Pallob Kundu

The interaction of cytoplasmic RNA viruses with the nucleus

Mammalian cells infected with poliovirus, the prototype member of the picornaviridae family, undergo rapid macromolecular and metabolic changes resulting in efficient replication and release of virus from infected cells. Although this virus is predominantly cytoplasmic, it does shut-off transcription of all three cellular transcription systems. Both biochemical and genetic studies have shown that a virally encoded protease, 3C(pro), is responsible for host cell transcription shut-off. The 3C protease cleaves a number of RNA polymerase II transcription factors including the TATA-binding protein (TBP), the cyclic AMP-responsive element binding protein (CREB), the Octamer binding protein (Oct-1), p53, and RNA polymerase III transcription factor IIICalpha, and Polymerase I factor SL-1. Most of these cleavages occur at glutamine-glycine bonds. Additionally, a second viral protease, 2A(pro), also cleaves TBP at a tyrosine-glycine bond. The latter cleavage could be responsible for shut-off of small nuclear RNA transcription. Recent studies indicate that the viral protease-polymerase precursor 3CD can enter nucleus in poliovirus-infected cells. The nuclear localization signal (NLS) present within the 3D sequence appears to play a role in the nuclear entry of 3CD. Thus, 3C may be delivered to the infected cell nucleus in the form the precursor 3CD or other 3C-containing precursors. Auto-proteolytic cleavage of these precursors could then generate 3C. Thus, for a small RNA virus that strictly replicates in the cytoplasm, a portion of its life cycle does include interaction with the host cell nucleus.

Slo1 Caveolin-binding Motif, a Mechanism of Caveolin-1-Slo1 Interaction Regulating Slo1 Surface Expression

The large conductance, voltage- and Ca2+-activated potassium (MaxiK, BK) channel and caveolin-1 play important roles in regulating vascular contractility. Here, we hypothesized that the MaxiK α-subunit (Slo1) and caveolin-1 may interact with each other. Slo1 and caveolin-1 physiological association in native vascular tissue is strongly supported by (i) detergent-free purification of caveolin-1-rich domains demonstrating a pool of aortic Slo1 co-migrating with caveolin-1 to light density sucrose fractions, (ii) reverse co-immunoprecipitation, and (iii) double immunolabeling of freshly isolated myocytes revealing caveolin-1 and Slo1 proximity at the plasmalemma. In HEK293T cells, Slo1-caveolin-1 association was unaffected by the smooth muscle MaxiK β1-subunit. Sequence analysis revealed two potential caveolin-binding motifs along the Slo1 C terminus, one equivalent, 1007YNMLCFGIY1015, and another mirror image, 537YTEYLSSAF545, to the consensus sequence, φXXXXφXXφ. Deletion of 1007YNMLCFGIY1015 caused ∼80% loss of Slo1-caveolin-1 association while preserving channel normal folding and overall Slo1 and caveolin-1 intracellular distribution patterns. 537YTEYLSSAF545 deletion had an insignificant dissociative effect. Interestingly, caveolin-1 coexpression reduced Slo1 surface and functional expression near 70% without affecting channel voltage sensitivity, and deletion of 1007YNMLCFGIY1015 motif obliterated channel surface expression. The results suggest 1007YNMLCFGIY1015 possible participation in Slo1 plasmalemmal targeting and demonstrate its role as a main mechanism for caveolin-1 association with Slo1 potentially serving a dual role: (i) maintaining channels in intracellular compartments downsizing their surface expression and/or (ii) serving as anchor of plasma membrane resident channels to caveolin-1-rich membranes. Because the caveolin-1 scaffolding domain is juxtamembrane, it is tempting to suggest that Slo1-caveolin-1 interaction facilitates the tethering of the Slo1 C-terminal end to the membrane.

