Compartment-specific aggregases direct distinct nuclear and cytoplasmic aggregate deposition.

Disruption of the functional protein balance in living cells activates protective quality control systems to repair damaged proteins or sequester potentially cytotoxic misfolded proteins into aggregates. The established model based on Saccharomyces cerevisiae indicates that aggregating proteins in the cytosol of eukaryotic cells partition between cytosolic juxtanuclear (JUNQ) and peripheral deposits. Substrate ubiquitination acts as the sorting principle determining JUNQ deposition and subsequent degradation. Here, we show that JUNQ unexpectedly resides inside the nucleus, defining a new intranuclear quality control compartment, INQ, for the deposition of both nuclear and cytosolic misfolded proteins, irrespective of ubiquitination. Deposition of misfolded cytosolic proteins at INQ involves chaperone-assisted nuclear import via nuclear pores. The compartment-specific aggregases, Btn2 (nuclear) and Hsp42 (cytosolic), direct protein deposition to nuclear INQ and cytosolic (CytoQ) sites, respectively. Intriguingly, Btn2 is transiently induced by both protein folding stress and DNA replication stress, with DNA surveillance proteins accumulating at INQ. Our data therefore reveal a bipartite, inter-compartmental protein quality control system linked to DNA surveillance via INQ and Btn2.

Efficient Pictet-Spengler Bioconjugation with N-Substituted Pyrrolyl Alanine Derivatives.

We discovered N-pyrrolyl alanine derivatives as efficient reagents for the fast and selective Pictet-Spengler reaction with aldehyde-containing biomolecules. Other aldehyde-labeling methods described so far have several drawbacks, like hydrolytic instability, slow reaction kinetics or not readily available labeling reagents. Pictet-Spengler cyclizations of pyrrolyl 2-ethylamine substituted at the pyrrole nitrogen are significantly faster than with analogues substituted at the α- and ß- position. Functionalized N-pyrrolyl alanine derivatives can be synthesized in only 2-3 steps from commercially available materials. The small size of the reagent, the high reaction rate, and the easy synthesis make pyrrolyl alanine Pictet-Spengler (PAPS) an attractive choice for bioconjugation reactions. PAPS was shown as an efficient strategy for the site-selective biotinylation of an antibody as well as for the condensation of nucleic-acid derivatives, demonstrating the versatility of this reagent.

A system for the expression and release of heterologous proteins from the core of Bacillus subtilis spores.

Methodologies that exploit the durability of Bacillus subtilis spores by displaying heterologous proteins or antigenic molecules on the spore surface for mucosal vaccine delivery and other applications are well established. Here we extend the concept by engineering spores intended as oral delivery vehicles for therapeutic proteins. The method is exemplified by the expression and deposition of human growth hormone in the developing spore core, where the protein is shielded from physicochemical and biological degradation by the protective spore structure. Lysates from physically disrupted spores are shown to stimulate differentiation of a pre-adipocyte cell line to mature adipocyte cells, indicating that the spore-core located human growth hormone is folded correctly and functional. We also introduce a methodology for controlled release of heterologous proteins from the spore core, which utilises components of the PBSX prophage to lyse spores during germination and outgrowth. With further development, spore core expression, coupled with an engineered autolytic germination mechanism, may permit the use of spores as oral delivery carriers of therapeutic proteins.