Alternative Splicing at N Terminus and Domain I Modulates CaV1.2 Inactivation and Surface Expression

The Ca_V1.2 L-type calcium channel is a key conduit for Ca²⁺ influx to initiate excitation-contraction coupling for contraction of the heart and vasoconstriction of the arteries and for altering membrane excitability in neurons. Its α_1C pore-forming subunit is known to undergo extensive alternative splicing to produce many Ca_V1.2 isoforms that differ in their electrophysiological and pharmacological properties. Here, we examined the structure-function relationship of human Ca_V1.2 with respect to the inclusion or exclusion of mutually exclusive exons of the N-terminus exons 1/1a and IS6 segment exons 8/8a. These exons showed tissue selectivity in their expression patterns: heart variant 1a/8a, one smooth-muscle variant 1/8, and a brain isoform 1/8a. Overall, the 1/8a, when coexpressed with Ca_Vβ_2a, displayed a significant and distinct shift in voltage-dependent activation and inactivation and inactivation kinetics as compared to the other three splice variants. Further analysis showed a clear additive effect of the hyperpolarization shift in V_1/2inact of Ca_V1.2 channels containing exon 1 in combination with 8a. However, this additive effect was less distinct for V_1/2act. However, the measured effects were β-subunit-dependent when comparing Ca_Vβ_2a with Ca_Vβ₃ coexpression. Notably, calcium-dependent inactivation mediated by local Ca²⁺-sensing via the N-lobe of calmodulin was significantly enhanced in exon-1-containing Ca_V1.2 as compared to exon-1a-containing Ca_V1.2 channels. At the cellular level, the current densities of the 1/8a or 1/8 variants were significantly larger than the 1a/8a and 1a/8 variants when coexpressed either with Ca_Vβ_2a or Ca_Vβ₃ subunit. This finding correlated well with a higher channel surface expression for the exon 1-Ca_V1.2 isoform that we quantified by protein surface-expression levels or by gating currents. Our data also provided a deeper molecular understanding of the altered biophysical properties of alternatively spliced human Ca_V1.2 channels by directly comparing unitary single-channel events with macroscopic whole-cell currents.     

The Leukocyte Nuclear Envelope Proteome Varies with Cell Activation and Contains Novel Transmembrane Proteins That Affect Genome Architecture

