The assembly of individual protein subunits into large-scale symmetrical structures is widespread in nature and confers new biological properties. Engineered protein assemblies have potential applications in nanotechnology and medicine; however, a major challenge in engineering assemblies de novo has been to design interactions between the protein subunits so that they specifically assemble into the desired structure. Here we demonstrate a simple, generalizable approach to assemble proteins into cage-like structures that uses short de novo designed coiled-coil domains to mediate assembly. We assembled eight copies of a C 3 -symmetric trimeric esterase into a well-defined octahedral protein cage by appending a C 4 -symmetric coiled-coil domain to the protein through a short, flexible linker sequence, with the approximate length of the linker sequence determined by computational modeling. The structure of the cage was verified using a combination of analytical ultracentrifugation, native electrospray mass spectrometry, and negative stain and cryoelectron microscopy. For the protein cage to assemble correctly, it was necessary to optimize the length of the linker sequence. This observation suggests that flexibility between the two protein domains is important to allow the protein subunits sufficient freedom to assemble into the geometry specified by the combination of C 4 and C 3 symmetry elements. Because this approach is inherently modular and places minimal requirements on the structural features of the protein building blocks, it could be extended to assemble a wide variety of proteins into structures with different symmetries.coiled coils | protein design | native mass spectrometry | analytical ultracentrifugation | cryoelectron microscopy

Histone deacetylases play important biological roles well beyond the deacetylation of histone tails. In particular, HDAC6 is involved in multiple cellular processes such as apoptosis, cytoskeleton reorganization, and protein folding, affecting substrates such as ɑ-tubulin, Hsp90 and cortactin proteins. We have applied a biochemical enzymatic assay to measure the activity of HDAC6 on a set of candidate unlabeled peptides. These served for the calibration of a structure-based substrate prediction protocol, Rosetta FlexPepBind, previously used for the successful substrate prediction of HDAC8 and other enzymes. A proteome-wide screen of reported acetylation sites using our calibrated protocol together with the enzymatic assay provide new peptide substrates and avenues to novel potential functional regulatory roles of this promiscuous, multi-faceted enzyme. In particular, we propose novel regulatory roles of HDAC6 in tumorigenesis and cancer cell survival via the regulation of EGFR/Akt pathway activation. The calibration process and comparison of the results between HDAC6 and HDAC8 highlight structural differences that explain the established promiscuity of HDAC6.

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Histone deacetylases play important biological roles well beyond the deacetylation of histone tails, and therefore have recently been renamed to acetyl-lysine deacetylases (KDACs). In particular, KDAC6 is involved in multiple cellular processes such as apoptosis, cytoskeleton reorganization, and protein folding, affecting substrates such as α-tubulin, Hsp90 and cortactin proteins. We have applied a biochemical enzymatic assay to measure the activity of KDAC6 on a set of candidate unlabeled peptides. These served for the calibration of a structure-based substrate prediction protocol, Rosetta FlexPepBind, previously used for the successful substrate prediction of KDAC8 and other enzymes. The calibration process and comparison of the results between KDAC6 and KDAC8 highlighted structural differences that explain the already reported promiscuity of KDAC6. A proteome-wide screen of reported acetylation sites using our calibrated protocol together with the enzymatic assay provide new peptide substrates and avenues to novel potential functional regulatory roles of this promiscuous, multi-faceted enzyme.

Acetylation is an important post‐translational modification (PTM). Aberrant acetylation, or deacetylation, has been linked to cancer, diabetes, neurodegeneration and auto‐immune disorders. Lysine acetylation is a reversible PTM, where deacetylation is catalyzed by histone deacetylases. Histone deacetylase function is crucial for a properly functioning cell, yet information about the specific biological pathways regulated by each isozyme is limited. HDAC1 is especially intriguing due to its known involvement in multiple stable nuclear complexes. However, the role of these complexes in regulating the deacetylase activity of HDAC1 is unclear. We have measured changes in activity and selectivity of HDAC1 upon addition of the following known protein binding partners; retinoblastoma protein 1 (Rb1), DNA methyltransferase 1 (DNMT1), rest corepressor 1 (CoREST), and lysine demethylase 1 (LSD1). We have reconstituted the HDAC1‐containing complexes with these proteins in vitro, as indicated by immunoprecipitation and size‐exclusion chromatography. Using substrate peptide analogs, we have measured the effect of binding partners on HDAC1 activity, demonstrating that these binding partners activate and increase HDAC1 activity to varying extents. Measurement of the reactivity of HDAC1 and HDAC1‐containing complexes with acetylated peptide substrates and full‐length protein substrates (i.e. histones) will provide insight into the role of binding partners in determining HDAC1 substrate selectivity. Support or Funding Information National Institutes of Health R01‐GM‐040602