The chemical compound 'Heatin' stimulates hypocotyl elongation and interferes with the Arabidopsis NIT1-subfamily of nitrilases.

Temperature passively affects biological processes involved in plant growth. Therefore, it is challenging to study the dedicated temperature signalling pathways that orchestrate thermomorphogenesis, a suite of elongation growth-based adaptations that enhance leaf-cooling capacity. We screened a chemical library for compounds that restored hypocotyl elongation in the pif4-2-deficient mutant background at warm temperature conditions in Arabidopsis thaliana to identify modulators of thermomorphogenesis. The small aromatic compound 'Heatin', containing 1-iminomethyl-2-naphthol as a pharmacophore, was selected as an enhancer of elongation growth. We show that ARABIDOPSIS ALDEHYDE OXIDASES redundantly contribute to Heatin-mediated hypocotyl elongation. Following a chemical proteomics approach, the members of the NITRILASE1-subfamily of auxin biosynthesis enzymes were identified among the molecular targets of Heatin. Our data reveal that nitrilases are involved in promotion of hypocotyl elongation in response to high temperature and Heatin-mediated hypocotyl elongation requires the NITRILASE1-subfamily members, NIT1 and NIT2. Heatin inhibits NIT1-subfamily enzymatic activity in vitro and the application of Heatin accordingly results in the accumulation of NIT1-subfamily substrate indole-3-acetonitrile in vivo. However, levels of the NIT1-subfamily product, bioactive auxin (indole-3-acetic acid), were also significantly increased. It is likely that the stimulation of hypocotyl elongation by Heatin might be independent of its observed interaction with NITRILASE1-subfamily members. However, nitrilases may contribute to the Heatin response by stimulating indole-3-acetic acid biosynthesis in an indirect way. Heatin and its functional analogues present novel chemical entities for studying auxin biology.

Liposome functionalization with copper-free "click chemistry".

The modification of liposomal surfaces is of interest for many different applications and a variety of chemistries are available that makes this possible. A major disadvantage of commonly used coupling chemistries (e.g. maleimide-thiol coupling) is the limited control over the site of conjugation in cases where multiple reactive functionalities are present, leading to heterogeneous products and in some cases dysfunctional conjugates. Bioorthogonal coupling approaches such as the well-established copper-catalyzed azide-alkyne cycloaddition (CuAAC) "click" reaction are attractive alternatives as the reaction kinetics are favorable and azide-containing reagents are widely available. In the work described here, we prepared lipids containing a reactive cyclooctyne group and, after incorporation into liposomes, demonstrated successful conjugation of both a small molecule dye (5'-TAMRA-azide) as well as a larger azide-containing model protein based upon a designed ankyrin repeat protein (azido-DARPin). By applying the strain-promoted azido-alkyne cycloaddition (SPAAC) the use of Cu(I) as a catalyst is avoided, an important advantage considering the known deleterious effects associated with copper in cell and protein studies. We demonstrate complete control over the number of ligands coupled per liposome when using a small molecule azide with conjugation occurring at a reasonable reaction rate. By comparison, the conjugation of a larger azide-modified protein occurs more slowly, however the number of protein ligands coupled was found to be sufficient for liposome targeting to cells. Importantly, these results provide a strong proof of concept for the site-specific conjugation of protein ligands to liposomal surfaces via SPAAC. Unlike conventional approaches, this strategy provides for the homogeneous coupling of proteins bearing a single site-specific azide modification and eliminates the chance of forming dysfunctional ligands on the liposome. Furthermore, the absence of copper in the reaction process should also make this approach much more compatible with cell-based and in vivo applications.

Charting the interactome of PDE3A in human cells using an IBMX based chemical proteomics approach.

In the cell the second messenger cyclic nucleotides cAMP and cGMP mediate a wide variety of external signals. Both signaling molecules are degraded by the superfamily of phosphodiesterases (PDEs) consisting of more than 50 different isoforms. Several of these PDEs are implicated in disease processes inspiring the quest for and synthesis of selective PDE inhibitors, that unfortunately have led to very mixed successes in clinical trials. This may be partially caused by their pharmacological action. Accumulating data suggests that small differences between different PDE isoforms may already result in specific tissue distributions, cellular localization and different involvement in higher order signal protein complexes. The role of PDEs in these higher order signal protein complexes has only been marginally addressed, as no screening methodology is available to address this in a more comprehensive way. Affinity based chemical proteomics is a relatively new tool to identify specific protein-protein interactions. Here, to study the interactome of PDEs, we synthesized a broad spectrum PDE-capturing resin based on the non-selective PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX). Chemical proteomics characterization of this resin in HeLa cell lysates led to the capture of several different PDEs. Combining the IBMX-resin with in-solution competition with the available more selective PDE inhibitors, cilostamide and papaverine, allowed us to selectively probe the interactome of PDE3A in HeLa cells. Besides known interactors such as the family of 14-3-3 proteins, PDE3A was found to associate with a PP2A complex composed of a regulatory, scaffold and catalytic subunit.