Lhs1 dependent ERAD is determined by transmembrane domain context.

Transmembrane proteins have unique requirements to fold and integrate into the endoplasmic reticulum (ER) membrane. Most notably, transmembrane proteins must fold in three separate environments: extracellular domains fold in the oxidizing environment of the ER lumen, transmembrane domains (TMDs) fold within the lipid bilayer, and cytosolic domains fold in the reducing environment of the cytosol. Moreover, each region is acted upon by a unique set of chaperones and monitored by components of the ER associated quality control machinery that identify misfolded domains in each compartment. One factor is the ER lumenal Hsp70-like chaperone, Lhs1. Our previous work established that Lhs1 is required for the degradation of the unassembled α-subunit of the epithelial sodium channel (αENaC), but not the homologous ß- and γENaC subunits. However, assembly of the ENaC heterotrimer blocked the Lhs1-dependent ER associated degradation (ERAD) of the α-subunit, yet the characteristics that dictate the specificity of Lhs1-dependent ERAD substrates remained unclear. We now report that Lhs1-dependent substrates share a unique set of features. First, all Lhs1 substrates appear to be unglycosylated, and second they contain two TMDs. Each substrate also contains orphaned or unassembled TMDs. Additionally, interfering with inter-subunit assembly of the ENaC trimer results in Lhs1-dependent degradation of the entire complex. Finally, our work suggests that Lhs1 is required for a subset of ERAD substrates that also require the Hrd1 ubiquitin ligase. Together, these data provide hints as to the identities of as-yet unconfirmed substrates of Lhs1 and potentially of the Lhs1 homolog in mammals, GRP170.

Protein folding on biosensor tips: folding of maltodextrin glucosidase monitored by its interactions with GroEL.

Protein folding has been extensively studied for the past six decades by employing solution-based methods such as solubility, enzymatic activity, secondary structure analysis, and analytical methods like FRET, NMR, and HD exchange. However, for rapid analysis of the folding process, solution-based approaches are often plagued with aggregation side reactions resulting in poor yields. In this work, we demonstrate that a bio-layer interferometry (BLI) chaperonin detection system can identify superior refolding conditions for denatured proteins. The degree of immobilized protein folding as a function of time can be detected by monitoring the binding of the high-affinity nucleotide-free form of the chaperonin GroEL. GroEL preferentially interacts with proteins that have hydrophobic surfaces exposed in their unfolded or partially folded form, so a decrease in GroEL binding can be correlated with burial of hydrophobic surfaces as folding progresses. The magnitude of GroEL binding to the protein immobilized on bio-layer interferometry biosensor inversely reflects the extent of protein folding and hydrophobic residue burial. We demonstrate conditions where accelerated folding can be observed for the aggregation-prone protein maltodextrin glucosidase (MalZ). Superior immobilized folding conditions identified on the bio-layer interferometry biosensor surface were reproduced on Ni-NTA sepharose bead surfaces and resulted in significant improvement in folding yields of released MalZ (measured by enzymatic activity) compared to bulk refolding conditions in solution.

Role of N-terminal region of Escherichia coli maltodextrin glucosidase in folding and function of the protein.

Maltodextrin glucosidase (MalZ) hydrolyses short malto-oligosaccharides from the reducing end releasing glucose and maltose in Escherichia coli. MalZ is a highly aggregation prone protein and molecular chaperonins GroEL and GroES assist in the folding of this protein to a substantial level. The N-terminal region of this enzyme appears to be a unique domain as seen in sequence comparison studies with other amylases as well as through homology modelling. The sequence and homology model analysis show a probability of disorder in the N-Terminal region of MalZ. The crystal structure of this enzyme has been reported in the present communication. Based on the crystallographic structure, it has been interpreted that the N-terminal region of the enzyme (Met1-Phe131) might be unstructured or flexible. To understand the role of the N-terminal region of MalZ in its enzymatic activity, and overall stability, a truncated version (Ala111-His616) of MalZ was created. The truncated version failed to fold into an active enzyme both in E. coli cytosol and in vitro even with the assistance of chaperonins GroEL and GroES. Furthermore, the refolding effort of N-truncated MalZ in the presence of isolated N-terminal domain didn't succeed. Our studies suggest that while the structural rigidity or orientation of the N-terminal region of the MalZ protein may not be essential for its stability and function, but the said domain is likely to play an important role in the formation of the native structure of the protein when present as an integral part of the protein.

Stable self-assembled nanostructured hen egg white lysozyme exhibits strong anti-proliferative activity against breast cancer cells.

Chemotherapy side effects have long been a matter of great concern. Here we describe a structurally stable self-assembled nanostructured lysozyme (snLYZ) synthesized using a simple desolvation technique that exhibited anticancer activity, as well as excellent hemocompatibility. Field emission scanning electron microscopy; atomic force microscopy and dynamic particle size analyzer were used for analyzing the synthesized snLYZ. The analysis revealed spherical shape with an average size of 300 nm. Circular dichroism and tryptophan fluorescence spectroscopic analysis revealed its gross change in secondary as well as the tertiary level of the structure. snLYZ also demonstrated excellent structural as well as the functional stability of LYZ in a wide range of pH and temperature with a fair level of protection against proteinase K digestion. When applied to MCF-7 breast cancer cells, it exhibited approximately 95% cell death within 24h, involving a reactive oxygen species (ROS) based mechanism, and showed excellent hemocompatibility. Fluorescence microscopy imaging revealed distinct cellular internalization of snLYZ and the formation of cytoplasmic granules, which initiated a cell-killing process through membrane damage. In order to mimic targeted therapy, we tagged folic acid with snLYZ, which further enhanced cytotoxicity against MCF-7 cells. Therefore, this is the first report of its kind where we demonstrated the preparation of a highly stable self-assembled nanostructured lysozyme with a strong anti-proliferative activity against breast cancer cells.