Starvation Induces Proteasome Autophagy with Different Pathways for Core and Regulatory Particles.

The proteasome is responsible for the degradation of many cellular proteins. If and how this abundant and normally stable complex is degraded by cells is largely unknown. Here we show that in yeast, upon nitrogen starvation, proteasomes are targeted for vacuolar degradation through autophagy. Using GFP-tagged proteasome subunits, we observed that autophagy of a core particle (CP) subunit depends on the deubiquitinating enzyme Ubp3, although a regulatory particle (RP) subunit does not. Furthermore, upon blocking of autophagy, RP remained largely nuclear, although CP largely localized to the cytosol as well as granular structures within the cytosol. In all, our data reveal a regulated process for the removal of proteasomes upon nitrogen starvation. This process involves CP and RP dissociation, nuclear export, and independent vacuolar targeting of CP and RP. Thus, in addition to the well characterized transcriptional up-regulation of genes encoding proteasome subunits, cells are also capable of down-regulating cellular levels of proteasomes through proteaphagy.

The proteasome-associated protein Ecm29 inhibits proteasomal ATPase activity and in vivo protein degradation by the proteasome.

Several proteasome-associated proteins regulate degradation by the 26 S proteasome using the ubiquitin chains that mark most substrates for degradation. The proteasome-associated protein Ecm29, however, has no ubiquitin-binding or modifying activity, and its direct effect on substrate degradation is unclear. Here, we show that Ecm29 acts as a proteasome inhibitor. Besides inhibiting the proteolytic cleavage of peptide substrates in vitro, it inhibits the degradation of ubiquitin-dependent and -independent substrates in vivo. Binding of Ecm29 to the proteasome induces a closed conformation of the substrate entry channel of the core particle. Furthermore, Ecm29 inhibits proteasomal ATPase activity, suggesting that the mechanism of inhibition and gate regulation by Ecm29 is through regulation of the proteasomal ATPases. Consistent with this, we identified through chemical cross-linking that Ecm29 binds to, or in close proximity to, the proteasomal ATPase subunit Rpt5. Additionally, we show that Ecm29 preferentially associates with both mutant and nucleotide depleted proteasomes. We propose that the inhibitory ability of Ecm29 is important for its function as a proteasome quality control factor by ensuring that aberrant proteasomes recognized by Ecm29 are inactive.

Assessing the feasibility, safety, and nutritional quality of using wild-caught pest flies in animal feed.

Studies have investigated the potential of using farmed insects in animal feeds; however, little research has been done using wild-caught insects for this purpose. Concerns about inadequate quantities collected, environmental impacts, and the spread of pathogens contribute to the preferred utilization of farmed insects. Nevertheless, by harvesting certain pest species from intensified agricultural operations, producers could provide their animals with affordable and sustainable protein sources while also reducing pest populations. This study explores the possibility of collecting large quantities of pest flies from livestock operations and analyzes the flies' nutritional content, potential pathogen load, and various disinfection methods. Using a newly designed mass collection-trapping device, we collected 5 kg of biomass over 13 wk, primarily house flies, from a poultry facility. While a substantial number of pests were removed from the environment, there was no reduction in the fly population. Short-read sequencing was used to compare the bacterial communities carried by flies from differing source populations, and the bacterial species present in the fly samples varied based on farm type and collection time. Drying and milling the wild-caught flies as well as applying an additional heat treatment significantly reduced the number of culturable bacteria present in or on the flies, though their pathogenicity remains unknown. Importantly, these disinfection methods did not affect the nutritional value of the processed flies. Further research is necessary to fully assess the safety and viability of integrating wild-caught insects into livestock feed; however, these data show promising results in favor of such a system.

Characterization of EHop-016, novel small molecule inhibitor of Rac GTPase.

