Damian B. van Rossum

Phospholipase Cγ1 controls surface expression of TRPC3 through an intermolecular PH domain

Many ion channels are regulated by lipids, but prominent motifs for lipid binding have not been identified in most ion channels. Recently, we reported that phospholipase Cγ1 (PLC-γ1) binds to and regulates TRPC3 channels, components of agonist-induced Ca2+ entry into cells. This interaction requires a domain in PLC-γ1 that includes a partial pleckstrin homology (PH) domain—a consensus lipid-binding and protein-binding sequence. We have developed a gestalt algorithm to detect hitherto ‘invisible’ PH and PH-like domains, and now report that the partial PH domain of PLC-γ1 interacts with a complementary partial PH-like domain in TRPC3 to elicit lipid binding and cell-surface expression of TRPC3. Our findings imply a far greater abundance of PH domains than previously appreciated, and suggest that intermolecular PH-like domains represent a widespread signalling mode.

Phospholipase C-γ Is Required for Agonist-Induced Ca2+ Entry

We report here that PLC-gamma isoforms are required for agonist-induced Ca2+ entry (ACE). Overexpressed wild-type PLC-gamma1 or a lipase-inactive mutant PLC-gamma1 each augmented ACE in PC12 cells, while a deletion mutant lacking the region containing the SH3 domain of PLC-gamma1 was ineffective. RNA interference to deplete either PLC-gamma1 or PLC-gamma2 in PC12 and A7r5 cells inhibited ACE. In DT40 B lymphocytes expressing only PLC-gamma2, overexpressed muscarinic M5 receptors (M5R) activated ACE. Using DT40 PLC-gamma2 knockout cells, M5R stimulation of ER Ca2+ store release was unaffected, but ACE was abolished. Normal ACE was restored by transient expression of PLC-gamma2 or a lipase-inactive PLC-gamma2 mutant. The results indicate a lipase-independent role of PLC-gamma in the physiological agonist-induced activation of Ca2+ entry.

HSP90 regulates cell survival via inositol hexakisphosphate kinase-2.

Heat-shock proteins (HSPs) are abundant, inducible proteins best known for their ability to maintain the conformation of proteins and to refold damaged proteins. Some HSPs, especially HSP90, can be antiapoptotic and the targets of anticancer drugs. Inositol hexakisphosphate kinase-2 (IP6K2), one of a family of enzymes generating the inositol pyrophosphate IP7 [diphosphoinositol pentakisphosphate (5-PP-IP5)], mediates apoptosis. Increased IP6K2 activity sensitizes cancer cells to stressors, whereas its depletion blocks cell death. We now show that HSP90 physiologically binds IP6K2 and inhibits its catalytic activity. Drugs and selective mutations that abolish HSP90–IP6K2 binding elicit activation of IP6K2, leading to cell death. Thus, the prosurvival actions of HSP90 reflect the inhibition of IP6K2, suggesting that selectively blocking this interaction could provide effective and safer modes of chemotherapy.

Glutamatergic regulation of serine racemase via reversal of PIP2 inhibition

D-serine is a physiologic coagonist with glutamate at NMDA-subtype glutamate receptors. As D-serine is localized in glia, synaptically released glutamate presumably stimulates the glia to form and release D-serine, enabling glutamate/D-serine cotransmission. We show that serine racemase (SR), which generates D-serine from L-serine, is physiologically inhibited by phosphatidylinositol (4,5)-bisphosphate (PIP2) presence in membranes where SR is localized. Activation of metabotropic glutamate receptors (mGluR5) on glia leads to phospholipase C-mediated degradation of PIP2, relieving SR inhibition. Thus mutants of SR that cannot bind PIP2 lose their membrane localizations and display a 4-fold enhancement of catalytic activity. Moreover, mGluR5 activation of SR activity is abolished by inhibiting phospholipase C.

The BRCA1 tumor suppressor binds to inositol 1,4,5-trisphosphate receptors to stimulate apoptotic calcium release

