Allosteric regulation of the proteasomes catalytic sites by the proteasome activator PA28gamma/REGgamma

Proteasome Activator 28γ (PA28γ) is a member of the 11S family of proteasomal regulators that is constitutively expressed in the nucleus and is implicated in certain cancers, lupus, rheumatoid arthritis, and Poly-glutamine neurodegenerative diseases. However, how PA28γ functions in protein degradation remains unclear. Though PA28γs mechanism has been investigated for some time, many alternative hypotheses have not been tested: e.g. 1) substrate selection, 2) allosteric upregulation of the Trypsin-like catalytic site, 3) allosteric inhibition of the Chymotrypsin- and Caspase-like catalytic sites, 4) conversion of the Chymotrypsin- or Caspase-like sites to new Trypsin-like catalytic sites, and 5) gate-opening in combination with these. The purpose of this study was to conclusively determine how PA28γ regulates proteasome function. Here, we rigorously and definitively show that PA28γ uses an allosteric mechanism to upregulate the proteolytic activity of the 20S proteasomes Trypsin-like catalytic site. Using a constitutively open channel proteasome, we were able to dissociate gating affects from catalytic affects demonstrating that the PA28γ-increases the affinity (Km) and Vmax for Trypsin-like peptide substrates. Mutagenesis of PA28γ also reveals that it does not select for (i.e. filter) peptide substrates, and does not change the specificity of the other active sites to trypsin-like. Further, using Cryo-EM we were able to visualize the C7 symmetric PA28γ-20S proteasome complex at 4.4A validating it's expected 11S-like quaternary structure and proteasome binding mode. The results of this study provide unambiguous evidence that PA28γ functions by allosterically upregulating the T-L like site in the 20S proteasome.

Plasma biomarkers of hemoglobin loss in Plasmodium falciparum-infected children identified by quantitative proteomics

Anemia is common among young children infected with Plasmodium falciparum (Pf) and severe malarial anemia (SMA) is a major cause of their mortality. Two major mechanisms cause malarial anemia: hemolysis of uninfected as well as infected erythrocytes and insufficient erythropoiesis. In a longitudinal birth cohort in Mali, we commonly observed marked hemoglobin reductions during Pf infections with a small proportion that progressed to SMA. We sought biomarkers of these processes using quantitative proteomic analysis on plasma samples from 9 P. falciparum-infected children, comparing those with reduced hemoglobin (with or without SMA) versus those with stable hemoglobin. We identified higher plasma levels of circulating 20S proteasome and lower IGF-1 levels in children with reduced hemoglobin. We confirmed these findings in independent ELISA-based validation studies of subsets of children from the same cohort (20S proteasome, N=71; IGF-1, N=78). We speculate that circulating 20S proteasome plays a role in digesting erythrocyte membrane proteins modified by oxidative stress, resulting in hemolysis, while decreased IGF-1, a critical factor for erythroid maturation, might contribute to insufficient erythropoiesis. Quantitative plasma proteomics identified soluble mediators that may contribute to the major mechanisms underlying malarial anemia.

Mitochondrial DNA metabolism is coupled with 20S proteasome function via regulation of deoxyribonucleotide homeostasis in Saccharomyces cerevisiae

The synthesis of mitochondrial DNA (mtDNA) is not coupled with cell cycle. Previous studies have shown that the size of deoxyribonucleoside triphosphate (dNTP) pools plays an important role in regulating mtDNA replication and amplification. In yeast, dNTPs are synthesized by the cytosolic ribonucleotide reductase (RNR). It is currently poorly understood as to how RNR activity is regulated in non-dividing or quiescent cells to finely tune mtDNA metabolism to cope with different metabolic states. Here, we show that defect in the 20S proteasome drastically destabilizes mtDNA. The mtDNA instability phenotype in 20S proteasome mutants is suppressed by overexpression of RNR3 or by the deletion of SML1, encoding a minor catalytic subunit and an intrinsic inhibitor of RNR respectively. We found that Sml1 is stabilized in the 20S proteasomal mutants, suggesting that 20S affects mtDNA stability by stabilizing Sml1. Interestingly, defect in the regulatory 19S proteasomal function has only subtle effect on mtDNA stability, supporting a role of the 20S proteasome in dNTP homeostasis independent of 19S. Finally, we found that when cells are transitioned from glycolytic to oxidative growth, Sml1 level is reduced in a 20S-dependent manner. In summary, our study establishes a link between cellular proteostasis and mtDNA metabolism through the regulation of dNTP homeostasis. We propose that increased degradation of Sml1 by the 20S proteasome under respiratory conditions provides a mechanism to stimulate dNTP synthesis and promote mtDNA amplification.

