Rapid flow-based synthesis of post-translationally modified peptides and proteins: a case study on MYC's transactivation domain.

Protein-protein interactions of c-Myc (MYC) are often regulated by post-translational modifications (PTMs), such as phosphorylation, and crosstalk thereof. Studying these interactions requires proteins with unique PTM patterns, which are challenging to obtain by recombinant methods. Standard peptide synthesis and native chemical ligation can produce such modified proteins, but are time-consuming and therefore typically limited to the study of individual PTMs. Herein, we report the development of flow-based methods for the rapid synthesis of phosphorylated MYC sequences (up to 84 AA), and demonstrate the versatility of this approach for the incorporation of other PTMs (N ε-methylation, sulfation, acetylation, glycosylation) and combinations thereof. Peptides containing up to seven PTMs and phosphorylation at up to five sites were successfully prepared and isolated in high yield and purity. We further produced ten PTM-decorated analogues of the MYC Transactivation Domain (TAD) to screen for binding to the tumor suppressor protein, Bin1, using heteronuclear NMR and native mass spectrometry. We determined the effects of phosphorylation and glycosylation on the strength of the MYC:Bin1 interaction, and reveal an influence of MYC sequence length on binding. Our platform for the rapid synthesis of MYC sequences up to 84 AA with distinct PTM patterns thus enables the systematic study of PTM function at a molecular level, and offers a convenient way for expedited screening of constructs.

Native mass spectrometry interrogation of complexes formed during targeted protein degradation.

RATIONALE: Protein degraders are small molecules that promote cellular degradation of a target protein. Degraders simultaneously bind to their target and an E3 ligase, bringing them into close spatial proximity, but the formation of this ternary complex is difficult to measure with many biophysical techniques. METHODS: Native mass spectrometry (nMS) is an effective label-free technique to identify the complexes formed by proteolysis-targeting chimeras (PROTACs). It can monitor the formation of ternary E3-PROTAC-target complexes and detect intermediate binary species. Experiments are described using a Synapt G2Si (Waters) equipped with a nano-electrospray ionisation source. RESULTS: The protocol describes nMS experiments for measuring the complexes formed by PROTAC molecules. It also describes how to investigate differences in the affinity of PROTAC complexes, whether a PROTAC shows specificity for a given target and whether a PROTAC shows cooperative behaviour. CONCLUSIONS: Here, we provide step-by-step instructions for the sample preparation of PROTAC complexes and their nMS interrogation to obtain optimal information on their binding modes.

Hyaluronan-carnosine conjugates inhibit Aß aggregation and toxicity.

Alzheimer's disease is the most common neurodegenerative disorder. Finding a pharmacological approach that cures and/or prevents the onset of this devastating disease represents an important challenge for researchers. According to the amyloid cascade hypothesis, increases in extracellular amyloid-ß (Aß) levels give rise to different aggregated species, such as protofibrils, fibrils and oligomers, with oligomers being the more toxic species for cells. Many efforts have recently been focused on multi-target ligands to address the multiple events that occur concurrently with toxic aggregation at the onset of the disease. Moreover, investigating the effect of endogenous compounds or a combination thereof is a promising approach to prevent the side effects of entirely synthetic drugs. In this work, we report the synthesis, structural characterization and Aß antiaggregant ability of new derivatives of hyaluronic acid (Hy, 200 and 700 kDa) functionalized with carnosine (Car), a multi-functional natural dipeptide. The bioactive substances (HyCar) inhibit the formation of amyloid-type aggregates of Aß42 more than the parent compounds; this effect is proportional to Car loading. Furthermore, the HyCar derivatives are able to dissolve the amyloid fibrils and to reduce Aß-induced toxicity in vitro. The enzymatic degradation of Aß is also affected by the interaction with HyCar.

Structural and functional evidence for citicoline binding and modulation of 20S proteasome activity: Novel insights into its pro-proteostatic effect.