Background: The non-nuclear BRCA1 tumor suppressor can stimulate cell death, but the mechanisms are unknown. Results: BRCA1 binds to the inositol 1,4,5-trisphophate receptor (IP3R) calcium channel at the endoplasmic reticulum to stimulate apoptotic calcium release. Conclusion: BRCA1 tumor suppressor activity includes direct stimulation of apoptotic cell death via increased IP3R activity. Significance: We identify a novel role for the tumor suppressor BRCA1. The inositol 1,4,5-trisphosphate receptor (IP3R) is a ubiquitously expressed endoplasmic reticulum (ER)-resident calcium channel. Calcium release mediated by IP3Rs influences many signaling pathways, including those regulating apoptosis. IP3R activity is regulated by protein-protein interactions, including binding to proto-oncogenes and tumor suppressors to regulate cell death. Here we show that the IP3R binds to the tumor suppressor BRCA1. BRCA1 binding directly sensitizes the IP3R to its ligand, IP3. BRCA1 is recruited to the ER during apoptosis in an IP3R-dependent manner, and, in addition, a pool of BRCA1 protein is constitutively associated with the ER under non-apoptotic conditions. This is likely mediated by a novel lipid binding activity of the first BRCA1 C terminus domain of BRCA1. These findings provide a mechanistic explanation by which BRCA1 can act as a proapoptotic protein.

Association of small ankyrin 1 with the sarcoplasmic reticulum

Small ankyrin 1, or sAnk1, is a small, alternatively spliced product of the erythroid ankyrin gene, ANK1, that is expressed in striated muscle and concentrated in the network sarcoplasmic reticulum (SR) surrounding the Z disks and M lines. We have characterized sAnk1 in muscle homogenates and SR vesicles, and have identified the region that targets it to the network SR. Selective extractions and partitioning into Triton X-114 show that sAnk1 behaves like the SR Ca-ATPase and so is an integral protein of the SR membrane. Mild proteolytic treatment of isolated SR vesicles indicates that sAnk1 is oriented with its hydrophilic, C-terminal sequence exposed to the solution, which is equivalent to the cytoplasmic face of the SR membrane in situ. SDS-PAGE in non-reducing gels suggests that sAnk1 is present as dimers and larger oligomers in the native SR. These results suggest that sAnk1 is oligomeric and oriented with its C-terminus exposed to the cytoplasm, where it may interact with proteins of the contractile apparatus. The N-terminal 29 amino acid hydrophobic sequence of sAnk1, which is predicted to span the SR membrane, is sufficient to target proteins to and anchor them in internal membranes of HEK 293 cells. It also targets reporter proteins to the network SR of skeletal myofibers and is thus the first example of a sequence that targets proteins to a particular compartment of the SR.

Functional evolution of Erg potassium channel gating reveals an ancient origin for IKr

Significance Ether-a-go-go related gene (Erg) family K+ channels regulate excitability of muscle and neurons. However, mammalian and Drosophila Erg channels have distinctive gating phenotypes, suggesting divergent physiological roles. We examined the origins of Erg channel gating properties by investigating the functional evolution of the Erg family in Eumetazoa. We find that the mammalian IKr-like Erg channel phenotype is ancestral. Thus, the gating features that specialize Erg1 for cardiac action potential repolarization may have had their origins in the slow wave contractions of early metazoans. These gating features, inactivation and slow deactivation, have been lost in the Drosophila Erg, Caenorhabditis elegans Erg, and some sea anemone (Nematostella) Ergs. Loss of the Ether-a-go-go domain, which regulates channel closing, accompanies and may in part explain phenotypic conversion. Mammalian Ether-a-go-go related gene (Erg) family voltage-gated K+ channels possess an unusual gating phenotype that specializes them for a role in delayed repolarization. Mammalian Erg currents rectify during depolarization due to rapid, voltage-dependent inactivation, but rebound during repolarization due to a combination of rapid recovery from inactivation and slow deactivation. This is exemplified by the mammalian Erg1 channel, which is responsible for IKr, a current that repolarizes cardiac action potential plateaus. The Drosophila Erg channel does not inactivate and closes rapidly upon repolarization. The dramatically different properties observed in mammalian and Drosophila Erg homologs bring into question the evolutionary origins of distinct Erg K+ channel functions. Erg channels are highly conserved in eumetazoans and first evolved in a common ancestor of the placozoans, cnidarians, and bilaterians. To address the ancestral function of Erg channels, we identified and characterized Erg channel paralogs in the sea anemone Nematostella vectensis. N. vectensis Erg1 (NvErg1) is highly conserved with respect to bilaterian homologs and shares the IKr-like gating phenotype with mammalian Erg channels. Thus, the IKr phenotype predates the divergence of cnidarians and bilaterians. NvErg4 and Caenorhabditis elegans Erg (unc-103) share the divergent Drosophila Erg gating phenotype. Phylogenetic and sequence analysis surprisingly indicates that this alternate gating phenotype arose independently in protosomes and cnidarians. Conversion from an ancestral IKr-like gating phenotype to a Drosophila Erg-like phenotype correlates with loss of the cytoplasmic Ether-a-go-go domain. This domain is required for slow deactivation in mammalian Erg1 channels, and thus its loss may partially explain the change in gating phenotype.

