Rpt5-Derived Analogs Stimulate Human Proteasome Activity in Cells and Degrade Proteins Forming Toxic Aggregates in Age-Related Diseases.

Aging and age-related diseases are associated with a decline in the capacity of protein turnover. Intrinsically disordered proteins, as well as proteins misfolded and oxidatively damaged, prone to aggregation, are preferentially digested by the ubiquitin-independent proteasome system (UIPS), a major component of which is the 20S proteasome. Therefore, boosting 20S activity constitutes a promising strategy to counteract a decrease in total proteasome activity during aging. One way to enhance the proteolytic removal of unwanted proteins appears to be the use of peptide-based activators of the 20S. In this study, we synthesized a series of peptides and peptidomimetics based on the C-terminus of the Rpt5 subunit of the 19S regulatory particle. Some of them efficiently stimulated human 20S proteasome activity. The attachment of the cell-penetrating peptide TAT allowed them to penetrate the cell membrane and stimulate proteasome activity in HEK293T cells, which was demonstrated using a cell-permeable substrate of the proteasome, TAS3. Furthermore, the best activator enhanced the degradation of aggregation-prone α-synuclein and Tau-441. The obtained compounds may therefore have the potential to compensate for the unbalanced proteostasis found in aging and age-related diseases.

Dissecting a role of a charge and conformation of Tat2 peptide in allosteric regulation of 20S proteasome.

Proteasome is a 'proteolytic factory' that constitutes an essential part of the ubiquitin-proteasome pathway. The involvement of proteasome in regulation of all major aspects of cellular physiology makes it an attractive drug target. So far, only inhibitors of the proteasome entered the clinic as anti-cancer drugs. However, proteasome regulators may also be useful for treatment of inflammatory and neurodegenerative diseases. We established in our previous studies that the peptide Tat2, comprising the basic domain of HIV-1 Tat protein: R(49) KKRRQRR(56) , supplemented with Q(66) DPI(69) fragment, inhibits the 20S proteasome in a noncompetitive manner. Mechanism of Tat2 likely involves allosteric regulation because it competes with the proteasome natural 11S activator for binding to the enzyme noncatalytic subunits. In this study, we performed alanine walking coupled with biological activity measurements and FTIR and CD spectroscopy to dissect contribution of a charge and conformation of Tat2 to its capability to influence peptidase activity of the proteasome. In solution, Tat2 and most of its analogs with a single Ala substitution preferentially adopted a conformation containing PPII/turn structural motifs. Replacing either Asp10 or two or more adjacent Arg/Lys residues induced a random coil conformation, probably by disrupting ionic interactions responsible for stabilization of the peptides ordered structure. The random coil Tat2 analogs lost their capability to activate the latent 20S proteasome. In contrast, inhibitory properties of the peptides more significantly depended on their positive charge. The data provide valuable clues for the future optimization of the Tat2-based proteasome regulators.

Peptidomimetics Based on C-Terminus of Blm10 Stimulate Human 20S Proteasome Activity and Promote Degradation of Proteins.

Degradation of misfolded, redundant and oxidatively damaged proteins constitutes one of the cellular processes which are influenced by the 20S proteasome. However, its activity is generally thought to decrease with age which leads to the gradual accumulation of abnormal proteins in cells and their subsequent aggregation. Therefore, increasing proteasomal degradation constitutes a promising strategy to delay the onset of various age-related diseases, including neurodegenerative disorders. In this study we designed and obtained a series of peptidomimetic stimulators of 20S comprising in their sequences the C-terminal fragment of Blm10 activator. Some of the compounds were capable of enhancing the degradation of natively unfolded and oxidatively damaged proteins, such as α-synuclein and enolase, whose applicability as proteasome substrates was evaluated by microscale thermophoresis (MST). Furthermore, they increased the ChT-L activity of the proteasome in HEK293T cell extracts. Our studies indicate that the 20S proteasome-mediated protein substrates hydrolysis may be selectively increased by peptide-based stimulators acting in an allosteric manner. These compounds, after further optimization, may have the potential to counteract proteasome impairment in patients suffering from age-related diseases.

Proteins, peptides and peptidomimetics as active agents in implant surface functionalization.

The recent impact of implants on improving the human life quality has been enormous. During the past two decades we witnessed major advancements in both material and structural development of implants. They were driven mainly by the increasing patients' demand and the need to address the major issues that come along with the initially underestimated complexity of the bone-implant interface. While both, the materials and design of implants reached a certain, balanced state, recent years brought a shift in focus towards the bone-implant interface as the weakest link in the increasing implant long-term usability. As a result, several approaches were developed. They aimed at influencing and enhancing the implant osseointegration and its proper behavior when under load and stress. With this review, we would like to discuss the recent advancements in the field of implant surface modifications, emphasizing the importance of chemical methods, focusing on proteins, peptides and peptidomimetics as promising agents for titanium surface coatings.

Proteasome Composition and Activity Changes in Cultured Fibroblasts Derived From Mucopolysaccharidoses Patients and Their Modulation by Genistein.