A favored hypothesis to explain the pathology underlying nuclear envelopathies is that mutations in nuclear envelope proteins alter genome/chromatin organization and thus gene expression. To identify nuclear envelope proteins that play roles in genome organization, we analyzed nuclear envelopes from resting and phytohemagglutinin-activated leukocytes because leukocytes have a particularly high density of peripheral chromatin that undergoes significant reorganization upon such activation. Thus, nuclear envelopes were isolated from leukocytes in the two states and analyzed by multidimensional protein identification technology using an approach that used expected contaminating membranes as subtractive fractions. A total of 3351 proteins were identified between both nuclear envelope data sets among which were 87 putative nuclear envelope transmembrane proteins (NETs) that were not identified in a previous proteomics analysis of liver nuclear envelopes. Nuclear envelope localization was confirmed for 11 new NETs using tagged fusion proteins and antibodies on spleen cryosections. 27% of the new proteins identified were unique to one or the other of the two leukocyte states. Differences in expression between activated and resting leukocytes were confirmed for some NETs by RT-PCR, and most of these proteins appear to only be expressed in certain types of blood cells. Several known proteins identified in both data sets have functions in chromatin organization and gene regulation. To test whether the novel NETs identified might include those that also regulate chromatin, nine were run through two screens for different chromatin effects. One screen found two NETs that can recruit a specific gene locus to the nuclear periphery, and the second found a different NET that promotes chromatin condensation. The variation in the protein milieu with pharmacological activation of the same cell population and consequences for gene regulation suggest that the nuclear envelope is a complex regulatory system with significant influences on genome organization.The nuclear envelope (NE)1 is a double membrane system consisting of the intermediate filament nuclear lamin polymer and associated proteins attached to the inner nuclear membrane (INM) (1), nuclear pore complexes (NPCs) that direct transport of soluble macromolecules in and out of the nucleus (2), and the outer nuclear membrane (ONM) and associated proteins. Structurally, the ONM is continuous with the endoplasmic reticulum (ER) and is studded with ribosomes (3), yet it also contains unique proteins, many of which connect the cytoskeleton to the NE (4). On the other side, lamins and many INM proteins directly connect chromatin to the NE. Lamins and an increasing number of nuclear envelope transmembrane proteins (NETs) have been linked to a similarly increasing number of diseases ranging from muscular dystrophy to neuropathy, dermopathy, lipodystrophy, bone disorders, and progeroid aging syndromes (5, 6).A favored hypothesis to explain how different NE proteins can produce such a wide range of disease pathologies is that chromatin-NE connections are disrupted with NE protein mutations, yielding changes in gene regulation. This hypothesis is supported by observations that the distribution of dense peripheral chromatin is affected in fibroblasts from patients with NE-linked muscular dystrophy, cardiomyopathy, mandibuloacral dysplasia, and progeria (7–10). Furthermore, many binding partners have been identified for NETs that are either chromatin proteins, enzymes that modify chromatin proteins, or regulators of gene expression (1, 11). These include markers of silent chromatin such as heterochromatin protein 1 (12) and proteins that modify chromatin to a silent conformation such as histone deacetylase 3 (13). The importance of the NE to global genome organization has been underscored by several recent studies that showed that affinity-based recruitment of a specific chromosome locus by the NE both pulled entire chromosomes to the periphery and affected gene regulation in complex ways (14–16).To identify NE proteins likely to be involved in genome organization, we turned to lymphocytes as a model system. Lymphocytes in the resting state tend to have massive amounts of dense peripheral chromatin as determined by electron microscopy studies. Upon activation with phytohemagglutinin, this dense chromatin largely dissipates as the cells actively express genes (17–20). Thus, to identify proteins that might be involved in tethering heterochromatin to the NE or in changing its organization, we analyzed the NE proteomes of leukocyte populations (∼70% lymphocytes) in both the resting and phytohemagglutinin (PHA)-activated states. The previously validated subtractive approach was applied (21) using microsomes and mitochondria, the principal membrane contaminants expected, as subtractive fractions.Many new NE proteins were identified that had not been identified in previous NE proteomics investigations using liver and neuroblastoma cells (21, 22). NE residence was confirmed for 12 novel NETs by expression of epitope-tagged versions and using antibodies on tissue cryosections.Roughly one-quarter of the proteins identified varied between the resting and activated states. Some NET differences between the two data sets were confirmed by RT-PCR. Among the known proteins identified were several that suggest that changes in NE composition associated with PHA activation contribute to gene regulation. Novel NETs identified also appear to play significant roles in genome organization/regulation as we found that several can either recruit a specific locus to the nuclear periphery or promote chromatin condensation. As several studies have implicated misregulation of chromatin organization in NE diseases (7, 8), these newly identified NETs may contribute to the diverse pathologies associated with NE diseases.     

A CACNA1C Variant Associated with Reduced Voltage-Dependent Inactivation,Increased CaV1.2 Channel Window Current,and Arrhythmogenesis