The Rho GTPase Rac regulates actin cytoskeleton reorganization to form cell surface extensions (lamellipodia) required for cell migration/invasion during cancer metastasis. Rac hyperactivation and overexpression are associated with aggressive cancers; thus, interference of the interaction of Rac with its direct upstream activators, guanine nucleotide exchange factors (GEFs), is a viable strategy for inhibiting Rac activity. We synthesized EHop-016, a novel inhibitor of Rac activity, based on the structure of the established Rac/Rac GEF inhibitor NSC23766. Herein, we demonstrate that EHop-016 inhibits Rac activity in the MDA-MB-435 metastatic cancer cells that overexpress Rac and exhibits high endogenous Rac activity. The IC(50) of 1.1 µM for Rac inhibition by EHop-016 is ∼100-fold lower than for NSC23766. EHop-016 is specific for Rac1 and Rac3 at concentrations of ≤5 µM. At higher concentrations, EHop-016 inhibits the close homolog Cdc42. In MDA-MB-435 cells that demonstrate high active levels of the Rac GEF Vav2, EHop-016 inhibits the association of Vav2 with a nucleotide-free Rac1(G15A), which has a high affinity for activated GEFs. EHop-016 also inhibits the Rac activity of MDA-MB-231 metastatic breast cancer cells and reduces Rac-directed lamellipodia formation in both cell lines. EHop-016 decreases Rac downstream effects of PAK1 (p21-activated kinase 1) activity and directed migration of metastatic cancer cells. Moreover, at effective concentrations (<5 µM), EHop-016 does not affect the viability of transformed mammary epithelial cells (MCF-10A) and reduces viability of MDA-MB-435 cells by only 20%. Therefore, EHop-016 holds promise as a targeted therapeutic agent for the treatment of metastatic cancers with high Rac activity.

Identification of the atypical MAPK Erk3 as a novel substrate for p21-activated kinase (Pak) activity.

The class I p21-activated kinases (Pak1-3) regulate many essential biological processes, including cytoskeletal rearrangement, cell cycle progression, apoptosis, and cellular transformation. Although many Pak substrates, including elements of MAPK signaling cascades, have been identified, it is likely that additional substrates remain to be discovered. Identification of such substrates, and determination of the consequences of their phosphorylation, is essential for a better understanding of class I Pak activity. To identify novel class I Pak substrates, we used recombinant Pak2 to screen high density protein microarrays. This approach identified the atypical MAPK Erk3 as a potential Pak2 substrate. Solution-based in vitro kinase assays using recombinant Erk3 confirmed the protein microarray results, and phospho-specific antisera identified serine 189, within the Erk3 activation loop, as a site directly phosphorylated by Pak2 in vitro. Erk3 protein is known to shuttle between the cytoplasm and the nucleus, and we showed that selective inhibition of class I Pak kinase activity in cells promoted increased nuclear accumulation of Erk3. Pak inhibition in cells additionally reduced the extent of Ser(189) phosphorylation and inhibited the formation of Erk3-Prak complexes. Collectively, our results identify the Erk3 protein as a novel class I Pak substrate and further suggest a role for Pak kinase activity in atypical MAPK signaling.

Loss of Rpt5 protein interactions with the core particle and Nas2 protein causes the formation of faulty proteasomes that are inhibited by Ecm29 protein.

