Polyubiquitin protein of Aedes aegypti as an interacting partner of dengue virus envelope protein.

Dengue virus (DENV) is an arbovirus that comprises four antigenically different serotypes. Aedes aegypti (Diptera: Culicidae) acts as the principal vector for DENV transmission, and vector control is crucial for dengue fever epidemic management. To design effective vector control strategies, a comprehensive understanding of the insect vector and virus interaction is required. Female Ae. aegypti ingests DENV during the acquisition of a blood meal from an infected human. DENV enters the insect midgut, replicates inside it and reaches the salivary gland for transmitting DENV to healthy humans during the subsequent feeding cycles. DENV must interact with the proteins present in the midgut and salivary glands to gain entry and accomplish successful replication and transmission. Ae. aegypti midgut cDNA library was prepared, and yeast two-hybrid screening was performed against the envelope protein domain III (EDIII) protein of DENV-2. The polyubiquitin protein was selected from the various candidate proteins for subsequent analysis. Polyubiquitin gene was amplified, and the protein was purified in a heterologous expression system for in vitro interaction studies. In vitro pull-down assay presented a clear interaction between polyubiquitin protein and EDIII. To further confirm this interaction, a dot blot assay was employed, and polyubiquitin protein was found to interact with DENV particles. Our results enable us to suggest that polyubiquitin plays an important role in DENV infection within mosquitoes.

The Ribosome-Sec61 Translocon Complex Forms a Cytosolically Restricted Environment for Early Polytopic Membrane Protein Folding.

Transmembrane topology of polytopic membrane proteins (PMPs) is established in the endoplasmic reticulum (ER) by the ribosome Sec61-translocon complex (RTC) through iterative cycles of translocation initiation and termination. It remains unknown, however, whether tertiary folding of transmembrane domains begins after the nascent polypeptide integrates into the lipid bilayer or within a proteinaceous environment proximal to translocon components. To address this question, we used cysteine scanning mutagenesis to monitor aqueous accessibility of stalled translation intermediates to determine when, during biogenesis, hydrophilic peptide loops of the aquaporin-4 (AQP4) water channel are delivered to cytosolic and lumenal compartments. Results showed that following ribosome docking on the ER membrane, the nascent polypeptide was shielded from the cytosol as it emerged from the ribosome exit tunnel. Extracellular loops followed a well defined path through the ribosome, the ribosome translocon junction, the Sec61-translocon pore, and into the ER lumen coincident with chain elongation. In contrast, intracellular loops (ICLs) and C-terminalresidues exited the ribosome into a cytosolically shielded environment and remained inaccessible to both cytosolic and lumenal compartments until translation was terminated. Shielding of ICL1 and ICL2, but not the C terminus, became resistant to maneuvers that disrupt electrostatic ribosome interactions. Thus, the early folding landscape of polytopic proteins is shaped by a spatially restricted environment localized within the assembled ribosome translocon complex.

Classification of chemical chaperones based on their effect on protein folding landscapes.

Various small molecules present in biological systems can assist protein folding in vitro and are known as chemical chaperones. De novo design of chemical chaperones with higher activity than currently known examples is desirable to ameliorate protein misfolding and aggregation in multiple contexts. However, this development has been hindered by limited knowledge of their activities. It is thought that chemical chaperones are typically poor solvents for a protein backbone and hence facilitate native structure formation. However, it is unknown if different chemical chaperones can act differently to modulate folding energy landscapes. Using a model slow folding protein, double-mutant Maltose-binding protein (DM-MBP), we show that a canonical chemical chaperone, trimethylamine-N-oxide (TMAO), accelerates refolding by decreasing the flexibility of the refolding intermediate (RI). Among a number of small molecules that chaperone DM-MBP folding, proline and serine stabilize the transition state (TS) enthalpically, while trehalose behaves like TMAO and increases the rate of barrier crossing through nonenthalpic processes. We propose a two-group classification of chemical chaperones based upon their thermodynamic effect on RI and TS, which is also supported by single molecule Förster resonance energy transfer (smFRET) studies. Interestingly, for a different test protein, the molecular mechanisms of the two groups of chaperones are not conserved. This provides a glimpse into the complexity of chemical chaperoning activity of osmolytes. Future work would allow us to engineer synergism between the two classes to design more efficient chemical chaperones to ameliorate protein misfolding and aggregation problems.

Chemical chaperones assist intracellular folding to buffer mutational variations.

Hidden genetic variations have the potential to lead to the evolution of new traits. Molecular chaperones, which assist protein folding, may conceal genetic variations in protein-coding regions. Here we investigate whether the chemical milieu of cells has the potential to alleviate intracellular protein folding, a possibility that could implicate osmolytes in concealing genetic variations. We found that the model osmolyte trimethylamine N-oxide (TMAO) can buffer mutations that impose kinetic traps in the folding pathways of two model proteins. Using this information, we rationally designed TMAO-dependent mutants in vivo, starting from a TMAO-independent protein. We show that different osmolytes buffer a unique spectrum of mutations. Consequently, the chemical milieu of cells may alter the folding pathways of unique mutant variants in polymorphic populations and lead to unanticipated spectra of genetic buffering.

Expression and molecular characterization of the Mycobacterium tuberculosis PII protein.

The signal transduction protein PII plays an important role in cellular nitrogen assimilation and regulation. The molecular characteristics of the Mycobacterium tuberculosis PII (Mtb PII) were investigated using biophysical experiments. The Mtb PII coding ORF Rv2919c was cloned and expressed in Escherichia coli. The binding characteristics of the purified protein with ATP and ADP were investigated using surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC). Mtb PII binds to ATP strongly with K(d) in the range 1.93-6.44 microM. This binding strength was not significantly affected by the presence of 2-ketoglutarate even in molar concentrations of 66 (ITC) or 636 (SPR) fold excess of protein concentration. However, an additional enthalpy of 0.3 kcal/mol was released in presence of 2-ketoglutarate. Binding of Mtb PII to ADP was weaker by an order of magnitude. Binding of ATP and 2-ketoglutarate were analysed by docking studies on the Mtb PII crystal structure (PDB id 3BZQ). We observed that hydrogen bonds involving the gamma-phosphate of ATP contribute to enhanced binding of ATP compared with ADP. Glutaraldehyde crosslinking showed that Mtb PII exists in homotrimeric state which is consistent with other PII proteins. Phylogenetic analysis showed that Mtb PII consistently grouped with other actinobacterial PII proteins.