Mutations in CACNA1C that increase current through the Ca_V1.2 L-type Ca²⁺ channel underlie rare forms of long QT syndrome (LQTS), and Timothy syndrome (TS). We identified a variant in CACNA1C in a male child of Filipino descent with arrhythmias and extracardiac features by candidate gene sequencing and performed functional expression studies to electrophysiologically characterize the effects of the variant on Ca_V1.2 channels. As a baby, the subject developed seizures and displayed developmental delays at 30 months of age. At age 5 years, he displayed a QTc of 520 ms and experienced recurrent VT. Physical exam at 17 years of age was notable for microcephaly, short stature, lower extremity weakness and atrophy with hyperreflexia, spastic diplegia, multiple dental caries and episodes of rhabdomyolysis. Candidate gene sequencing identified a G>C transversion at position 5731 of CACNA1C (rs374528680) predicting a glycine>arginine substitution at residue 1911 (p.G1911R) of Ca_V1.2. The allele frequency of this variant is 0.01 in Malays, but absent in 984 Caucasian alleles and in the 1000 genomes project. In electrophysiological analyses, the variant decreased voltage-dependent inactivation, thus causing a gain of function of Ca_V1.2. We also observed a negative shift of V_1/2 of activation and positive shift of V_1/2 of channel inactivation, resulting in an increase of the window current. Together, these suggest a gain-of-function effect on Ca_V1.2 and suggest increased susceptibility for arrhythmias in certain clinical settings. The p.G1911R variant was also identified in a case of sudden unexplained infant death (SUID), for which an increasing number of clinical observations have demonstrated can be associated with arrhythmogenic mutations in cardiac ion channels. In summary, the combined effects of the CACNA1C variant to diminish voltage-dependent inactivation of Ca_V1.2 and increase window current expand our appreciation of mechanisms by which a gain of function of Ca_V1.2 can contribute to QT prolongation.     

ADAMTSL-6 Is a Novel Extracellular Matrix Protein That Binds to Fibrillin-1 and Promotes Fibrillin-1 Fibril Formation

ADAMTS (A disintegrin and metalloproteinase with thrombospondin motifs)-like (ADAMTSL) proteins, a subgroup of the ADAMTS superfamily, share several domains with ADAMTS proteinases, including thrombospondin type I repeats, a cysteine-rich domain, and an ADAMTS spacer, but lack a catalytic domain. We identified two new members of ADAMTSL proteins, ADAMTSL-6α and -6β, that differ in their N-terminal amino acid sequences but have common C-terminal regions. When transfected into MG63 osteosarcoma cells, both isoforms were secreted and deposited into pericellular matrices, although ADAMTSL-6α, in contrast to -6β, was barely detectable in the conditioned medium. Immunolabeling at the light and electron microscopic levels showed their close association with fibrillin-1-rich microfibrils in elastic connective tissues. Surface plasmon resonance analyses demonstrated that ADAMTSL-6β binds to the N-terminal half of fibrillin-1 with a dissociation constant of ∼80 nm. When MG63 cells were transfected or exogenously supplemented with ADAMTSL-6, fibrillin-1 matrix assembly was promoted in the early but not the late stage of the assembly process. Furthermore, ADAMTSL-6 transgenic mice exhibited excessive fibrillin-1 fibril formation in tissues where ADAMTSL-6 was overexpressed. All together, these results indicated that ADAMTSL-6 is a novel microfibril-associated protein that binds directly to fibrillin-1 and promotes fibrillin-1 matrix assembly.     

Coupled and Independent Contributions of Residues in IS6 and IIS6 to Activation Gating of CaV1.2