The proteasome is a large and complex protease formed by 66 polypeptides. The assembly of the proteasome is assisted by at least nine chaperones. One of these chaperones, Nas2/p27, binds to the C-terminal region of the AAA-ATPase Rpt5. We report here that the tail of Rpt5 provides two functions. First, it facilitates the previously reported interaction with the proteasome core particle (CP). Second, it is essential for the interaction with Nas2. Deletion of the C-terminal amino acid of Rpt5 disrupts the CP interaction, but not the binding to Nas2. The latter is surprising considering Nas2 contains a PDZ domain, which is often involved in binding to C termini. Interestingly, deletion of the last three amino acids interferes with both functions. The disruption of the Rpt5-CP interactions gave distinct phenotypes different from disruption of the Nas2-Rpt5 interaction. Additionally, proteasomes purified from a Saccharomyces cerevisiae rpt5-Δ3 strain show a strong enrichment of Ecm29. The function of Ecm29, a proteasome-associated protein, is not well understood. Our data show that Ecm29 can inhibit proteasomes, because our Ecm29-containing proteasomes have reduced suc-LLVY-AMC hydrolytic activity. Consistent with this apparent role as negative regulator, the deletion of ECM29 rescues the phenotypes of rpt5-Δ3 and nas2Δ in an hsm3Δ background. In sum, the interactions facilitated by the tail of Rpt5 act synergistically to minimize the formation of faulty proteasomes, thereby preventing recognition and inhibition by Ecm29.

Novel inhibitors of Rac1 in metastatic breast cancer.

OBJECTIVE: Rho family GTPases are molecular switches that control signaling pathways regulating a myriad of cellular functions. Rac1, a Rho family member, plays a critical role in several aspects of tumorigenesis, cancer progression, invasion, and metastasis. Rac proteins are not mutated in most invasive human cancers but are found to be overactive or over-expressed. Since Rho GTPases are activated by guanine nucleotide exchange factors (GEFs), inhibition of the interaction of Rac with its GEFs is a targeted strategy for blocking Rac activation. METHODS: The IC50 of NSC23766, an inhibitor of the interaction of Rac1 with a subset of GEFs, is too high for therapeutic use and more efficacious inhibitors are desired. Therefore, we initiated the synthesis of new derivatives of NSC23766 with modifications of the substituents connected to the central pyrimidine ring, and tested their Rac1 inhibitory activity. RESULTS: Several of the NSC23766 derivatives were shown to inhibit Rac1 activity of cancer cells with higher efficiency (20-50% more) than NSC23766. The new compounds are not toxic to normal mammary epithelial cells and are more efficient (60-70%) than NSC23766 in inhibiting cell migration and reducing cell spreading and extension of lamellipodia, cell functions regulated by Rac that contribute to cancer invasion. CONCLUSION: Based on the results, we conclude that the novel compounds show promise of further development as small molecule inhibitors of invasive breast cancer progression.

Individual and combined soy isoflavones exert differential effects on metastatic cancer progression.

To investigate the effects soy isoflavones in established cancers, the role of genistein, daidzein, and combined soy isoflavones was studied on progression of subcutaneous tumors in nude mice created from green fluorescent protein (GFP) tagged-MDA-MB-435 cells. Following tumor establishment, mice were gavaged with vehicle or genistein or daidzein at 10 mg/kg body weight (BW) or a combination of genistein (10 mg/kg BW), daidzein (9 mg/kg BW), and glycitein (1 mg/kg BW) three times per week. Tumor progression was quantified by whole body fluorescence image analysis followed by microscopic image analysis of excised organs for metastases. Results show that daidzein increased while genistein decreased mammary tumor growth by 38 and 33% respectively, compared to vehicle. Daidzein increased lung and heart metastases while genistein decreased bone and liver metastases. Combined soy isoflavones did not affect primary tumor growth but increased metastasis to all organs tested, which include lung, liver, heart, kidney, and bones. Phosphoinositide-3-kinase (PI3-K) pathway real time PCR array analysis and western blotting of excised tumors demonstrate that genistein significantly downregulated 10/84 genes, including the Rho GTPases RHOA, RAC1, and CDC42 and their effector PAK1. Daidzein significantly upregulated 9/84 genes that regulate proliferation and protein synthesis including EIF4G1, eIF4E, and survivin protein levels. Combined soy treatment significantly increased gene and protein levels of EIF4E and decreased TIRAP gene expression. Differential regulation of Rho GTPases, initiation factors, and survivin may account for the disparate responses of breast cancers to genistein and daidzein diets. This study indicates that consumption of soy foods may increase metastasis.