Cystathionine Is a Novel Substrate of Cystine/Glutamate Transporter: IMPLICATIONS FOR IMMUNE FUNCTION*

The cystine/glutamate transporter, designated as system x_c⁻, is important for maintaining intracellular glutathione levels and extracellular redox balance. The substrate-specific component of system x_c⁻, xCT, is strongly induced by various stimuli, including oxidative stress, whereas it is constitutively expressed only in specific brain regions and immune tissues, such as the thymus and spleen. Although cystine and glutamate are the well established substrates of system x_c⁻ and the knockout of xCT leads to alterations of extracellular redox balance, nothing is known about other potential substrates. We thus performed a comparative metabolite analysis of tissues from xCT-deficient and wild-type mice using capillary electrophoresis time-of-flight mass spectrometry. Although most of the analyzed metabolites did not show significant alterations between xCT-deficient and wild-type mice, cystathionine emerged as being absent specifically in the thymus and spleen of xCT-deficient mice. No expression of either cystathionine β-synthase or cystathionine γ-lyase was observed in the thymus and spleen of mice. In embryonic fibroblasts derived from wild-type embryos, cystine uptake was significantly inhibited by cystathionine in a concentration-dependent manner. Wild-type cells showed an intracellular accumulation of cystathionine when incubated in cystathionine-containing buffer, which concomitantly stimulated an increased release of glutamate into the extracellular space. By contrast, none of these effects could be observed in xCT-deficient cells. Remarkably, unlike knock-out cells, wild-type cells could be rescued from cystine deprivation-induced cell death by cystathionine supplementation. We thus conclude that cystathionine is a novel physiological substrate of system x_c⁻ and that the accumulation of cystathionine in immune tissues is exclusively mediated by system x_c⁻.     

An Integrated Approach for Analysis of the DNA Damage Response in Mammalian Cells: NUCLEOTIDE EXCISION REPAIR,DNA DAMAGE CHECKPOINT,AND APOPTOSIS*

DNA damage by UV and UV-mimetic agents elicits a set of inter-related responses in mammalian cells, including DNA repair, DNA damage checkpoints, and apoptosis. Conventionally, these responses are analyzed separately using different methodologies. Here we describe a unified approach that is capable of quantifying all three responses in parallel using lysates from the same population of cells. We show that a highly sensitive in vivo excision repair assay is capable of detecting nucleotide excision repair of a wide spectrum of DNA lesions (UV damage, chemical carcinogens, and chemotherapeutic drugs) within minutes of damage induction. This method therefore allows for a real-time measure of nucleotide excision repair activity that can be monitored in conjunction with other components of the DNA damage response, including DNA damage checkpoint and apoptotic signaling. This approach therefore provides a convenient and reliable platform for simultaneously examining multiple aspects of the DNA damage response in a single population of cells that can be applied for a diverse array of carcinogenic and chemotherapeutic agents.     

An Immunomodulating Motif of the HIV-1 Fusion Protein Is Chirality-independent: IMPLICATIONS FOR ITS MODE OF ACTION*

An immunosuppressive motif was recently found within the HIV-1 gp41 fusion protein (termed immunosuppressive loop-associated determinant core motif (ISLAD CM)). Peptides containing the motif interact with the T-cell receptor (TCR) complex; however, the mechanism by which the motif exerts its immunosuppressive activity is yet to be determined. Recent studies showed that interactions between protein domains in the membrane milieu are not always sterically controlled. Therefore, we utilized the unique membrane leniency toward association between d- and l-stereoisomers to investigate the detailed mechanism by which ISLAD CM inhibits T-cell activation. We show that a d-enantiomer of ISLAD CM (termed ISLAD d-CM) inhibited the proliferation of murine myelin oligodendrocyte glycoprotein (MOG)-(35–55)-specific line T-cells to the same extent as the l-motif form. Moreover, the d- and l-forms preferentially bound spleen-derived T-cells over B-cells by 13-fold. Furthermore, both forms of ISLAD CM co-localized with the TCR on activated T-cells and interacted with the transmembrane domain of the TCR. FRET experiments revealed the importance of basic residues for the interaction between ISLAD CM forms and the TCR transmembrane domain. Ex vivo studies demonstrated that ISLAD d-CM administration inhibited the proliferation (72%) and proinflammatory cytokine secretion of pathogenic MOG(35–55)-specific T-cells. This study provides insights into the immunosuppressive mechanism of gp41 and demonstrates that chirality-independent interactions in the membrane can take place in diverse biological systems. Apart from HIV pathogenesis, the d-peptide reported herein may serve as a potential tool for treating T-cell-mediated pathologies.     