Voltage dependence and kinetics of Ca_V1.2 activation are affected by structural changes in pore-lining S6 segments of the α₁-subunit. Significant effects are induced by either proline or threonine substitutions in the lower third of segment IIS6 (“bundle crossing region”), where S6 segments are likely to seal the channel in the closed conformation (Hohaus, A., Beyl, S., Kudrnac, M., Berjukow, S., Timin, E. N., Marksteiner, R., Maw, M. A., and Hering, S. (2005) J. Biol. Chem. 280, 38471–38477). Here we report that S435P in IS6 results in a large shift of the activation curve (-25.9 ± 1.2 mV) and slower current kinetics. Threonine substitutions at positions Leu-429 and Leu-434 induced a similar kinetic phenotype with shifted activation curves (L429T by -6.6 ± 1.2 and L434T by -12.1 ± 1.7 mV). Inactivation curves of all mutants were shifted to comparable extents as the activation curves. Interdependence of IS6 and IIS6 mutations was analyzed by means of mutant cycle analysis. Double mutations in segments IS6 and IIS6 induce either additive (L429T/I781T, -34.1 ± 1.4 mV; L434T/I781T, -40.4 ± 1.3 mV; L429T/L779T, -12.6 ± 1.3 mV; and L434T/L779T, -22.4 ± 1.3 mV) or nonadditive shifts of the activation curves along the voltage axis (S435P/I781T, -33.8 ± 1.4 mV). Mutant cycle analysis revealed energetic coupling between residues Ser-435 and Ile-781, whereas other paired mutations in segments IS6 and IIS6 had independent effects on activation gating.Ca²⁺ current through Ca_V1.2 channels initiates muscle contraction, release of hormones and neurotransmitters, and affects physiological processes such as vision, hearing, and gene expression (1). Their pore-forming α₁-subunit is composed of four homologous domains formed by six transmembrane segments (S1–S6) (2). The signal of the voltage-sensing machinery, consisting of multiple charged amino acids (located in segments S4 and adjacent structures of each domain), is transmitted to the pore region (3). Conformational changes in pore lining S6 and adjacent segments finally lead to pore openings (activation) and closures (inactivation).Our understanding of how Ca_V1.2 channels open and close is largely based on extrapolations of structural information from potassium channels. The crystal structures of the closed conformation of two bacterial potassium channels (KcsA and MlotiK) (4, 5) show a gate located at the intracellular channel mouth formed by tightly packed S6 helices. The crystal structure of the open conformation of Kv1.2 (6, 7) revealed a bent S6 with the highly conserved PXP motif apparently acting as a hinge (see 8). The activation mechanism proposed for MthK channels involves helix bending at a highly conserved glycine at position 83 (see Ref. 9, “glycine gating hinge” hypothesis).Compared with potassium channels, the pore of Ca_V is asymmetric, and none of the four S6 segments has a putative helix-bending PXP motif. Furthermore, the conserved glycine (corresponding to position 83 in MthK, see Ref. 10) is only present in segments IS6 and IIS6 (for review see Ref. 11). We have shown that substituting proline for this glycine in IIS6 of Ca_V1.2 does not significantly affect gating (12).Zhen et al. (13) investigated the pore lining S6 segments of Ca_V2.1 using the substituted cysteine accessibility method. The accessibility of cysteines was changed by opening and closing the channel, consistent with the gate being on the intracellular side. The general picture of a channel gate close to the inner channel mouth of Ca_V1.2 was recently supported by pharmacological studies (14).Substitution of hydrophilic residues in the lower third of segment IIS6 of Ca_V1.2 (LAIA motif, 779–784, see Ref. 12) induces pronounced changes in channel gating as follows: a shift in the voltage dependence of activation accompanied by a slowing of the activation kinetics near the footstep of the m_∞(V) curve and a slowing of deactivation at all potentials. Interestingly, these changes in channel gating resemble the effects of proline substitution of Gly-219 in the bacterial sodium channel from Bacillus halodurans (“Gly-219 gating hinge,” see Ref. 15).The strongest shifts of the activation curve reported so far were observed for proline substitutions (12). As prolines in an α-helix cause a rigid kink with an angle of about 26° (16), we hypothesized that these mutants were causing a kink in helix IIS6 similar to a bend that would normally occur flexibly during the activation process (12).Here we extend our previous study by systematically substituting residues in segment IS6 of Ca_V1.2 by proline or the small and polar threonine. Several functional IS6 mutants with shifted activation and inactivation characteristics were identified (S435P, L429T, and L434T), and the interdependence of IS6 and IIS6 mutations was analyzed. Mutant cycle analysis revealed both mutually independent and energetically coupled contributions of IS6 and IIS6 residues on activation gating.     

