Flexibility of NS5 Methyltransferase-Polymerase Linker Region Is Essential for Dengue Virus Replication

We examined the function of the conserved Val/Ile residue within the dengue virus NS5 interdomain linker (residues 263 to 272) by site-directed mutagenesis. Gly substitution or Gly/Pro insertion after the conserved residue increased the linker flexibility and created slightly attenuated viruses. In contrast, Pro substitution abolished virus replication by imposing rigidity in the linker and restricting NS5''s conformational plasticity. Our biochemical and reverse genetics experiments demonstrate that NS5 utilizes conformational regulation to achieve optimum viral replication.     

Motor neuron degeneration in a mouse model of seipinopathy

Heterozygosity for missense mutations (N88S/S90L) in BSCL2 (Berardinelli–Seip congenital lipodystrophy type 2)/Seipin is associated with a broad spectrum of motoneuron diseases. To understand the underlying mechanisms how the mutations lead to motor neuropathy, we generated transgenic mice with neuron-specific expression of wild-type (tgWT) or N88S/S90L mutant (tgMT) human Seipin. Transgenes led to the broad expression of WT or mutant Seipin in the brain and spinal cord. TgMT, but not tgWT, mice exhibited late-onset altered locomotor activities and gait abnormalities that recapitulate symptoms of seipinopathy patients. We found loss of alpha motor neurons in tgMT spinal cord. Mild endoreticular stress was present in both tgMT and tgWT neurons; however, only tgMT mice exhibited protein aggregates and disrupted Golgi apparatus. Furthermore, autophagosomes were significantly increased, along with elevated light chain 3 (LC3)-II level in tgMT spinal cord, consistent with the activation of autophagy pathway in response to mutant Seipin expression and protein aggregation. These results suggest that induction of autophagy pathway is involved in the cellular response to mutant Seipin in seipinopathy and that motoneuron loss is a key pathogenic process underlying the development of locomotor abnormalities.     

Mutations in the DG Loop of Adenovirus Type 5 Fiber Knob Protein Abolish High-Affinity Binding to Its Cellular Receptor CAR

The amino acid residues in adenovirus type 5 (Ad5) fiber that interact with its cellular receptor, the coxsackie B virus and Ad receptor (CAR), have not been defined. To investigate this, multiple mutations were constructed in the region between residues 479 and 497 in Ad5 fiber (beta-strands E and F and the adjacent region of the DG loop). The effects of these mutations on binding to CAR were determined by use of cell-binding competition experiments, surface plasmon resonance, and direct binding studies. The mutation effects on the overall folding and secondary structure of the protein were assessed by circular dichroism (CD) spectroscopy. Deletions of two consecutive amino acids between residues 485 and 493 abolished high-affinity binding to CAR; the CD spectra indicated that although there was no disruption of the overall folding and secondary structure of the protein, local conformational changes did occur. Moreover, single site mutations in this region of residues with exposed, surface-accessible side chains, such as Thr492, Asn493, and Val495, had no effect on receptor binding, which demonstrates that these residues are not in contact with CAR themselves. This implies the involvement of residues in neighboring loop regions. Replacement of the segment containing the two very short beta-strands E and F and the turn between them (residues 479 to 486) with the corresponding sequence from Ad3 (betaEFAd3-->5 mutation) resulted in the loss of receptor binding. The identical CD spectra for betaEFAd3-->5 and wild-type proteins suggest that these substitutions caused no conformational rearrangement and that the loss of binding may thus be due to the substitution of one or more critical contact residues. These findings have implications for our understanding of the interaction of Ad5 fiber with CAR and for the construction of targeted recombinant Ad5 vectors for gene therapy purposes.     

Development of an Efficient Targeted Cell-SELEX Procedure for DNA Aptamer Reagents

Background

DNA aptamers generated by cell-SELEX offer an attractive alternative to antibodies, but generating aptamers to specific, known membrane protein targets has proven challenging, and has severely limited the use of aptamers as affinity reagents for cell identification and purification.

Methodology

We modified the BJAB lymphoblastoma cell line to over-express the murine c-kit cell surface receptor. After six rounds of cell-SELEX, high-throughput sequencing and bioinformatics analysis, we identified aptamers that bound BJAB cells expressing c-kit but not wild-type BJAB controls. One of these aptamers also recognizes c-kit endogenously expressed by a mast cell line or hematopoietic progenitor cells, and specifically blocks binding of the c-kit ligand stem cell factor (SCF). This aptamer enables better separation by fluorescence-activated cell sorting (FACS) of c-kit⁺ hematopoietic progenitor cells from mixed bone marrow populations than a commercially available antibody, suggesting that this approach may be broadly useful for rapid isolation of affinity reagents suitable for purification of other specific cell types.