Stress-induced Condensation of Bacterial Genomes Results in Re-pairing of Sister Chromosomes: IMPLICATIONS FOR DOUBLE STRAND DNA BREAK REPAIR*

Genome condensation is increasingly recognized as a generic stress response in bacteria. To better understand the physiological implications of this response, we used fluorescent markers to locate specific sites on Escherichia coli chromosomes following exposure to cytotoxic stress. We find that stress-induced condensation proceeds through a nonrandom, zipper-like convergence of sister chromosomes, which is proposed to rely on the recently demonstrated intrinsic ability of identical double-stranded DNA molecules to specifically identify each other. We further show that this convergence culminates in spatial proximity of homologous sites throughout chromosome arms. We suggest that the resulting apposition of homologous sites can explain how repair of double strand DNA breaks might occur in a mechanism that is independent of the widely accepted yet physiologically improbable genome-wide search for homologous templates. We claim that by inducing genome condensation and orderly convergence of sister chromosomes, diverse stress conditions prime bacteria to effectively cope with severe DNA lesions such as double strand DNA breaks.     

Structure of Shigella IpgB2 in Complex with Human RhoA: IMPLICATIONS FOR THE MECHANISM OF BACTERIAL GUANINE NUCLEOTIDE EXCHANGE FACTOR MIMICRY*

A common theme in bacterial pathogenesis is the manipulation of eukaryotic cells by targeting the cytoskeleton. This is in most cases achieved either by modifying actin, or indirectly via activation of key regulators controlling actin dynamics such as Rho-GTPases. A novel group of bacterial virulence factors termed the WXXXE family has emerged as guanine nucleotide exchange factors (GEFs) for these GTPases. The precise mechanism of nucleotide exchange, however, has remained unclear. Here we report the structure of the WXXXE-protein IpgB2 from Shigella flexneri and its complex with human RhoA. We unambiguously identify IpgB2 as a bacterial RhoA-GEF and dissect the molecular mechanism of GDP release, an essential prerequisite for GTP binding. Our observations uncover that IpgB2 induces conformational changes on RhoA mimicking DbI- but not DOCK family GEFs. We also show that dissociation of the GDP·Mg²⁺ complex is preceded by the displacement of the metal ion to the α-phosphate of the nucleotide, diminishing its affinity to the GTPase. These data refine our understanding of the mode of action not only of WXXXE GEFs but also of mammalian GEFs of the DH/PH family.     

NUCLEOTIDE SEQUENCES OF THE SMALL-SUBUNIT RIBOSOMAL RNA GENES FROM SELECTED LAMINARIALES (PHAEOPHYTA): IMPLICATIONS FOR KELP EVOLUTION1

The entire small-subunit (SSU) ribosomal ribonucleic acid sequence was inferred for kelp representing seven genera: Alaria marginata Postels and Ruprecht (1824 bp), Egregia menziesii (Turner) Areschoug (1825 bp), Lessoniopsis littoralis (Tilden) Reinke (1825 bp), Macrocystis integrifolia Bory (1825 bp), Nereocystis leutkeana (Mertens) Postels and Ruprecht (1824 bp), Postelsia palmaeformis Ruprecht (1826 bp), and Pterygophora californica Ruprecht (1825 bp). We obtained a partial sequence for Eisenia arborea Areschoug (1669 bp) from a single clone of polymerase chain reaction-amplified product. The SSU sequence was too conserved among these morphologically distinct taxa to permit phylogenetic analysis. The divergence between the most distant taxa was only 0.66%. This value was used in a SSU molecular clock to suggest that the most distantly related kelp investigated in this study diverged between 16 and 30 (more probably 16 and 20) million years ago.     

