine Interactions with the Polyspecific Organic Cation Transporter OCT3 [Membrane Biology]

September 24th, 2015 by Li, D. C., Nichols, C. G., Sala-Rabanal, M.

Organic Cation Transporter 3 (OCT3, SLC22A3) is a polyspecific, facilitative transporter expressed in astrocytes and in placental, intestinal and blood-brain barrier epithelia, and thus elucidating the molecular mechanisms underlying OCT3 substrate recognition is critical for the rational design of drugs targeting these tissues. The pharmacology of OCT3 is distinct from that of other OCTs, and here we investigated the role of a hydrophobic cavity tucked within the translocation pathway in OCT3 transport properties. Replacement of an absolutely conserved Asp by charge reversal (D478E), neutralization (D478N), or even exchange (D478E) abolished MPP+ uptake, demonstrating this residue to be obligatory for OCT3-mediated transport. Mutations at non-conserved residues lining the putative binding pocket of OCT3 to the corresponding residue in OCT1 (L166F, F450L, and E451Q) reduced the rate of MPP+ transport, but recapitulated the higher-sensitivity pharmacological profile of OCT1. Thus, interactions of natural polyamines (putrescine, spermidine, spermine) and polyamine-like potent OCT1 blockers (1,10-diaminodecane, decamethonium, bis-triethylaminodecane, and 1,10 bis-quinuclidinedecane) with wild-type OCT3 were weak, but were significantly potentiated in the mutant OCT3s. Conversely, a reciprocal mutation in OCT1 (F161L) shifted the polyamine-sensitivity phenotype towards that of OCT3. Further analysis indicated that OCT1 and OCT3 can recognize essentially the same substrates, but the strength of substrate-transporter interactions is weaker in OCT3, as informed by the distinct makeup of the hydrophobic cleft. The residues identified here are key contributors to both the observed differences between OCT3 and OCT1 and to the mechanisms of substrate recognition by OCTs in general.

Molecular Interactions and Cellular Itinerary of the Yeast RAVE (Regulator of the H+-ATPase of Vacuolar and Endosomal Membranes) Complex [Cell Biology]

September 24th, 2015 by Smardon, A. M., Nasab, N. D., Tarsio, M., Diakov, T. T., Kane, P. M.

The RAVE complex (regulator of the H+-ATPase of vacuolar and endosomal mem-branes) is required for biosynthetic assembly and glucose-stimulated reassembly of the yeast vacuolar H+-ATPase (V-ATPase). Yeast RAVE contains three subunits, Rav1, Rav2 and Skp1. Rav1 is the largest subunit, and it binds Rav2 and Skp1 of RAVE, the E, G, and C subunits of the V-ATPase peripheral V1 sector and Vph1 of the membrane Vo sector. We identified Rav1 regions required for interaction with its binding partners through deletion analysis, co-immunoprecipitation, two-hybrid assay, and pull-down assays with expressed proteins. We find that Skp1 binding requires sequences near the C-terminus of Rav1, V1 subunits E and C bind to a conserved region in the C-terminal half of Rav1, and the cytosolic domain of Vph1 binds near the junction of the Rav1 N- and C-terminal halves. In contrast, Rav2 binds to the N-terminal domain of Rav1, which can be modeled as a double β-propeller. Only the V1 C subunit binds to both Rav1 and Rav2. Using GFP-tagged RAVE subunits in vivo, we demonstrate glucose-dependent association of RAVE with the vacuolar membrane, consistent with its role in glucose-dependent V-ATPase assembly. It is known that V1 subunit C localizes to the V1-Vo interface in assembled V-ATPase complexes and is important in regulated disassembly of V-ATPases. We propose that RAVE cycles between cytosol and vacuolar membrane in a glucose-dependent manner, positioning V1 and V0 subcomplexes and orienting the V1 C subunit to promote assembly.
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The replication initiation protein Sld3/Treslin orchestrates the assembly of the replication fork helicase during S phase [DNA and Chromosomes]

September 24th, 2015 by Bruck, I., Kaplan, D. L.

The initiation of DNA replication is a highly regulated process in eukaryotic cells, and central to the process of initiation is the assembly and activation of the replication fork helicase. The replication fork helicase is comprised of CMG (Cdc45, Mcm2-7, and GINS) in eukaryotic cells, and the mechanism underlying assembly of the CMG during S phase was studied in this manuscript. We identified a point mutation of Sld3 that is specifically defective for Mcm3 and Mcm5 interaction (sld3-m10), and we also identified a point mutation of Sld3 that is specifically defective for single-stranded DNA (ssDNA) interaction (sld3-m9). Expression of wild-type levels of sld3-m9 resulted in a severe DNA replication defect with no recruitment of GINS to Mcm2-7, while expression of wild-type levels of sld3-m10 resulted in a severe replication defect with no Cdc45 recruitment to Mcm2-7. We propose a model for Sld3-mediated control of replication initiation, wherein Sld3 manages the proper assembly of the CMG during S phase. We also find that the biochemical functions identified for Sld3 are conserved in human Treslin, suggesting that Treslin orchestrates assembly of the CMG in human cells.
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Cell polarity kinase MST4 cooperates with cAMP-dependent kinase to orchestrate histamine-stimulated acid secretion in gastric parietal cells [Cell Biology]