Precursor of the Inactive 2S Seed Storage Protein from the Indian Mustard Brassica juncea Is a Novel Trypsin Inhibitor CHARACTERIZATION, POST-TRANSLATIONAL PROCESSING STUDIES, AND TRANSGENIC EXPRESSION TO DEVELOP INSECT-RESISTANT PLANTS ,

A number of trypsin inhibitor (TI) genes have been used to generate insect-resistant plants. Here we report a novel trypsin inhibitor from Indian mustard Brassica juncea(BjTI) that is unique in being the precursor of a 2S seed storage protein. The inhibitory activity is lost upon processing. The predicted amino acid sequence of the precursor based on the B. juncea2S albumin (Bj2S) gene cloned and sequenced in this laboratory (Bj2Sc; GenBankTM accession number X65972) showed a soybean-TI active site-like motif GPFRI at the expected processing site. The BjTI was found to be a thermostable Kunitz type TI that inhibits trypsin at a molar ratio of 1:1. The 20-kDa BjTI was purified from midmature seeds and found to be processed in vitro to 9- and 4-kDa subunits upon incubation with seed extract. The Bj2Sc sequence was expressed in Escherichia coli pET systems as the inhibitor precursor. The radiolabeled gene product was expressedin vitro in a coupled transcription-translation system and showed the expected processing into subunits. Two in vitroexpressed pre-2S proteins, mutated at Gly and Asp residues, were processed normally to mature subunits, showing thereby no absolute requirement of Gly and Asp residues for processing. Finally, the 2S gene was introduced into tobacco and tomato plants. Third generation transgenics expressing BjTI at 0.28–0.83% of soluble leaf proteins showed remarkable resistance against the tobacco cutworm,Spodoptera litura. This novel TI can be used in transforming seed crops for protection to their vegetative parts and early seed stages, when insect damage is maximal; as the seeds mature, the TI will be naturally processed to the inactive storage protein that is safe for consumption.

Regulation of mouse Slo gene expression: multiple promoters, transcription start sites, and genomic action of estrogen.

The large conductance, voltage- and Ca2+-activated K+ channel plays key roles in diverse body functions influenced by estrogen, including smooth muscle and neural activities. In mouse (m), estrogen up-regulates the transcript levels of its pore-forming α-subunit (Slo, KCNMA1), yet the underlying genomic mechanism(s) is (are) unknown. We first mapped the promoters and regulatory motifs within the mSlo 5′-flanking sequence to subsequently identify genomic regions and mechanisms required for estrogen regulation. mSlo gene has at least two TATA-less promoters with distinct potencies that may direct mSlo transcription from multiple transcription start sites. These qualities mark mSlo as a prototype gene with promoter plasticity capable of generating multiple mRNAs and the potential to adapt to organismal needs. mSlo promoters contain multiple estrogen-responsive sequences, e.g. two quasi-perfect estrogen-responsive elements, ERE1 and ERE2, and Sp1 sites. Accordingly, mSlo promoter activity was highly enhanced by estrogen and blocked by estrogen antagonist ICI 182,780. When promoters are embedded in a 4.91-kb backbone, estrogen responsiveness involves a classical genomic mechanism, via ERE1 and ERE2, that may be complemented by Sp factors, particularly Sp1. Simultaneous but not individual ERE1 and ERE2 mutations caused significant loss of estrogen action. ERE2, which is closer to the proximal promoter, up-regulates this promoter via a classical genomic mechanism. ERE2 strategic position together with ERE1 and ERE2 independence and Sp contribution should ensure mSlo estrogen responsiveness. Thus, the mSlo gene seems to have uniquely evolved to warrant estrogen regulation. Estrogen-mediated mSlo genomic regulation has important implications on long term estrogenic effects affecting smooth muscle and neural functions.

Shutoff of RNA Polymerase II Transcription by Poliovirus Involves 3C Protease-Mediated Cleavage of the TATA-Binding Protein at an Alternative Site: Incomplete Shutoff of Transcription Interferes with Efficient Viral Replication