PLK1是DNA内切酶CtIP的新相互作用蛋白

CtIP是DNA双链断裂修复中的关键蛋白之一,它能够促进断裂DNA末端切割,并且是一种已知的抑癌基因,与许多参与癌变过程的蛋白质如BRCA1,Rb等相互作用。为了更好地理解CtIP的分子网络,我们用在线工具PrePPI预测CtIP相互作用蛋白,发现PLK1是新的CtIP相互作用蛋白。PLK1在有丝分裂和癌症进展中发挥重要作用。我们进一步通过免疫沉淀法验证了它们的相互作用。 结果显示PLK1与CtIP有较强相互作用。此外,还采用Frodock 2.0工具对接CtIP和PLK1之间的蛋白质相互作用。最后,免疫沉淀测定和免疫荧光染色结果显示这两种蛋白质之间的相互作用与DNA损伤相关。基于这些结果,我们提出CtIP-PLK1相互作用可能在DNA损伤反应以及其他生物过程中发挥重要作用。     

Characterization of the Stability and Bio-functionality of Tethered Proteins on Bioengineered Scaffolds: IMPLICATIONS FOR STEM CELL BIOLOGY AND TISSUE REPAIR*

Various engineering applications have been utilized to deliver molecules and compounds in both innate and biological settings. In the context of biological applications, the timely delivery of molecules can be critical for cellular and organ function. As such, previous studies have demonstrated the superiority of long-term protein delivery, by way of protein tethering onto bioengineered scaffolds, compared with conventional delivery of soluble protein in vitro and in vivo. Despite such benefits little knowledge exists regarding the stability, release kinetics, longevity, activation of intracellular pathway, and functionality of these proteins over time. By way of example, here we examined the stability, degradation and functionality of a protein, glial-derived neurotrophic factor (GDNF), which is known to influence neuronal survival, differentiation, and neurite morphogenesis. Enzyme-linked immunosorbent assays (ELISA) revealed that GDNF, covalently tethered onto polycaprolactone (PCL) electrospun nanofibrous scaffolds, remained present on the scaffold surface for 120 days, with no evidence of protein leaching or degradation. The tethered GDNF protein remained functional and capable of activating downstream signaling cascades, as revealed by its capacity to phosphorylate intracellular Erk in a neural cell line. Furthermore, immobilization of GDNF protein promoted cell survival and differentiation in culture at both 3 and 7 days, further validating prolonged functionality of the protein, well beyond the minutes to hours timeframe observed for soluble proteins under the same culture conditions. This study provides important evidence of the stability and functionality kinetics of tethered molecules.     

Novel Zn2+-binding Sites in Human Transthyretin: IMPLICATIONS FOR AMYLOIDOGENESIS AND RETINOL-BINDING PROTEIN RECOGNITION*

Human transthyretin (TTR) is a homotetrameric protein involved in several amyloidoses. Zn²⁺ enhances TTR aggregation in vitro, and is a component of ex vivo TTR amyloid fibrils. We report the first crystal structure of human TTR in complex with Zn²⁺ at pH 4.6–7.5. All four structures reveal three tetra-coordinated Zn²⁺-binding sites (ZBS 1–3) per monomer, plus a fourth site (ZBS 4) involving amino acid residues from a symmetry-related tetramer that is not visible in solution by NMR. Zn²⁺ binding perturbs loop E-α-helix-loop F, the region involved in holo-retinol-binding protein (holo-RBP) recognition, mainly at acidic pH; TTR affinity for holo-RBP decreases ∼5-fold in the presence of Zn²⁺. Interestingly, this same region is disrupted in the crystal structure of the amyloidogenic intermediate of TTR formed at acidic pH in the absence of Zn²⁺. HNCO and HNCA experiments performed in solution at pH 7.5 revealed that upon Zn²⁺ binding, although the α-helix persists, there are perturbations in the resonances of the residues that flank this region, suggesting an increase in structural flexibility. While stability of the monomer of TTR decreases in the presence of Zn²⁺, which is consistent with the tertiary structural perturbation provoked by Zn²⁺ binding, tetramer stability is only marginally affected by Zn²⁺. These data highlight structural and functional roles of Zn²⁺ in TTR-related amyloidoses, as well as in holo-RBP recognition and vitamin A homeostasis.     