Phylogenetic profiles reveal evolutionary relationships within the “twilight zone” of sequence similarity

Inferring evolutionary relationships among highly divergent protein sequences is a daunting task. In particular, when pairwise sequence alignments between protein sequences fall <25% identity, the phylogenetic relationships among sequences cannot be estimated with statistical certainty. Here, we show that phylogenetic profiles generated with the Gestalt Domain Detection Algorithm–Basic Local Alignment Tool (GDDA-BLAST) are capable of deriving, ab initio, phylogenetic relationships for highly divergent proteins in a quantifiable and robust manner. Notably, the results from our computational case study of the highly divergent family of retroelements accord with previous estimates of their evolutionary relationships. Taken together, these data demonstrate that GDDA-BLAST provides an independent and powerful measure of evolutionary relationships that does not rely on potentially subjective sequence alignment. We demonstrate that evolutionary relationships can be measured with phylogenetic profiles, and therefore propose that these measurements can provide key insights into relationships among distantly related and/or rapidly evolving proteins.

TRP_2, a Lipid/Trafficking Domain That Mediates Diacylglycerol-induced Vesicle Fusion

We recently modeled transient receptor potential (TRP) channels using the Gestalt Domain Detection Algorithm-Basic Local Alignment Tool (GDDA-BLAST), which derives structural, functional, and evolutionary information from primary amino acid sequences using phylogenetic profiles ( Ko, K. D., Hong, Y., Chang, G. S., Bhardwaj, G., van Rossum, D. B., and Patterson, R. L. (2008) Physics Arch. Quant. Methods arXiv: 0806.2394v1 ). Herein we test our functional predictions for the TRP_2 domain of TRPC3; a domain of unknown function that is conserved in all TRPC channels. Our functional models of this domain identify both lipid binding and trafficking activities. In this study, we reveal: (i) a novel structural determinant of ion channel sensitivity to lipids, (ii) a molecular mechanism for the difference between diacylglycerol (DAG)-sensitive and DAG-insensitive TRPC subfamilies, and (iii) evidence that TRPC3 can comprise part of the vesicle fusion machinery. Indeed, the TRPC3 TRP_2 domain mediates channel trafficking to the plasma membrane and binds to plasma membrane lipids. Further, mutations in TRP_2, which alter lipid binding, also disrupt the DAG-mediated fusion of TRPC3-containing vesicles with the plasma membrane without disrupting SNARE interactions. Importantly, these data agree with the known role of DAG in membrane destabilization, which facilitates SNARE-dependent synaptic vesicle fusion ( Villar, A. V., Goni, F. M., and Alonso, A. (2001) FEBS Lett. 494, 117-120 and Goni, F. M., and Alonso, A. (1999) Prog. Lipid Res. 38, 1-48 ). Taken together, functional models generated by GDDA-BLAST provide a computational platform for deriving domain functionality, which can have in vivo and mechanistic relevance.

Conserved GXXXG- and S/T-Like Motifs in the Transmembrane Domains of NS4B Protein Are Required for Hepatitis C Virus Replication

ABSTRACT Hepatitis C virus (HCV) nonstructural protein 4B (NS4B) is an integral membrane protein, which plays an important role in the organization and function of the HCV replication complex (RC). Although much is understood about its amphipathic N-terminal and C-terminal domains, we know very little about the role of the transmembrane domains (TMDs) in NS4B function. We hypothesized that in addition to anchoring NS4B into host membranes, the TMDs are engaged in intra- and intermolecular interactions required for NS4B structure/function. To test this hypothesis, we have engineered a chimeric JFH1 genome containing the Con1 NS4B TMD region. The resulting virus titers were greatly reduced from those of JFH1, and further analysis indicated a defect in genome replication. We have mapped this incompatibility to NS4B TMD1 and TMD2 sequences, and we have defined putative TMD dimerization motifs (GXXXG in TMD2 and TMD3; the S/T cluster in TMD1) as key structural/functional determinants. Mutations in each of the putative motifs led to significant decreases in JFH1 replication. Like most of the NS4B chimeras, mutant proteins had no negative impact on NS4B membrane association. However, some mutations led to disruption of NS4B foci, implying that the TMDs play a role in HCV RC formation. Further examination indicated that the loss of NS4B foci correlates with the destabilization of NS4B protein. Finally, we have identified an adaptive mutation in the NS4B TMD2 sequence that has compensatory effects on JFH1 chimera replication. Taken together, these data underscore the functional importance of NS4B TMDs in the HCV life cycle.