In this study, we have asked whether proteasome composition and function are affected in cells derived from patients suffering from all types of mucopolysaccharidosis (MPS), an inherited metabolic disease caused by accumulation of undegraded glycosaminoglycans (GAGs). Moreover, we have tested if genistein, a small molecule proposed previously as a potential therapeutic agent in MPS, can modulate proteasomes, which might shed a new light on the molecular mechanisms of action of this isoflavone as a potential drug for macromolecule storage diseases. Significant changes in expression of various proteasome-linked genes have been detected during transcriptomic (RNA-seq) analyses in vast majority of MPS types. These results were corroborated by demonstration of increased proteasomal activities in MPS cells. However, GAGs were not able to stimulate the 26S proteasome in vitro, suggesting that the observed activation in cells is indirect rather than arising from direct GAG-proteasome interactions. Genistein significantly reduced proteasomal activities in fibroblasts derived from patients suffering from all MPS types, while its effects on in vitro 26S proteasome activity were negligible. Unexpectedly, levels of many proteasomal subunits were increased in genistein-treated MPS cells. On the other hand, this ostensible discrepancy between results of experiments designed for estimation of effects of genistein on proteasome activities and abundance of proteasomal subunits can be explained by demonstration that in the presence of this isoflavone, levels of ubiquitinated proteins were decreased. The genistein-mediated reduction of proteasomal activities might have beneficial effects in cells of MPS patients due to potential increasing of residual activities of defective lysosomal enzymes which would otherwise be subjected to efficient ubiquitination and proteasomal degradation as misfolded proteins. These results indicate another activity of genistein (apart from previously demonstrated reduction of GAG synthesis efficiency, stimulation of lysosomal biogenesis, and activation of the autophagy process) which can be beneficial in the use of this small molecule in treatment of MPS.

Proline- and Arginine-Rich Peptides as Flexible Allosteric Modulators of Human Proteasome Activity.

Proline- and arginine-rich peptide PR11 is an allosteric inhibitor of 20S proteasome. We modified its sequence inter alia by introducing HbYX, RYX, or RHbX C-terminal extensions (Hb, hydrophobic moiety; R, arginine; Y, tyrosine; X, any residue). Consequently, we were able to improve inhibitory potency or to convert inhibitors into strong activators: the former with an aromatic penultimate Hb residue and the latter with the HbYX motif. The PR peptide activator stimulated 20S proteasome in vitro to efficiently degrade protein substrates, such as α-synuclein and enolase, but also activated proteasome in cultured fibroblasts. The positive and negative PR modulators differently influenced the proteasome conformational dynamics and affected opening of the substrate entry pore. The resolved crystal structure showed PR inhibitor bound far from the active sites, at the proteasome outer face, in the pocket used by natural activators. Our studies indicate the opportunity to tune proteasome activity by allosteric regulators based on PR peptide scaffold.

Crystal structure of a low molecular weight activator Blm-pep with yeast 20S proteasome - insights into the enzyme activation mechanism.

Proteasomes are responsible for protein turnover in eukaryotic cells, degrading short-lived species but also removing improperly folded or oxidatively damaged ones. Dysfunction of a proteasome results in gradual accumulation of misfolded/damaged proteins, leading to their aggregation. It has been postulated that proteasome activators may facilitate removal of such aggregation-prone proteins and thus prevent development of neurodegenerative disorders. However, the discovery of pharmacologically relevant compounds is hindered by insufficient structural understanding of the activation process. In this study we provide a model peptidic activator of human proteasome and analyze the structure-activity relationship within this novel scaffold. The binding mode of the activator at the relevant pocket within the proteasome has been determined by X-ray crystallography. This crystal structure provides an important basis for rational design of pharmacological compounds. Moreover, by providing a novel insight into the proteasome gating mechanism, our results allow the commonly accepted model of proteasome regulation to be revisited.

Interplay between Structure and Charge as a Key to Allosteric Modulation of Human 20S Proteasome by the Basic Fragment of HIV-1 Tat Protein.

The proteasome is a giant protease responsible for degradation of the majority of cytosolic proteins. Competitive inhibitors of the proteasome are used against aggressive blood cancers. However, broadening the use of proteasome-targeting drugs requires new mechanistic approaches to the enzyme's inhibition. In our previous studies we described Tat1 peptide, an allosteric inhibitor of the proteasome derived from a fragment of the basic domain of HIV-Tat1 protein. Here, we attempted to dissect the structural determinants of the proteasome inhibition by Tat1. Single- and multiple- alanine walking scans were performed. Tat1 analogs with stabilized beta-turn conformation at positions 4-5 and 8-9, pointed out by the molecular dynamics modeling and the alanine scan, were synthesized. Structure of Tat1 analogs were analyzed by circular dichroism, Fourier transform infrared and nuclear magnetic resonance spectroscopy studies, supplemented by molecular dynamics simulations. Biological activity tests and structural studies revealed that high flexibility and exposed positive charge are hallmarks of Tat1 peptide. Interestingly, stabilization of a beta-turn at the 8-9 position was necessary to significantly improve the inhibitory potency.