Sarcoglycan Complex: IMPLICATIONS FOR METABOLIC DEFECTS IN MUSCULAR DYSTROPHIES*

The sarcoglycans are known as an integral subcomplex of the dystrophin glycoprotein complex, the function of which is best characterized in skeletal muscle in relation to muscular dystrophies. Here we demonstrate that the white adipocytes, which share a common precursor with the myocytes, express a cell-specific sarcoglycan complex containing β-, δ-, and ϵ-sarcoglycan. In addition, the adipose sarcoglycan complex associates with sarcospan and laminin binding dystroglycan. Using multiple sarcoglycan null mouse models, we show that loss of α-sarcoglycan has no consequence on the expression of the adipocyte sarcoglycan complex. However, loss of β- or δ-sarcoglycan leads to a concomitant loss of the sarcoglycan complex as well as sarcospan and a dramatic reduction in dystroglycan in adipocytes. We further demonstrate that β-sarcoglycan null mice, which lack the sarcoglycan complex in adipose tissue and skeletal muscle, are glucose-intolerant and exhibit whole body insulin resistance specifically due to impaired insulin-stimulated glucose uptake in skeletal muscles. Thus, our data demonstrate a novel function of the sarcoglycan complex in whole body glucose homeostasis and skeletal muscle metabolism, suggesting that the impairment of the skeletal muscle metabolism influences the pathogenesis of muscular dystrophy.Muscle fat infiltration is recognized as a hallmark pathological feature in dystrophin glycoprotein complex (DGC)³-related muscular dystrophies (1) that include dystrophinopathies (2, 3) and sarcoglycanopathies (LGMD2C-F) (4). In agreement, magnetic resonance imaging measurements of fat infiltration allow accurate assessments of disease severity in Duchenne muscular dystrophy patients (3). Association of adipose tissue development with degenerative/regenerative or atrophic changes in skeletal muscle is also supported by the finding that adipogenesis-competent cells within the skeletal muscle are activated during muscle regeneration (5). However, the molecular mechanism(s) underlying muscle fatty metamorphosis remain unclear.Ectopic fat deposition in skeletal muscles is primarily described in animals and humans with lipodystrophy and sarcopenia. In these conditions, the accumulation of lipids and adipocytes in skeletal muscle is often accompanied by hyperglycemia and insulin resistance (6–11), both of which are strong indicators of muscle metabolic defects (12, 13) and deregulated adipogenesis (14). Furthermore, both adipose-derived and muscle-derived stem cells differentiate into adipocytes upon exposure to high levels of glucose (15), linking impaired muscle metabolism with muscle fat deposition.It is long held that the biogenesis of a basement membrane takes place in the earliest steps of adipogenesis and that extensive extracellular matrix (ECM) remodeling occurs throughout adipogenesis (16, 17). The concept that cell surface receptors play a role in the regulation of adipogenesis and thus may underlie metabolic disorders just recently emerged with a study of the integrin complexes (18). Given that the DGC in its capacity as an ECM receptor is critical for muscle integrity (19, 20) and that white adipocytes and skeletal muscle cells originate from the same mesenchymal precursor cells (21, 22), we set out to determine whether components of the skeletal muscle DGC are expressed in white adipocytes. Herein, we describe a unique adipose sarcoglycan (SG) complex that includes β-, δ-, and ϵ-SG. This complex is tightly associated with sarcospan (Sspn) and dystroglycan (DG). Moreover, we show that DG functions as a novel ECM receptor in white adipocytes. Because adipose tissue and skeletal muscle play critical roles in the maintenance of normal glucose homeostasis and whole body insulin sensitivity (23), we examined the metabolic consequences of the SG complex disruption in both adipose tissue and skeletal muscle. Using in vivo approaches, we observed that the β-SG null mouse (24), a mouse model of muscular dystrophy, is glucose-intolerant and exhibits whole body insulin resistance specifically due to impaired insulin-stimulated glucose uptake in skeletal muscle.     