Screening and Characterization of a Novel RNA Aptamer That Specifically Binds to Human Prostatic Acid Phosphatase and Human Prostate Cancer Cells

Prostatic acid phosphatase (PAP) expression increases proportionally with prostate cancer progression, making it useful in prognosticating intermediate to high-risk prostate cancers. A novel ligand that can specifically bind to PAP would be very helpful for guiding prostate cancer therapy. RNA aptamers bind to target molecules with high specificity and have key advantages such as low immunogenicity and easy synthesis. Here, human PAP-specific aptamers were screened from a 2′-fluoropyrimidine (FY)-modified RNA library by SELEX. The candidate aptamer families were identified within six rounds followed by analysis of their sequences and PAP-specific binding. A gel shift assay was used to identify PAP binding aptamers and the 6N aptamer specifically bound to PAP with a Kd value of 118 nM. RT-PCR and fluorescence labeling analyses revealed that the 6N aptamer bound to PAP-positive mammalian cells, such as PC-3 and LNCaP. IMR-90 negative control cells did not bind the 6N aptamer. Systematic minimization analyses revealed that 50 nucleotide sequences and their two hairpin structures in the 6N 2′-FY RNA aptamer were equally important for PAP binding. Renewed interest in PAP combined with the versatility of RNA aptamers, including conjugation of anti-cancer drugs and nano-imaging probes, could open up a new route for early theragnosis of prostate cancer.     

BLyS对噬菌体随机12肽库的筛选

用B淋巴细胞刺激因子（BLyS）对噬菌体随机12肽库进行亲和淘洗,3轮筛选后阳性噬菌体得到富集。用ELISA鉴定噬菌体克隆,多个阳性克隆测序后得到了同一个小肽序列(RHKIQLRQNIIT)。将该小肽与GST融合,在大肠杆菌中进行表达及纯化,ELISA实验进一步验证了其具有与BLyS特异结合的活性。该小肽有可能成为其天然受体的拮抗剂。      

Isolation, Characterization, and Molecular Cloning of a Protein (Abp2) That Binds to a Schizosaccharomyces pombe Origin of Replication (ars3002)

The autonomously replicating sequence (ARS) element ars3002 is associated with the most active replication origin within a cluster of three closely spaced origins on chromosome III of Schizosaccharomyces pombe. A 361-bp portion of ars3002 containing detectable ARS activity includes multiple near matches to the S. pombe ARS consensus sequence previously reported by Maundrell et al. (K. Maundrell, A. Hutchison, and S. Shall, EMBO J. 7:2203–2209, 1988). Using a gel shift assay with a multimer of an oligonucleotide containing three overlapping matches to the Maundrell ARS consensus sequence, we have detected several proteins in S. pombe crude extracts that bind to the oligonucleotide and ars3002. One of these proteins, ARS binding protein 1, was previously described (Abp1 [Y. Murakami, J. A. Huberman, and J. Hurwitz, Proc. Natl. Acad. Sci. USA 93:502–507, 1996]). In this report the isolation, characterization, and cloning of a second binding activity, designated ARS binding protein 2 (Abp2), are described. Purified Abp2 has an apparent molecular mass of 75 kDa. Footprinting analyses revealed that it binds preferentially to overlapping near matches to the Maundrell ARS consensus sequence. The gene abp2 was isolated, sequenced, and overexpressed in Escherichia coli. The DNA binding activity of overexpressed Abp2 was similar to that of native Abp2. The deduced amino acid sequence contains a region similar to a proline-rich motif (GRP) present in several proteins that bind A+T-rich DNA sequences. Replacement of amino acids within this motif with alanine either abolished or markedly reduced the DNA binding activity of the mutated Abp2 protein, indicating that this motif is essential for the DNA binding activity of Abp2. Disruption of the abp2 gene showed that the gene is not essential for cell viability. However, at elevated temperatures the null mutant was less viable than the wild type and exhibited changes in nuclear morphology. The null mutant entered mitosis with delayed kinetics when DNA replication was blocked with hydroxyurea, and advancement through mitosis led to the loss of cell viability and aberrant formation of septa. The null mutant was also sensitive to UV radiation, suggesting that Abp2 may play a role in regulating the cell cycle response to stress signals.     