Plasma Exosomes of Patients with Breast and Ovarian Tumors Contain an Inactive 20S Proteasome

Exosomes are directly involved in governing of physiological and pathological conditions of an organism through the transfer of information from producing to receiving cells. It can be assumed that exosomes are one of the key players of tumor dissemination since they are very stable and small enough to penetrate from various tissues into biological fluids and then back, thus interacting with tissue target cells. We evaluated the enzymatic activity and the level of 20S proteasome in tissue and exosomes of healthy females (n = 39) and patients with ovarian (n = 50) and breast (n = 108) tumors to reveal the critical role of exosomal cargo in the mediation of different types of metastases. Exosomes from plasma and ascites were isolated and characterized in according to International Society for Extracellular Vesicles guidelines. The level of 20S proteasome in tissue and exosomes was determined using Western blot analysis. Chymotrypsin- and caspase-like (ChTL and CL, respectively) peptidase activities of the proteasomes were determined using fluorogenic Suc-LLVY-AMC and Cbz-LLG-AMC substrates, respectively. We observed increased levels of 20S proteasome in ovarian cancer tissue and luminal B subtype breast cancer tissue as well as in plasma exosomes from cancer patients. Moreover, the level of the 20S proteasome in plasma exosomes and ascites exosomes in patients with ovarian tumors is comparable and higher in ovarian cancer patients with low volume ascites than in patients with moderate and high-volume ascites. We also found increased ChTL and CL activities in breast cancer and ovarian cancer tissues, as well as in peritoneal metastases in ovarian cancer, while proteasomal activity in exosomes from plasma of healthy females and all patients, as well as from ascites of ovarian tumor patients were lower than detection limit of assay. Thus, regardless of the type of tumor metastasis (lymphogenous or peritoneal), the exosomes of cancer patients were characterized by an increased level of 20S proteasome, which do not exhibit enzymatic activity.

Clinical Relevance of Elevated Soluble ST2, HSP27 and 20S Proteasome at Hospital Admission in Patients with COVID-19

Although, severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) represents one of the biggest challenges in the world today, the exact immunopathogenic mechanism that leads to severe or critical Coronavirus Disease 2019 (COVID-19) has remained incompletely understood. Several studies have indicated that high systemic plasma levels of inflammatory cytokines result in the so-called “cytokine storm”, with subsequent development of microthrombosis, disseminated intravascular coagulation, and multiorgan-failure. Therefore, we reasoned those elevated inflammatory molecules might act as prognostic factors. Here, we analyzed 245 serum samples of patients with COVID-19, collected at hospital admission. We assessed the levels of heat shock protein 27 (HSP27), soluble suppressor of tumorigenicity-2 (sST2) and 20S proteasome at hospital admission and explored their associations with overall-, 30-, 60-, 90-day- and in-hospital mortality. Moreover, we investigated their association with the risk of ventilation. We demonstrated that increased serum sST2 was uni- and multivariably associated with all endpoints. Furthermore, we also identified 20S proteasome as independent prognostic factor for in-hospital mortality (sST2, AUC = 0.73; HSP27, AUC = 0.59; 20S proteasome = 0.67). Elevated sST2, HSP27, and 20S proteasome levels at hospital admission were univariably associated with higher risk of invasive ventilation (OR = 1.8; p < 0.001; OR = 1.1; p = 0.04; OR = 1.03, p = 0.03, respectively). These findings could help to identify high-risk patients early in the course of COVID-19.

Temporal disruption of neuromuscular communication and muscle atrophy following non-invasive ACL injury in rats

Many patients with anterior cruciate ligament (ACL) injuries have persistent quadriceps muscle atrophy, even after considerable time in rehabilitation. Understanding the factors that regulate muscle mass, and the time course of atrophic events, is important for identifying therapeutic interventions. Using a non-invasive animal model of ACL injury, a longitudinal study was performed to elucidate key parameters underlying quadriceps muscle atrophy. Male Long-Evans rats were euthanized at 6, 12, 24, 48-hrs and 1, 2, 4-wks after ACL injury that was induced via tibial compression overload; controls were not injured. Vastus Lateralis muscle size was determined by wet weight and fiber CSA. Evidence of disrupted neuromuscular communication was assessed via the expression of NCAM and genes associated with denervation and neuromuscular junction instability. Abundance of MuRF-1, MAFbx, and 45s pre-rRNA along with 20S proteasome activity were determined to investigate mechanisms related to muscle atrophy. Lastly, muscle damage-related parameters were assessed by measuring IgG permeability, centronucleation, CD68 mRNA and satellite cell abundance. Compared to controls, we observed a greater percentage of NCAM positive fibers at 6-hrs post-injury, followed by higher MAFbx abundance 48-hrs post-injury, and higher 20S proteasome activity at 1-wk post-injury. A loss of muscle wet weight, smaller fiber CSA and the elevated expression of Runx1 were also observed at the 1-wk post-injury time point relative to controls. There also were no differences observed in any damage markers. These results indicate that alterations in neuromuscular communication precede the upregulation of atrophic factors that regulate quadriceps muscle mass early after non-invasive ACL injury.