Phosphorylation of Threonine 3: IMPLICATIONS FOR HUNTINGTIN AGGREGATION AND NEUROTOXICITY*

Huntingtin (Htt) is a widely expressed protein that causes tissue-specific degeneration when mutated to contain an expanded polyglutamine (poly(Q)) domain. Although Htt is large, 350 kDa, the appearance of amino-terminal fragments of Htt in extracts of postmortem brain tissue from patients with Huntington disease (HD), and the fact that an amino-terminal fragment, Htt exon 1 protein (Httex1p), is sufficient to cause disease in models of HD, points to the importance of the amino-terminal region of Htt in the disease process. The first exon of Htt encodes 17 amino acids followed by a poly(Q) repeat of variable length and culminating with a proline-rich domain of 50 amino acids. Because modifications to this fragment have the potential to directly affect pathogenesis in several ways, we have surveyed this fragment for potential post-translational modifications that might affect Htt behavior and detected several modifications of Httex1p. Here we report that the most prevalent modifications of Httex1p are NH₂-terminal acetylation and phosphorylation of threonine 3 (pThr-3). We demonstrate that pThr-3 occurs on full-length Htt in vivo, and that this modification affects the aggregation and pathogenic properties of Htt. Thus, therapeutic strategies that modulate these events could in turn affect Htt pathogenesis.Aberrant behavior of mutant Huntingtin protein (Htt),² caused by an expansion of the CAG triplet repeat sequence within the first exon of the huntingtin (IT15) gene, results in neurodegeneration and leads to Huntington disease (HD) (1). Full-length Htt protein is 350 kDa in size, but a truncated form of Htt (Httex1p), which includes the expanded polyglutamine region, is sufficient to cause pathology in animal models (2–4). Moreover, an amino-terminal fragment of Htt is detected in nuclear extracts from patient brain and is not detected in control cortex samples (5). In fact, recent studies suggest that production of truncated fragments is essential for disease (6, 7).The first 17 amino acids of Htt, MATLEKLMKAFESLKSF, are highly conserved throughout mammalian evolution (8, 9), suggesting an important function for these residues. It is well established that post-translational modifications of a protein can affect activity state, intracellular localization, turnover rate, and protein-protein interactions. Several modifications of Htt, without the addition of exogenous modifiers, have been identified (10–18) and implicated in HD (18, 19), but to date, none of these occur within the pathogenic Httex1p fragment. Given that this domain is sufficient to cause HD-like phenotypes, modifications that occur within this pathologic fragment may directly affect either its biophysical properties or its interaction with cellular components that affect pathology. Within the first 17 amino acids of Httex1p, there are several candidate amino acids for post-translational modification. Whereas genetic mutation of the lysines in this region alters HD pathology (20, 21), direct evidence for modifications of the amino-terminal fragment, e.g. by mass spectrometry, and identification of the modified residues, remains undocumented.In addition to affecting interactions with cellular components, recent reports indicate that mutations in the first 17 amino acids can alter the intrinsic structure of the peptide and modulate the propensity of Htt to aggregate (8, 22). The role of Htt-containing aggregates in HD remains unclear, with recent studies suggesting that visible aggregates may be protective and function as a coping response to toxic mutant Htt (22, 23). An increasingly popular notion is that oligomer/protofibrillar soluble intermediates formed during the aggregation process are the pathogenic structures (24). Post-translational modification of the first 17 amino acids could influence Httex1p aggregation behavior by changing the properties of the modified residue much like the amino acid substitutions reported (8, 22).In this study, we use mass spectrometry to present the first direct physical evidence for post-translational modification of the pathogenic exon 1 fragment of Htt without overexpressing modifying moieties or enzymes. We find that Htt is modified by the native cellular machinery and that the most common modifications of Httex1p are amino (NH₂)-terminal acetylation and phosphorylation of threonine 3 (Thr³). Furthermore, we show that Thr-3 phosphorylation occurs in vivo on full-length, endogenous Htt, that the length of the poly(Q) tract affects the relative abundance of this modification, and that Thr-3 phosphorylation affects HD pathology and the propensity for Htt aggregation in vitro and in vivo.     

