Minimal mechanistic component of proteasome activation and prevention of impairment by pathological oligomers

Impairment of proteasomal function has been implicated in neurodegenerative diseases, justifying the need to understand how the proteasome is activated for protein degradation. Here, using biochemical and structural (cryo-EM) strategies in both archaeal and mammalian proteasomes, we further determine the HbYX(-motif)-dependent mechanism of proteasomal activation used by multiple proteasome-activating complexes including the 19S Particle. We identify multiple proteasome α subunit residues involved in HbYX-dependent activation, a point mutation that activates the proteasome by partially mimicking a HbYX-bound state, and conformational changes involved in gate-opening with a 2.0A structure. Through an iterative process of peptide synthesis, we successfully design a HbYX-like dipeptide mimetic as a robust tool to elucidate how the motif autonomously activates the proteasome. The mimetic induces near complete gate-opening at saturating concentration, activating mammalian proteasomal degradation of peptides and proteins. Findings using our peptide mimetic suggest the HbYX-dependent mechanism requires cooperative binding in at least two intersubunit pockets of the α ring. Collectively, the results presented here unambiguously demonstrate the lone role of the HbYX tyrosine in the allosteric mechanism of proteasome activation and offer proof of concept for the robust potential of HbYX-like small molecules to activate the proteasome.

Impact of Storage Conditions on the Breast Milk Peptidome

Human donor milk (HDM) provides appropriate nutrition and offers protective functions in preterm infants. The aim of the study is to examine the impact of different storage conditions on the stability of the human breast milk peptidome. HDM was directly frozen at −80 °C or stored at −20 °C (120 h), 4 °C (6 h), or room temperature (RT for 6 or 24 h). The milk peptidome was profiled by mass spectrometry after peptide collection by ultrafiltration. Profiling of the peptidome covered 3587 peptides corresponding to 212 proteins. The variance of the peptidome increased with storage temperature and time and varied for different peptides. The highest impact was observed when samples were stored at RT. Smaller but significant effects were still observed in samples stored at 4 °C, while samples showed highest similarity to those immediately frozen at −80 °C when stored at −20 °C. Peptide structures after storage at RT for 24 h point to the increased activity of thrombin and other proteases cleaving proteins at lysine/arginine. The results point to an ongoing protein degradation/peptide production by milk-derived proteases. They underline the need for immediate freezing of HDM at −20 °C or −80 °C to prevent degradation of peptides and enable reproducible investigation of prospectively collected samples.

Monitoring Stepwise Proteolytic Degradation of Peptides by Supramolecular Domino Tandem Assays and Mass Spectrometry for Trypsin and Leucine Aminopeptidase

A label-free optical detection method has been designed that allows direct monitoring of enzymatic peptide digestion in vitro. The method is based on the addition of a reporter pair, composed of the macrocyclic host cucurbit[7]uril (CB7) and the fluorescent dye acridine orange (AO), to detect the proteolytic degradation of peptides. The enzymatic activity of trypsin and leucine aminopeptidase (LAP) was investigated using H-LSRFSWGA-OH as a substrate. The substrate as well as the intermediary and final products (i.e., H-FSWGA-OH and phenylalanine) formed during its enzymatic hydrolysis differ in their binding affinity to the receptor CB7, which results in varying degrees of dye displacement and, therefore, different fluorescence intensities. CB7 showed a relatively weak binding constant of K ≈ 104 M–1 with the substrate, a relatively strong binding constant of K ≥ 106 M–1 with H-FSWGA-OH (which is a final product formed by trypsin digestion and the intermediary product formed during the enzymatic activity of LAP), and a moderate binding constant of K ≤ 105 M–1 with phenylalanine. Owing to this differential binding affinity of CB7 with the substrate and the corresponding products, the digestion of a peptide by trypsin was followed as a decrease in fluorescence signal, while the complete degradation of the peptide by LAP was monitored as a decrease and a subsequent increase in fluorescence signal. The kcat/ KM value for trypsin (2.0 × 107 min–1M–1) was derived from the change in fluorescence signal with time. Additionally, the complete degradation of the peptide by LAP was also followed by mass spectrometry. The use of a supramolecular sensing ensemble (macrocyclic host and dye) as a fluorescent reporter pair gives this method the flexibility to adapt for monitoring the stepwise degradation of different biologically relevant peptides by other proteases.