A Bacteriophage Capsid Protein Provides a General Amyloid Interaction Motif (GAIM) That Binds and Remodels Misfolded Protein Assemblies

Misfolded protein aggregates, characterized by a canonical amyloid fold, play a central role in the pathobiology of neurodegenerative diseases. Agents that bind and sequester neurotoxic intermediates of amyloid assembly, inhibit the assembly or promote the destabilization of such protein aggregates are in clinical testing. Here, we show that the gene 3 protein (g3p) of filamentous bacteriophage mediates potent generic binding to the amyloid fold. We have characterized the amyloid binding and conformational remodeling activities using an array of techniques, including X-ray fiber diffraction and NMR. The mechanism for g3p binding with amyloid appears to reflect its physiological role during infection of Escherichia coli, which is dependent on temperature-sensitive interdomain unfolding and cis–trans prolyl isomerization of g3p. In addition, a natural receptor for g3p, TolA-C, competitively interferes with Aβ binding to g3p. NMR studies show that g3p binding to Aβ fibers is predominantly through middle and C-terminal residues of the Aβ subunit, indicating β strand–g3p interactions. A recombinant bivalent g3p molecule, an immunoglobulin Fc (Ig) fusion of the two N-terminal g3p domains, (1) potently binds Aβ fibers (fAβ) (K_D = 9.4 nM); (2); blocks fAβ assembly (IC₅₀ ~ 50 nM) and (3) dissociates fAβ (EC₅₀ = 40–100 nM). The binding of g3p to misfolded protein assemblies is generic, and amyloid-targeted activities can be demonstrated using other misfolded protein systems. Taken together, our studies show that g3p(N1N2) acts as a general amyloid interaction motif.     

A Bacillus anthracis S-Layer Homology Protein That Binds Heme and Mediates Heme Delivery to IsdC

