Protein kinase C activator bryostatin‐1 modulates proteasome function

Abstract

Proteasome activity in ubiquitin-proteasome pathway plays a pivotal role in degradation and clearance of aggregated, oxidized, damaged, and misfolded unwanted proteins to control protein homeostasis or proteostasis. Proteasome activity decreases with cellular senescence, aging, and age-related diseases. Therefore, enhancement of impaired proteasome function by molecular biological and/or pharmacological intervention is an active area of research. Bryostatin-1, a naturally occurring macrocyclic lactone, activates PKC isozymes (specifically, -α and -ϵ) at sub-nanomolar concentrations, but downregulates at higher concentrations. Here, we present bryostatin-1 increased chymotrypsin-like proteasome activity of 20S assembly at sub-nanomolar to nanomolar concentrations (0.3-30 nM). However, proteasome activity decreased at a micromolar concentration of bryostatin-1 (AG08044 cultured skin: P < 0.005; differentiated SH-SY5Y cells: P < 0.02). Modulation of proteasome function by bryostatin-1 was studied in six dermal fibroblast primary cell lines developed both from freshly taken biopsies from healthy donors (n = 2) and obtained from well-characterized cell repositories (n = 4; without any diseases). Bryostatin-1 enhanced proteasome activity in cultured skin fibroblasts obtained from banked and freshly isolated skin fibroblasts from skin biopsies at the sub-nanomolar concentration (P < 0.015). Modulation of proteasome function by bryostatin-1 was confirmed in neuron-like differentiated SH-SY5Y cells. Direct additions of bryostatin-1 into cell lysates prepared from neuron-like differentiated SH-SY5Y, Jurkat cells, and cultured skin fibroblasts were unable to increase proteasome activity indicating that bryostatin-1 can only modulate proteasome activity when added to live cell culture systems. Standard PKC inhibitors blocked bryostatin-1 induced proteasome activity modulation suggesting that enhancement of proteasome activity was mediated by PKC modulation.

Latency reversal agents modulate HIV antigen processing and presentation to CD8 T cells

“…hours to reach maximum effect both for HDACi and PKCa, suggesting a transcriptional effect either on the expression of the antigen processing machinery or on proteins regulating its activity rather than a direct binding to peptidases' catalytic sites. Bryostatin can enhance proteasome activities in fibroblasts [43] while disulfiram effect on NF-κB inhibited proteasome activities in cell lines [42]. The RNA expression of some proteasome subunits was modulated in an in vitro latency model upon LRA treatment [55] but neither proteasome expression and hydrolytic activities, nor the impact on antigen processing were assessed.…”

“…Peptides displayed by MHC-I to CD8 T or NK cells derive from the intracellular degradation of proteins by the antigen processing machinery, including proteasomes and post-proteasomal peptidases in the cytosol and endoplasmic reticulum (ER) where LRA-induced HIV antigens may be found [31]. The expression and hydrolytic activities of the antigen processing machineries are modulated by various stimuli such as cytokines [32], viral infection [33], TLR ligands [34][35][36], TCR-induced cellular activation [37], oxidative stress [38] or enzyme inhibitors [39][40][41][42][43]. We previously showed that TCR-mediated cellular activation enhanced peptidase activities, modulated protein degradation patterns into peptides and CTL recognition [37].…”

Unlocking the Drug Potential of the Bryostatin Family: Recent Advances in Product Synthesis and Biomedical Applications

“…Interestingly, the work of Khan et al work demonstrated that bryo 1 could increasep roteasome function through PKC modulation in cells. [43] 2.6 Challenges related to the biological activity of bryostatin 1…”

Modulation of proteasome activity by curcumin and didemethylcurcumin

“…The actual procedure of measurement of proteasome activity using fluorometric assay has been published elsewhere (Khan and Nelson, 2018). In principle, the assay monitored the chymotrypsin-like activity of the 20S proteasome assembly using a 7-amino-4-methylcoumarin (AMC)-tagged peptide substrate (Succ-LLVY-AMC) (BioVision (Milpitus, CA).…”

“…The procedure of standardization of the proteasome activity assay has been published elsewhere (Khan and Nelson, 2018). Briefly, standardizing reagent, we used a positive control sample (Jurkat Cell lysate; BioVision, Milpitas, CA) with significant proteasome activity for standardization of the fluorescence spectrometer.…”