N-METHYL-N'-NITRO-N-NITROSOGUANIDINE AS A MUTAGEN FOR BLUE-GREEN ALGAE: EVIDENCE FOR REPAIR1

Survival and mutagenesis have been examined in the marine coccoid blue-green alga, Agmenellum quadruplicatum, after treatment with the chemical mutagen, N-methyl-N'-nitro-N-nitrosoguanidine (NTG). When cells were immediately returned to growth conditions following NTG treatment, the survival level was consistently low and essentially independent of the treatment conditions. The types of mutants found paralleled those previously described in the freshwater coccoid, Anacystis nidulans. If, however, cells were kept under very low light intensity, a nongrowth condition, following NTG treatment, viable cell recovery was dramatically increased. This “dim light” repair in A. quadruplicatum has characteristics similar to those reported for the dark repair systems of bacteria and yeast.     

Structure of a Proteasome Pba1-Pba2 Complex: IMPLICATIONS FOR PROTEASOME ASSEMBLY,ACTIVATION, AND BIOLOGICAL FUNCTION*

The 20S proteasome is an essential, 28-subunit protease that sequesters proteolytic sites within a central chamber, thereby repressing substrate degradation until proteasome activators open the entrance/exit gate. Two established activators, Blm10 and PAN/19S, induce gate opening by binding to the pockets between proteasome α-subunits using C-terminal HbYX (hydrophobic-tyrosine-any residue) motifs. Equivalent HbYX motifs have been identified in Pba1 and Pba2, which function in proteasome assembly. Here, we demonstrate that Pba1-Pba2 proteins form a stable heterodimer that utilizes its HbYX motifs to bind mature 20S proteasomes in vitro and that the Pba1-Pba2 HbYX motifs are important for a physiological function of proteasomes, the maintenance of mitochondrial function. Other factors that contribute to proteasome assembly or function also act in the maintenance of mitochondrial function and display complex genetic interactions with one another, possibly revealing an unexpected pathway of mitochondrial regulation involving the Pba1-Pba2 proteasome interaction. Our determination of a proteasome Pba1-Pba2 crystal structure reveals a Pba1 HbYX interaction that is superimposable with those of known activators, a Pba2 HbYX interaction that is different from those reported previously, and a gate structure that is disrupted but not sufficiently open to allow entry of even small peptides. These findings extend understanding of proteasome interactions with HbYX motifs and suggest multiple roles for Pba1-Pba2 interactions throughout proteasome assembly and function.     

Regulated Secretion of Acid Sphingomyelinase: IMPLICATIONS FOR SELECTIVITY OF CERAMIDE FORMATION*