September 24th, 2015 by

The digestive function of the stomach depends on acidification of the gastric lumen. Acid secretion into the lumen is triggered by activation of PKA cascade, which ultimately results in the insertion of gastric H,K-ATPases into the apical plasma membranes of parietal cells. A coupling protein is ezrin whose phosphorylation at Ser66 by PKA is required for parietal cell activation. However, little is known regarding the molecular mechanism(s) by which this signaling pathway operates in gastric acid secretion. Here we show PKA cooperates with MST4 to orchestrate histamine-elicited acid secretion by phosphorylating ezrin at Ser66 and Thr567, respectively. Histamine stimulation activates PKA which phosphorylates MST4 at Thr178 and then promotes MST4 kinase activity. Interestingly, activated MST4 then phosphorylates ezrin pre-phosphorylated by PKA. Importantly, MST4 is important for acid secretion in parietal cells because either suppression of MST4 or overexpression of non-phosphorylatable MST4 prevents the apical membrane reorganization and proton pump translocation elicited by histamine stimulation. In addition, overexpressing MST4 phosphorylation-deficient ezrin results in an inhibition of gastric acid secretion. Taken together, these results define a novel molecular mechanism linking PKA-MST4-ezrin signaling cascade to polarized epithelial secretion in gastric parietal cells.
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Structural Studies of IRF4 Reveal a Flexible Autoinhibitory Region and a Compact Linker Domain [Gene Regulation]

September 24th, 2015 by Remesh, S. G., Santosh, V., Escalante, C. R.

IRF4 is a unique member of the IRF family playing critical regulatory roles in immune cell development, regulation of obesity-induced inflammation and control of thermogenic gene expression. The ability of IRF4 to control diverse transcriptional programs arises from its proficiency to interact with numerous transcriptional partners. In this study we present the structural characterization of full length IRF4. Using a combination of X-ray and SAXS studies, we reveal unique features of the Interferon Activation Domain (IAD) including a set of β-sheets and loops that serve as the binding site for PU.1 and also show that unlike other IRF members, IRF4 has a flexible autoinhibitory region. In addition, we have determined the SAXS solution structure of full length IRF4 that together with circular dichroism studies suggest that the linker region is not extended but folds into a domain structure.

Genomic survey and biochemical analysis of recombinant candidate cyanobacteriochromes reveals enrichment for near UV/Violet sensors in the halotolerant and alkaliphilic cyanobacterium Microcoleus IPPAS B353 [Genomics and Proteomics]

September 24th, 2015 by

Cyanobacteriochromes (CBCRs), which are exclusive to and widespread among cyanobacteria, are photoproteins that sense the entire range of near-UV and visible light. CBCRs are related to the red/far-red phytochromes that utilize linear tetrapyrrole (bilin) chromophores. Best characterized from the unicellular cyanobacterium Synechocystis sp. PCC 6803 and the multicellular heterocyst forming filamentous cyanobacteria Nostoc punctiforme ATCC 29133 and Anabaena sp. PCC 7120, CBCRs have been poorly investigated in mat-forming, nonheterocystous cyanobacteria. In this study, we sequenced the genome of one of such species, Microcoleus IPPAS B353 (Microcoleus B353), and identified two phytochromes and seven CBCRs with one or more bilin-binding cGMP-specific phosphodiesterase, Adenylyl cyclase and FhlA (GAF) domains. Biochemical and spectroscopic measurements of 23 purified GAF proteins from phycocyanobilin (PCB) producing recombinant E. coli indicated that 13 of these proteins formed near-UV and visible light-absorbing covalent adducts: ten GAFs contained PCB chromophores whereas three contained the PCB isomer, phycoviolobilin (PVB). Furthermore, the complement of Microcoleus B353 CBCRs is enriched in near-UV and violet sensors, but lacks red/green and green/red CBCRs that are widely distributed in other cyanobacteria. We hypothesize that enrichment in short wavelength-absorbing CBCRs is critical for acclimation to high-light environments where this organism is found.
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Assembling a correctly folded and functional heptahelical membrane protein by protein trans-splicing [Membrane Biology]

September 24th, 2015 by

Protein trans-splicing using split inteins is well established as useful tool for protein engineering. Here we show for the first time, that this method can be applied to a membrane protein under native conditions. We provide compelling evidence that the heptahelical proteorhodopsin can be assembled from two separate fragments consisting of helical bundles A-B and C-D-E-F-G via a splicing site located in the BC loop. The procedure presented here is based on dual expression and ligation in vivo. Global fold, stability and photodynamic were analyzed in detergent by CD-, stationary as well as time-resolved optical spectroscopy. Its fold within lipid bilayers has been probed by high field and DNP-enhanced solid-state NMR utilizing a 13Clabeled retinal co-factor and extensively 13C-15N labeled protein. Our data show unambiguously that the ligation product is identical to its non-ligated counterpart. Furthermore, our data highlight effects of BC loop modifications onto the photocycle kinetics of proteorhodopsin. Our data demonstrate that a correctly folded and functionally intact protein can be produced in this artificial way. Our findings are of high relevance for a general understanding of the assembly of membrane proteins, for elucidating intramolecular interactions and they offer the possibility towards developing novel labeling schemes for spectroscopic applications.