The sequestration of iron by mammalian hosts represents a significant obstacle to the establishment of a bacterial infection. In response, pathogenic bacteria have evolved mechanisms to acquire iron from host heme. Bacillus anthracis, the causative agent of anthrax, utilizes secreted hemophores to scavenge heme from host hemoglobin, thereby facilitating iron acquisition from extracellular heme pools and delivery to iron-regulated surface determinant (Isd) proteins covalently attached to the cell wall. However, several Gram-positive pathogens, including B. anthracis, contain genes that encode near iron transporter (NEAT) proteins that are genomically distant from the genetically linked Isd locus. NEAT domains are protein modules that partake in several functions related to heme transport, including binding heme and hemoglobin. This finding raises interesting questions concerning the relative role of these NEAT proteins, relative to hemophores and the Isd system, in iron uptake. Here, we present evidence that a B. anthracis S-layer homology (SLH) protein harboring a NEAT domain binds and directionally transfers heme to the Isd system via the cell wall protein IsdC. This finding suggests that the Isd system can receive heme from multiple inputs and may reflect an adaptation of B. anthracis to changing iron reservoirs during an infection. Understanding the mechanism of heme uptake in pathogenic bacteria is important for the development of novel therapeutics to prevent and treat bacterial infections.Pathogenic bacteria need to acquire iron to survive in mammalian hosts (12). However, the host sequesters most iron in the porphyrin heme, and heme itself is often bound to proteins such as hemoglobin (14, 28, 85). Circulating hemoglobin can serve as a source of heme-iron for replicating bacteria in infected hosts, but the precise mechanisms of heme extraction, transport, and assimilation remain unclear (25, 46, 79, 86). An understanding of how bacterial pathogens import heme will lead to the development of new anti-infectives that inhibit heme uptake, thereby preventing or treating infections caused by these bacteria (47, 68).The mechanisms of transport of biological molecules into a bacterial cell are influenced by the compositional, structural, and topological makeup of the cell envelope. Gram-negative bacteria utilize specific proteins to transport heme through the outer membrane, periplasm, and inner membrane (83, 84). Instead of an outer membrane and periplasm, Gram-positive bacteria contain a thick cell wall (59, 60). Proteins covalently anchored to the cell wall provide a functional link between extracellular heme reservoirs and intracellular iron utilization pathways (46). In addition, several Gram-positive and Gram-negative bacterial genera also contain an outermost structure termed the S (surface)-layer (75). The S-layer is a crystalline array of protein that surrounds the bacterial cell and may serve a multitude of functions, including maintenance of cell architecture and protection from host immune components (6, 7, 18, 19, 56). In bacterial pathogens that manifest an S-layer, the “force field” function of this structure raises questions concerning how small molecules such as heme can be successfully passed from the extracellular milieu to cell wall proteins for delivery into the cell cytoplasm.Bacillus anthracis is a Gram-positive, spore-forming bacterium that is the etiological agent of anthrax disease (30, 33). The life cycle of B. anthracis begins after a phagocytosed spore germinates into a vegetative cell inside a mammalian host (2, 40, 69, 78). Virulence determinants produced by the vegetative cells facilitate bacterial growth, dissemination to major organ systems, and eventually host death (76-78). The release of aerosolized spores into areas with large concentrations of people is a serious public health concern (30).Heme acquisition in B. anthracis is mediated by the action of IsdX1 and IsdX2, two extracellular hemophores that extract heme from host hemoglobin and deliver the iron-porphyrin to cell wall-localized IsdC (21, 45). Both IsdX1 and IsdX2 harbor near iron transporter domains (NEATs), a conserved protein module found in Gram-positive bacteria that mediates heme uptake from hemoglobin and contributes to bacterial pathogenesis upon infection (3, 8, 21, 31, 44, 46, 49, 50, 67, 81, 86). Hypothesizing that B. anthracis may contain additional mechanisms for heme transport, we provide evidence that B. anthracis S-layer protein K (BslK), an S-layer homology (SLH) and NEAT protein (32, 43), is surface localized and binds and transfers heme to IsdC in a rapid, contact-dependent manner. These results suggest that the Isd system is not a self-contained conduit for heme trafficking and imply that there is functional cross talk between differentially localized NEAT proteins to promote heme uptake during infection.     

Cyclin E2, a Novel G1 Cyclin That Binds Cdk2 and Is Aberrantly Expressed in Human Cancers

A novel cyclin gene was discovered by searching an expressed sequence tag database with a cyclin box profile. The human cyclin E2 gene encodes a 404-amino-acid protein that is most closely related to cyclin E. Cyclin E2 associates with Cdk2 in a functional kinase complex that is inhibited by both p27^Kip1 and p21^Cip1. The catalytic activity associated with cyclin E2 complexes is cell cycle regulated and peaks at the G₁/S transition. Overexpression of cyclin E2 in mammalian cells accelerates G₁, demonstrating that cyclin E2 may be rate limiting for G₁ progression. Unlike cyclin E1, which is expressed in most proliferating normal and tumor cells, cyclin E2 levels were low to undetectable in nontransformed cells and increased significantly in tumor-derived cells. The discovery of a novel second cyclin E family member suggests that multiple unique cyclin E-CDK complexes regulate cell cycle progression.     

Role of the C terminus of the alpha 1C (CaV1.2) subunit in membrane targeting of cardiac L-type calcium channels