Citicoline or CDP-choline is a drug, made up by a cytidine 5'-diphosphate moiety and choline, which upon adsorption is rapidly hydrolyzed into cytidine 5'-diphosphate and choline, easily bypassing the blood-brain barrier. Once in the brain, these metabolites are used to re-synthesize citicoline in neurons and in the other cell histo-types which uptake them. Citicoline administration finds broad therapeutic application in the treatment of glaucoma as well as other retinal disorders by virtue of its safety profile and neuro-protective and neuroenhancer activity, which significantly improves the visual function. Further, though supported by limited clinical studies, this molecule finds therapeutic application in neurodegenerative disease, delaying the cognitive decline in Alzheimer's Disease (AD) and Parkinson's Disease (PD) subjects. In this work we show that citicoline greatly affects the proteolytic activity of the 20S proteasome on synthetic and natural substrates, functioning as a bimodal allosteric modulator, likely binding at multiple sites. In silico binding simulations identify several potential binding sites for citicoline on 20S proteasome, and their topology envisages the possibility that, by occupying some of these pockets, citicoline may induce a conformational shift of the 20S proteasome, allowing to sketch a working hypothesis for the structural basis of its function as allosteric modulator. In addition, we show that over the same concentration range citicoline affects the distribution of assembled proteasome populations and turn-over of ubiquitinated proteins in SH-SY5Y and SK-N-BE human neuroblastoma cells, suggesting its potential role as a regulator of proteostasis in nervous cells.

Acrolein and Copper as Competitive Effectors of α-Synuclein.

Mounting evidence supports the role of amyloidogenesis, oxidative stress, and metal dyshomeostasis in the development of neurodegenerative disorders. Parkinson's Disease is characterized by α-synuclein (αSyn) accumulation and aggregation in brain regions, also promoted by Cu2+ . αSyn is modified by reactive carbonyl species, including acrolein (ACR). Notwithstanding these findings, the interplay between ACR, copper, and αSyn has never been investigated. Therefore, we explored more thoroughly the effects of ACR on αSyn using an approach based on LC-MS/MS analysis. We also evaluated the influence of Cu2+ on the protein carbonylation and how the ACR modification impacts the Cu2+ binding and the production of Reactive Oxygen Species (ROS). Finally, we investigated the effects of ACR and Cu2+ ions on the αSyn aggregation by dynamic light scattering and fluorescence assays. Cu2+ regioselectively inhibits the modification of His50 by ACR, the carbonylation lowers the affinity of His50 for Cu2+ and ACR inhibits αSyn aggregation both in the presence and in the absence of Cu2+ .

Pyrazolones Activate the Proteasome by Gating Mechanisms and Protect Neuronal Cells from ß-Amyloid Toxicity.

Proteasome malfunction parallels abnormal amyloid accumulation in Alzheimer's Disease (AD). Here we scrutinize a small library of pyrazolones by assaying their ability to enhance proteasome activity and protect neuronal cells from amyloid toxicity. Tube tests evidenced that aminopyrine and nifenazone behave as 20S proteasome activators. Enzyme assays carried out on an "open gate" mutant (α3ΔN) proteasome demonstrated that aminopyrine activates proteasome through binding the α-ring surfaces and influencing gating dynamics. Docking studies coupled with STD-NMR experiments showed that H-bonds and π-π stacking interactions between pyrazolones and the enzyme play a key role in bridging α1 to α2 and, alternatively, α5 to α6 subunits of the outer α-ring. Aminopyrine and nifenazone exhibit neurotrophic properties and protect differentiated human neuroblastoma SH-SY5Y cells from ß-amyloid (Aß) toxicity. ESI-MS studies confirmed that aminopyrine enhances Aß degradation by proteasome in a dose-dependent manner. Our results suggest that some pyrazolones and, in particular, aminopyrine are promising compounds for the development of proteasome activators for AD treatment.