In all cells, proteins are continuously synthesized and degraded in order to maintain protein homeostasis and modify gene expression levels in response to stimuli. Collectively, the processes of protein synthesis and degradation are referred to as protein turnover. At steady state, protein turnover is constant to maintain protein homeostasis, but in dynamic responses, proteins change their rates of synthesis and degradation in order to adjust their proteomes to internal or external stimuli. Thus, probing the kinetics and dynamics of protein turnover lends insight into how cells regulate essential processes such as growth, differentiation, and stress response. Here we outline historical and current approaches to measuring the kinetics of protein turnover on a proteome-wide scale in both steady-state and dynamic systems, with an emphasis on metabolic tracing using stable-isotope-labeled amino acids. We highlight important considerations for designing proteome turnover experiments, key biological findings regarding the conserved principles of proteome turnover regulation, and future perspectives for both technological and biological investigation.

Activation of translation initiation is a common trait of cancer cells. Formation of the heterotrimeric eukaryotic initiation factor F (eIF4F) complex is the rate-limiting step in 5′ m7GpppN cap-dependent translation. This trimeric complex includes the eIF4E cap binding protein, the eIF4G scaffolding protein, and the DEAD box RNA helicase eIF4A. eIF4A is an ATP-dependent helicase and because it is the only enzyme in the eIF4F complex, it has been shown to be a potential therapeutic target for a variety of malignancies. To this end, we have used a simple ATPase biochemical screen to survey several hundred marine and terrestrial derived natural products. Herein, we report the discovery of two natural products from marine sources, elisabatin A (1) and allolaurinterol (2), which show low µM inhibition of eIF4A ATPase activity. Enzymological analyses revealed 1 and 2 to be ATP-competitive, and cellular evaluations showed reasonable cytotoxicity against A549 (lung cancer) and MDA-MA-468 (breast cancer) cell lines. However, only compound 2 showed potent inhibition of helicase activity congruent with its ATPase inhibitory activity.

Glucose regulated protein 78 (GRP78) is the ER resident 70 kDa heat shock protein 70 (HSP70) and has been hypothesized to be a therapeutic target for various forms of cancer due to its role in mitigating proteotoxic stress in the ER, its elevated expression in some cancers, and the correlation between high levels for GRP78 and a poor prognosis. Herein we report the development and use of a high throughput fluorescence polarization-based peptide binding assay as an initial step toward the discovery and development of GRP78 inhibitors. This assay was used in a pilot screen to discover the anti-infective agent, hexachlorophene, as an inhibitor of GRP78. Through biochemical characterization we show that hexachlorophene is a competitive inhibitor of the GRP78-peptide interaction. Biological investigations showed that this molecule induces the unfolded protein response, induces autophagy, and leads to apoptosis in a colon carcinoma cell model, which is known to be sensitive to GRP78 inhibition.

A library of natural products and their derivatives was screened for inhibition of protein tyrosine phosphatase (PTP) 1B, which is a validated drug target for the treatment of obesity and type II diabetes. Of those active in the preliminary assay, the most promising was compound 2 containing a novel pyrrolopyrazoloisoquinolone scaffold derived by treating radicicol (1) with hydrazine. This nitrogen-atom augmented radicicol derivative was found to be PTP1B selective relative to other highly homologous nonreceptor PTPs. Biochemical evaluation, molecular docking, and mutagenesis revealed 2 to be an allosteric inhibitor of PTP1B with a submicromolar K i. Cellular analyses using C2C12 myoblasts indicated that 2 restored insulin signaling and increased glucose uptake.

Certain long non-coding RNAs (lncRNAs) are known to contain small open reading frames that can be translated. Here we describe a much larger 25 kDa human protein, “ R ibosomal I GS E ncoded P rotein” (RIEP), that remarkably is encoded by the well-characterized RNA polymerase (RNAP) II-transcribed nucleolar “promoter and pre-rRNA antisense” lncRNA ( PAPAS ). Strikingly, RIEP, which is conserved throughout primates but not found in other species, predominantly localizes to the nucleolus as well as mitochondria, but both exogenously expressed and endogenous RIEP increase in the nuclear and perinuclear regions upon heat shock (HS). RIEP associates specifically with the rDNA locus, increases levels of the RNA:DNA helicase Senataxin, and functions to sharply reduce DNA damage induced by heat shock. Proteomics analysis identified two mitochondrial proteins, C1QBP and CHCHD2, both known to have mitochondrial and nuclear functions, that we show interact directly, and relocalize following heat shock, with RIEP. Finally, it is especially notable that the rDNA sequences encoding RIEP are multifunctional, giving rise to an RNA that functions both as RIEP messenger RNA (mRNA) and as PAPAS lncRNA, as well as containing the promoter sequences responsible for rRNA synthesis by RNAP I. Our work has thus not only shown that a nucleolar “non-coding” RNA in fact encodes a protein, but also established a novel link between mitochondria and nucleoli that contributes to the cellular stress response.