ABSTRACT The TATA-binding protein (TBP) plays a crucial role in cellular transcription catalyzed by all three DNA-dependent RNA polymerases. Previous studies have shown that TBP is targeted by the poliovirus (PV)-encoded protease 3Cpro to bring about shutoff of cellular RNA polymerase II-mediated transcription in PV-infected cells. The processing of the majority of viral precursor proteins by 3Cpro involves cleavages at glutamine-glycine (Q-G) sites. We present evidence that suggests that the transcriptional inactivation of TBP by 3Cpro involves cleavage at the glutamine 104-serine 105 (Q104-S105) site of TBP and not at the Q18-G19 site as previously thought. The TBP Q104-S105 cleavage by 3Cpro is greatly influenced by the presence of an aliphatic amino acid at the P4 position, a hallmark of 3Cpro-mediated proteolysis. To examine the importance of host cell transcription shutoff in the PV life cycle, stable HeLa cell lines were created that express recombinant TBP resistant to cleavage by the viral proteases, called GG rTBP. Transcription shutoff was significantly impaired and delayed in GG rTBP cells upon infection with poliovirus compared with the cells that express wild-type recombinant TBP (wt rTBP). Infection of GG rTBP cells with poliovirus resulted in small plaques, significantly reduced viral RNA synthesis, and lower viral yields compared to the wt rTBP cell line. These results suggest that a defect in transcription shutoff can lead to inefficient replication of poliovirus in cultured cells.

c-Src tyrosine kinase, a critical component for 5-HT2A receptor-mediated contraction in rat aorta

Serotonin (5‐hydroxytryptamine, 5‐HT) receptors (5‐HTRs) play critical roles in brain and cardiovascular functions. In the vasculature, 5‐HT induces potent vasoconstrictions, which in aorta are mainly mediated by activation of the 5‐HT2AR subtype. We previously proposed that one signalling mechanism of 5‐HT‐induced vasoconstriction could be c‐Src, a member of the Src tyrosine kinase family. We now provide evidence for a central role of c‐Src in 5‐HT2AR‐mediated contraction. Inhibition of Src kinase activity with 10 μm 4‐amino‐5‐(4‐chlorophenyl)‐7‐(t‐butyl)pyrazolo[3,4‐d]pyrimidine (PP2) prior to contraction resulted in ∼90–99% inhibition of contractions induced by 5‐HT or by α‐methyl‐5‐HT (5‐HT2R agonist). In contrast, PP2 pretreatment only partly inhibited contractions induced by angiotensin II and the thromboxane A2 mimetic, U46619, and had no significant action on phenylephrine‐induced contractions. 5‐Hydroxytryptamine increased Src kinase activity and PP2‐sensitive tyrosine‐phosphorylated proteins. As expected for c‐Src identity, PP2 pretreatment inhibited 5‐HT‐induced contraction with an IC50 of ∼1 μm. Ketanserin (10 nm), a 5‐HT2A antagonist, but not antagonists of 5‐HT2BR (100 nm SB204741) or 5‐HT2CR (20 nm RS102221), prevented 5‐HT‐induced contractions, mimicking PP2 and implicating 5‐HT2AR as the major receptor subtype coupled to c‐Src. In HEK 293T cells, c‐Src and 5‐HT2AR were reciprocally co‐immunoprecipitated and co‐localized at the cell periphery. Finally, 5‐HT‐induced Src activity was unaffected by inhibition of Rho kinase, supporting a role of c‐Src upstream of Rho kinase. Together, the results highlight c‐Src activation as one of the early and pivotal mechanisms in 5‐HT2AR contractile signalling in aorta.

Regulation of Poliovirus 3C Protease by the 2C Polypeptide

ABSTRACT Poliovirus-encoded nonstructural polypeptide 2C is a multifunctional protein that plays an important role in viral RNA replication. 2C interacts with both intracellular membranes and virus-specific RNAs and has ATPase and GTPase activities. Extensive computer analysis of the 2C sequence revealed that in addition to the known ATPase-, GTPase-, membrane-, and RNA-binding domains it also contains several “serpin” (serine protease inhibitor) motifs. We provide experimental evidence suggesting that 2C is indeed capable of regulating virus-encoded proteases. The purified 2C protein inhibits 3Cpro-catalyzed cleavage of cellular transcription factors at Q-G sites in vitro. It also inhibits cleavage of a viral precursor by the other viral protease, 2Apro. However, at least three cellular proteases appear not to be inhibited by 2C in vitro. The 2C-associated protease inhibitory activity can be depleted by anti-2C antibody. A physical interaction between 2C and His-tagged 3Cpro can be demonstrated in vitro by coimmunoprecipitation of 2C with anti-His antibody. Deletion analysis suggests that the 2C central and C-terminal domains that include several serpin motifs are important for 3Cpro-inhibitory activity. To examine the 2C protease inhibitory activity in vivo, stable HeLa cell lines were made that express 2C in an inducible fashion. Infection of 2C-expressing cells with poliovirus led to incomplete (or inefficient) processing of viral precursor polypeptides compared to control cell lines containing the vector alone. These results suggest that 2C can negatively regulate the viral protease 3Cpro. The possible role of the 2C protease inhibitory activity in viral RNA replication is discussed.