We have previously demonstrated that formation of a complex between L-type calcium (Ca(2+)) channel alpha(1C) (Ca(V)1.2) and beta subunits was necessary to target the channels to the plasma membrane when expressed in tsA201 cells. In the present study, we identified a region in the C terminus of the alpha(1C) subunit that was required for membrane targeting. Using a series of C-terminal deletion mutants of the alpha(1C) subunit, a domain consisting of amino acid residues 1623-1666 ("targeting domain") in the C terminus of the alpha(1C) subunit has been identified to be important for correct targeting of L-type Ca(2+) channel complexes to the plasma membrane. Although cells expressing the wild-type alpha(1C) and beta(2a) subunits exhibited punctate clusters of channel complexes along the plasma membrane with little intracellular staining, co-expression of deletion mutants of the alpha(1C) subunit that lack the targeting domain with the beta(2a) subunit resulted in an intracellular localization of the channels. In addition, three other regions in the C terminus of the alpha(1C) subunit that were downstream of residues 1623-1666 were found to contribute to membrane targeting of the L-type channels. Deletion of these domains in the alpha(1C) subunit resulted in a reduction of plasma membrane-localized channels, and a concomitant increase in channels localized intracellularly. Taken together, these results have demonstrated that a targeting domain in the C terminus of the alpha(1C) subunit was required for proper plasma membrane localization of the L-type Ca(2+) channels.     

The Voltage-dependent Anion Channel (VDAC) Binds Tissue-type Plasminogen Activator and Promotes Activation of Plasminogen on the Cell Surface

The voltage-dependent anion channel (VDAC), a major pore-forming protein in the outer membrane of mitochondria, is also found in the plasma membrane of a large number of cells where in addition to its role in regulating cellular ATP release and volume control it is important for maintaining redox homeostasis. Cell surface VDAC is a receptor for plasminogen kringle 5, which promotes partial closure of the channel. In this study, we demonstrate that VDAC binds tissue-type plasminogen activator (t-PA) on human neuroblastoma SK-N-SH cells. Binding of t-PA to VDAC induced a decrease in K_m and an increase in the V_max for activation of its substrate, plasminogen (Pg). This resulted in accelerated Pg activation when VDAC, t-PA, and Pg were bound together. VDAC is also a substrate for plasmin; hence, it mimics fibrin activity. Binding of t-PA to VDAC occurs between a t-PA fibronectin type I finger domain located between amino acids Ile⁵ and Asn³⁷ and a VDAC region including amino acids ²⁰GYGFG²⁴. These VDAC residues correspond to a GXXXG repeat motif commonly found in amyloid β peptides that is necessary for aggregation when these peptides form fibrillar deposits on the cell surface. Furthermore, we also show that Pg kringle 5 is a substrate for the NADH-dependent reductase activity of VDAC. This ternary complex is an efficient proteolytic complex that may facilitate removal of amyloid β peptide deposits from the normal brain and cell debris from injured brain tissue.     

C-terminal fragments of the alpha 1C (CaV1.2) subunit associate with and regulate L-type calcium channels containing C-terminal-truncated alpha 1C subunits

L-type Ca(2+) channels in native tissues have been found to contain a pore-forming alpha(1) subunit that is often truncated at the C terminus. However, the C terminus contains many important domains that regulate channel function. To test the hypothesis that C-terminal fragments may associate with and regulate C-terminal-truncated alpha(1C) (Ca(V)1.2) subunits, we performed electrophysiological and biochemical experiments. In tsA201 cells expressing either wild type or C-terminal-truncated alpha(1C) subunits in combination with a beta(2a) subunit, truncation of the alpha(1C) subunit by as little as 147 amino acids led to a 10-15-fold increase in currents compared with those obtained from control, full-length alpha(1C) subunits. Dialysis of cells expressing the truncated alpha(1C) subunits with C-terminal fragments applied through the patch pipette reconstituted the inhibition of the channels seen with full-length alpha(1C) subunits. In addition, C-terminal deletion mutants containing a tethered C terminus also exhibited the C-terminal-induced inhibition. Immunoprecipitation assays demonstrated the association of the C-terminal fragments with truncated alpha(1C) subunits. In addition, glutathione S-transferase pull-down assays demonstrated that the C-terminal inhibitory fragment could associate with at least two domains within the C terminus. The results support the hypothesis the C- terminal fragments of the alpha(1C) subunit can associate with C-terminal-truncated alpha(1C) subunits and inhibit the currents through L-type Ca(2+) channels.