The acid sphingomyelinase (aSMase) gene gives rise to two distinct enzymes, lysosomal sphingomyelinase (L-SMase) and secretory sphingomyelinase (S-SMase), via differential trafficking of a common protein precursor. However, the regulation of S-SMase and its role in cytokine-induced ceramide formation remain ill defined. To determine the role of S-SMase in cellular sphingolipid metabolism, MCF7 breast carcinoma cells stably transfected with V5-aSMase^WT were treated with inflammatory cytokines. Interleukin-1β and tumor necrosis factor-α induced a time- and dose-dependent increase in S-SMase secretion and activity, coincident with selective elevations in cellular C₁₆-ceramide. To establish a role for S-SMase, we utilized a mutant of aSMase (S508A) that is shown to retain L-SMase activity, but is defective in secretion. MCF7 expressing V5-aSMase^WT exhibited increased S-SMase and L-SMase activity, as well as elevated cellular levels of specific long-chain and very long-chain ceramide species relative to vector control MCF7. Interestingly, elevated levels of only certain very long-chain ceramides were evident in V5-aSMase^S508A MCF7. Secretion of the S508A mutant was also defective in response to IL-1β, as was the regulated generation of C₁₆-ceramide. Taken together, these data support a crucial role for Ser⁵⁰⁸ in the regulation of S-SMase secretion, and they suggest distinct metabolic roles for S-SMase and L-SMase.     

Mycobacterium tuberculosis DinG Is a Structure-specific Helicase That Unwinds G4 DNA: IMPLICATIONS FOR TARGETING G4 DNA AS A NOVEL THERAPEUTIC APPROACH*

The significance of G-quadruplexes and the helicases that resolve G4 structures in prokaryotes is poorly understood. The Mycobacterium tuberculosis genome is GC-rich and contains >10,000 sequences that have the potential to form G4 structures. In Escherichia coli, RecQ helicase unwinds G4 structures. However, RecQ is absent in M. tuberculosis, and the helicase that participates in G4 resolution in M. tuberculosis is obscure. Here, we show that M. tuberculosis DinG (MtDinG) exhibits high affinity for ssDNA and ssDNA translocation with a 5′ → 3′ polarity. Interestingly, MtDinG unwinds overhangs, flap structures, and forked duplexes but fails to unwind linear duplex DNA. Our data with DNase I footprinting provide mechanistic insights and suggest that MtDinG is a 5′ → 3′ polarity helicase. Notably, in contrast to E. coli DinG, MtDinG catalyzes unwinding of replication fork and Holliday junction structures. Strikingly, we find that MtDinG resolves intermolecular G4 structures. These data suggest that MtDinG is a multifunctional structure-specific helicase that unwinds model structures of DNA replication, repair, and recombination as well as G4 structures. We finally demonstrate that promoter sequences of M. tuberculosis PE_PGRS2, mce1R, and moeB1 genes contain G4 structures, implying that G4 structures may regulate gene expression in M. tuberculosis. We discuss these data and implicate targeting G4 structures and DinG helicase in M. tuberculosis could be a novel therapeutic strategy for culminating the infection with this pathogen.     

N Terminus Is Key to the Dominant Negative Suppression of CaV2 Calcium Channels: IMPLICATIONS FOR EPISODIC ATAXIA TYPE 2*

Expression of the calcium channels Ca_V2.1 and Ca_V2.2 is markedly suppressed by co-expression with truncated constructs containing Domain I. This is the basis for the phenomenon of dominant negative suppression observed for many of the episodic ataxia type 2 mutations in Ca_V2.1 that predict truncated channels. The process of dominant negative suppression has been shown previously to stem from interaction between the full-length and truncated channels and to result in downstream consequences of the unfolded protein response and endoplasmic reticulum-associated protein degradation. We have now identified the specific domain that triggers this effect. For both Ca_V2.1 and Ca_V2.2, the minimum construct producing suppression was the cytoplasmic N terminus. Suppression was enhanced by tethering the N terminus to the membrane with a CAAX motif. The 11-amino acid motif (including Arg⁵² and Arg⁵⁴) within the N terminus, which we have previously shown to be required for G protein modulation, is also essential for dominant negative suppression. Suppression is prevented by addition of an N-terminal tag (XFP) to the full-length and truncated constructs. We further show that suppression of Ca_V2.2 currents by the N terminus-CAAX construct is accompanied by a reduction in Ca_V2.2 protein level, and this is also prevented by mutation of Arg⁵² and Arg⁵⁴ to Ala in the truncated construct. Taken together, our evidence indicates that both the extreme N terminus and the Arg⁵², Arg⁵⁴ motif are involved in the processes underlying dominant negative suppression.