Antimicrobial Peptides: Insights into Membrane Permeabilization, Lipopolysaccharide Fragmentation and Application in Plant Disease Control

The recent increase in multidrug resistance against bacterial infections has become a major concern to human health and global food security. Synthetic antimicrobial peptides (AMPs) have recently received substantial attention as potential alternatives to conventional antibiotics because of their potent broad-spectrum antimicrobial activity. These peptides have also been implicated in plant disease control for replacing conventional treatment methods that are polluting and hazardous to the environment and to human health. Here, we report de novo design and antimicrobial studies of VG16, a 16-residue active fragment of Dengue virus fusion peptide. Our results reveal that VG16KRKP, a non-toxic and non-hemolytic analogue of VG16, shows significant antimicrobial activity against Gram-negative E. coli and plant pathogens X. oryzae and X. campestris, as well as against human fungal pathogens C. albicans and C. grubii. VG16KRKP is also capable of inhibiting bacterial disease progression in plants. The solution-NMR structure of VG16KRKP in lipopolysaccharide features a folded conformation with a centrally located turn-type structure stabilized by aromatic-aromatic packing interactions with extended N- and C-termini. The de novo design of VG16KRKP provides valuable insights into the development of more potent antibacterial and antiendotoxic peptides for the treatment of human and plant infections.

Thromboxane A2 receptor and MaxiK-channel intimate interaction supports channel trans-inhibition independent of G-protein activation

Large conductance voltage- and calcium-activated potassium channels (MaxiK, BKCa) are well known for sustaining cerebral and coronary arterial tone and for their linkage to vasodilator β-adrenergic receptors. However, how MaxiK channels are linked to counterbalancing vasoconstrictor receptors is unknown. Here, we show that vasopressive thromboxane A2 receptors (TP) can intimately couple with and inhibit MaxiK channels. Activation of the receptor with its agonist trans-inhibits MaxiK independently of G-protein activation. This unconventional mechanism is supported by independent lines of evidence: (i) inhibition of MaxiK current by thromboxane A2 mimetic, U46619, occurs even when G-protein activity is suppressed; (ii) MaxiK and TP physically associate and display a high degree of proximity; and (iii) Förster resonance energy transfer occurs between fluorescently labeled MaxiK and TP, supporting a direct interaction. The molecular mechanism of MaxiK–TP intimate interaction involves the receptors first intracellular loop and C terminus, and it entails the voltage-sensing conduction cassette of MaxiK channel. Further, physiological evidence of MaxiK–TP physical interaction is given in human coronaries and rat aorta, and by confirming TP role (with antagonist SQ29,548) in the U46619-induced MaxiK inhibition in human coronaries. We propose that vasoconstrictor TP receptor and MaxiK-channel direct interaction facilitates G-protein–independent TP to MaxiK trans-inhibition, which would promote vasoconstriction.

Hormonal regulation of cardiac KCNE2 gene expression

The KCNE2 gene encodes a single transmembrane domain protein that modulates a variety of K+ channel functions in various tissues. Here we show that cardiac KCNE2 transcript levels are approximately 10-fold upregulated at the end of pregnancy. This upregulation was mimicked by 17-beta estradiol but not by 5alpha-dihydrotestosterone treatments in ovariectomized mice. To investigate the mechanism of KCNE2 transcriptional regulation by estrogen, we experimentally identified KCNE2 transcription start sites, delineated its gene structure and characterized its promoter region. Estrogen treatment stimulated KCNE2 promoter activity in a dose-dependent manner and ICI 182,780 blocked estrogen stimulation. A direct genomic mechanism was demonstrated by (i) the loss of estrogen responsiveness in the presence of a DNA-binding domain mutant estrogen receptor alpha or mutant KCNE2 ERE and (ii) binding of ERalpha to the KCNE2 ERE. These findings show that a genomic mechanism of estrogen action alters KCNE2 expression, which may have important physiological implications.