The BAP1/ASXL2 Histone H2A Deubiquitinase Complex Regulates Cell Proliferation and is Disrupted in Cancer [Cell Biology]

September 28th, 2015 by

The deubiquitinase (DUB) and tumor suppressor BAP1 catalyzes ubiquitin removal from histone H2A K119 and coordinates cell proliferation, but how BAP1 partners modulate its function remains poorly understood. Here, we report that BAP1 forms two mutually exclusive complexes with the transcriptional regulators ASXL1 and ASXL2, which are necessary for maintaining proper protein levels of this DUB. Conversely, BAP1 is essential for maintaining ASXL2, but not ASXL1 protein stability. Notably, cancer-associated loss of BAP1 expression results in ASXL2 destabilization and hence loss of its function. ASXL1 and ASXL2 use their ASXM domains to interact with the C-terminal domain (CTD) of BAP1 and these interactions are required for ubiquitin binding and H2A deubiquitination. The deubiquitination promoting effect of ASXM requires intramolecular interactions between catalytic and non-catalytic domains of BAP1 which generate a composite ubiquitin binding interface (CUBI). Notably, the CUBI engages multiple interactions with ubiquitin involving, (i) the ubiquitin carboxyl hydrolase (UCH) catalytic domain of BAP1 which interacts with the hydrophobic patch of ubiquitin and (ii) the CTD domain which interacts with a charged patch of ubiquitin. Significantly, we identified cancer-associated mutations of BAP1 that disrupt the CUBI, and notably an in frame deletion in the CTD that inhibits its interaction with ASXL1/2, DUB activity and deregulates cell proliferation. Moreover, we demonstrated that BAP1 interaction with ASXL2 regulates cell senescence and that ASXL2 cancer-associated mutations disrupt BAP1 DUB activity. Thus, inactivation of BAP1/ASXL2 axis might contribute to cancer development.

Truncated Amyloid-{beta}(11-40/42) from Alzheimer’s Disease Binds Copper2+ with a Femtomolar Affinity and Influences Fibre Assembly [Molecular Biophysics]

September 25th, 2015 by Barritt, J. D., Viles, J. H.

Alzheimer's disease (AD) coincides with the formation of extracellular amyloid plaques composed of the amyloid-β (Aβ) peptide. Aβ is typically forty residues long (Aβ(1-40)) but can have variable C- and N- termini. Naturally occurring N-terminally truncated Aβ(11-40/42) is found in the cerebrospinal fluid and has a similar abundance to Aβ(1-42), constituting one fifth of the plaque load. Based on its specific N-terminal sequence we hypothesized that truncated Aβ(11-40/42) would have an elevated affinity for Cu2+. Various spectroscopic techniques, complimented with transmission electron microscopy, were used to determine the properties of the Cu2+Aβ(11-40/42) interaction and how Cu2+ influences amyloid fibre formation. We show, Cu2+Aβ(11-40) forms a tetragonal complex with a 34 ± 5 femtomolar dissociation constant at pH 7.4. This affinity is three orders of magnitude tighter than Cu2+ binding to Aβ(1-40/42) and more than an order of magnitude tighter than that of serum albumin, the extracellular Cu2+ transport protein. Furthermore, Aβ(11-40/42) forms fibres twice as fast as Aβ(1-40) with a very different morphology; forming bundles of very short amyloid rods. Substoichiometric Cu2+ drastically perturbs Aβ(11-40/42) assembly, stabilizing much longer fibres. The very tight femtomolar affinity of Cu2+ for Aβ(11-40/42) explains the high levels of Cu2+ observed in AD plaques.
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IL-10 mediated immunosuppression: March-I induction regulates antigen presentation by macrophages but not dendritic cells [Immunology]

September 25th, 2015 by Mittal, S. K., Cho, K.-J., Ishido, S., Roche, P. A.

Efficient immune responses require regulated antigen presentation to CD4 T cells. IL-10 inhibits the ability of DCs and macrophages to stimulate antigen-specific CD4 T cells, however the mechanisms by which IL-10 suppresses antigen presentation remain poorly understood. We now report that IL-10 stimulates expression of the E3 ubiquitin-ligase March-I in activated macrophages, thereby down-regulating MHC-II, CD86, and antigen presentation to CD4 T cells. By contrast, IL-10 does not stimulate March-I expression in DCs, does not suppress MHC-II or CD86 expression on either resting or activated DCs, and does not affect antigen presentation by activated DCs. IL-10 does, however, inhibit the process of DC activation itself, thereby reducing the efficiency of antigen presentation in a March-I-independent manner. Thus, IL-10 suppression of APC function in macrophages is March-I-dependent whereas in DCs suppression is March-I-independent.

Plant Protochlorophyllide Oxidoreductases A and B – Catalytic Efficiency and Initial Reaction Steps [Plant Biology]

September 25th, 2015 by

The enzyme protochlorophyllide oxidoreductase (POR, EC has a key role in plant development. It catalyzes one of the later steps in chlorophyll synthesis, the light-induced reduction of protochlorophyllide (PChlide) into chlorophyllide (Chlide) in the presence of NADPH. Two isozymes of plant POR, POR A and POR B from barley, which differ in their function during plant life, are compared with respect to their substrate binding affinity, catalytic efficiency, and catalytic mechanism. POR B as compared to POR A shows an 5-fold higher binding affinity for protochlorophyllide a (PChlide) and an about 6-fold higher catalytic efficiency measured as ratio kcat/KM. Based on the reaction intermediates, which can be trapped at low temperatures the same reaction mechanism operates in both POR A and POR B. In contrast to results reported for POR enzymes from cyanobacteria, the initial light-driven step, which occurs at temperatures below 180 K already involves the full chemistry of the photoreduction and yields the reaction product, Chlide, in an enzyme-bound form. The subsequent dark reactions, which include cofactor (NADP+) release and cofactor (NADPH) rebinding, show different temperature dependencies for POR A and POR B and suggest a higher conformational flexibility of POR B in the surrounding of the active center. Both the higher substrate binding affinity and well-adapted enzyme dynamics are held responsible for the increased catalytic activity of POR B as compared to POR A.

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.

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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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.