Conclusions/Significance

Here we describe a novel procedure for the efficient generation of DNA aptamers that bind to specific cell membrane proteins and can be used as high affinity reagents. We have named the procedure STACS (Specific TArget Cell-SELEX).     

Comparison of plastid DNA replication in different cells and tissues of the rice plant

In a previous study, we mapped replication origin regions of the plastid DNA around the 3 end of the 23S rRNA gene in rice suspension-cultured cells. Here, we examined initiation of the plastid DNA replication in different rice cells by two-dimensional agarose gel electrophoresis. We show for the first time, to our knowledge, that the replication origin region of the plastid DNA differs among cultured cells, coleoptiles and mature leaves. In addition, digestion of the replication intermediates from the rice cultured cells with mung bean nuclease, a single-strand-specific nuclease, revealed that both two single strands of the double-stranded parental DNA were simultaneously replicated in the origin region. This was further confirmed by two-dimensional agarose gel analysis with single-stranded RNA probes. Thus, the mode of plastid DNA replication presented here differs from the unidirectional replication started by forming displacement loops (D-loops), in which the two D-loops on the opposite strands expand toward each other and only one parental strand serves as a template.     

Increased expression of co-chaperone HOP with HSP90 and HSC70 and complex formation in human colonic carcinoma

Co-chaperone HOP (also called stress-inducible protein 1) is a co-chaperone that interacts with the cytosolic 70-kDa heat shock protein (HSP70) and 90-kDa heat shock protein (HSP90) families using different tetratricopeptide repeat domains. HOP plays crucial roles in the productive folding of substrate proteins by controlling the chaperone activities of HSP70 and HSP90. Here, we examined the levels of HOP, HSC70 (cognate of HSP70, also called HSP73), and HSP90 in the tumor tissues from colon cancer patients, in comparison with the non-tumor tissues from the same patients. Expression level of HOP was significantly increased in the tumor tissues (68% of patients, n = 19). Levels of HSC70 and HSP90 were also increased in the tumor tissues (95% and 74% of patients, respectively), and the HOP level was highly correlated with those of HSP90 (r = 0.77, p < 0.001) and HSC70 (r = 0.68, p < 0.01). Immunoprecipitation experiments indicated that HOP complexes with HSC70 or HSP90 in the tumor tissues. These data are consistent with increased formation of co-chaperone complexes in colon tumor specimens compared to adjacent normal tissue and could reflect a role for HOP in this process.     

Identification of Multifaceted Binding Modes for Pyrin and ASC Pyrin Domains Gives Insights into Pyrin Inflammasome Assembly

Inflammasomes are macromolecular complexes that mediate inflammatory and cell death responses to pathogens and cellular stress signals. Dysregulated inflammasome activation is associated with autoinflammatory syndromes and several common diseases. During inflammasome assembly, oligomerized cytosolic pattern recognition receptors recruit procaspase-1 and procaspase-8 via the adaptor protein ASC. Inflammasome assembly is mediated by pyrin domains (PYDs) and caspase recruitment domains, which are protein interaction domains of the death fold superfamily. However, the molecular details of their interactions are poorly understood. We have studied the interaction between ASC and pyrin PYDs that mediates ASC recruitment to the pyrin inflammasome, which is implicated in the pathogenesis of familial Mediterranean fever. We demonstrate that both the ASC and pyrin PYDs have multifaceted binding modes, involving three sites on pyrin PYD and two sites on ASC PYD. Molecular docking of pyrin-ASC PYD complexes showed that pyrin PYD can simultaneously interact with up to three ASC PYDs. Furthermore, ASC PYD can self-associate and interact with pyrin, consistent with previous reports that pyrin promotes ASC clustering to form a proinflammatory complex. Finally, the effects of familial Mediterranean fever-associated mutations, R42W and A89T, on structural and functional properties of pyrin PYD were investigated. The R42W mutation had a significant effect on structure and increased stability. Although the R42W mutant exhibited reduced interaction with ASC, it also bound less to the pyrin B-box domain responsible for autoinhibition and hence may be constitutively active. Our data give new insights into the binding modes of PYDs and inflammasome architecture.