The Function of Immunoproteasomes—An Immunologists’ Perspective

Proteasomes are responsible for intracellular proteolysis and play an important role in cellular protein homeostasis. Cells of the immune system assemble a specialized form of proteasomes, known as immunoproteasomes, in which the constitutive catalytic sites are replaced for cytokine-inducible homologues. While immunoproteasomes may fulfill all standard proteasome’ functions, they seem specially adapted for a role in MHC class I antigen processing and CD8+ T-cell activation. In this way, they may contribute to CD8+ T-cell-mediated control of intracellular infections, but also to the immunopathogenesis of autoimmune diseases. Starting at the discovery of its catalytic subunits in the genome, here, we review the observations shaping our current understanding of immunoproteasome function, and the consequential novel opportunities for immune intervention.

Small-Molecule Inhibitors Targeting Proteasome-Associated Deubiquitinases

The 26S proteasome is the principal protease for regulated intracellular proteolysis. This multi-subunit complex is also pivotal for clearance of harmful proteins that are produced throughout the lifetime of eukaryotes. Recent structural and kinetic studies have revealed a multitude of conformational states of the proteasome in substrate-free and substrate-engaged forms. These conformational transitions demonstrate that proteasome is a highly dynamic machinery during substrate processing that can be also controlled by a number of proteasome-associated factors. Essentially, three distinct family of deubiquitinases–USP14, RPN11, and UCH37–are associated with the 19S regulatory particle of human proteasome. USP14 and UCH37 are capable of editing ubiquitin conjugates during the process of their dynamic engagement into the proteasome prior to the catalytic commitment. In contrast, RPN11-mediated deubiquitination is directly coupled to substrate degradation by sensing the proteasome’s conformational switch into the commitment steps. Therefore, proteasome-bound deubiquitinases are likely to tailor the degradation events in accordance with substrate processing steps and for dynamic proteolysis outcomes. Recent chemical screening efforts have yielded highly selective small-molecule inhibitors for targeting proteasomal deubiquitinases, such as USP14 and RPN11. USP14 inhibitors, IU1 and its progeny, were found to promote the degradation of a subset of substrates probably by overriding USP14-imposed checkpoint on the proteasome. On the other hand, capzimin, a RPN11 inhibitor, stabilized the proteasome substrates and showed the anti-proliferative effects on cancer cells. It is highly conceivable that these specific inhibitors will aid to dissect the role of each deubiquitinase on the proteasome. Moreover, customized targeting of proteasome-associated deubiquitinases may also provide versatile therapeutic strategies for induced or repressed protein degradation depending on proteolytic demand and cellular context.

Flow Cytometric Assessment of the Viability and Functionality of Uterine Polymorphonuclear Leukocytes in Postpartum Dairy Cows

Postpartum dairy cows experience impaired peripheral polymorphonuclear leukocyte (PMN) functionality, which has been associated with reproductive tract inflammatory diseases. However, it has not been elucidated yet whether endometrial PMN functionality is (equally) impaired. We developed a method for endometrial PMN isolation and flow cytometric assessment of their viability and functionality. We also evaluated PMN immunolabeling, using a specific bovine granulocyte marker, CH138A. Blood and endometrial cytobrush samples were collected in duplicate from seventeen clinically healthy Holstein-Friesian cows between 9 and 37 days in milk. The proportion of viable, apoptotic, and necrotic PMN in endometrial samples roughly ranged from 10 to 80%, indicating highly dynamic endometrial PMN populations in the postpartum uteri. Endometrial PMN functionality testing revealed that PMN immunolabeling increased the accuracy, although this protocol might influence the median fluorescence intensity of the sample. Phagocytosis seemed the most stable and reliable endometrial PMN function and could be assessed satisfactorily without prior CH138A immunolabeling. However, the interpretation of oxidative burst and intracellular proteolysis tests remains challenging. The correlation between peripheral and endometrial PMN functionality was poor. Further research is warranted to unravel the role of uterine PMN viability and functionality in bovine uterine health.

Calpain-Associated Proteolytic Regulation of the Stromal Microenvironment in Cancer

Background: Normalization of the stromal microenvironment is a promising strategy for cancer control. Cancer-associated fibroblasts, tumor-associated macrophages, and mesenchymal stromal cells have a central role in stromal functions. Accordingly, understanding these stromal cells is indispensable for the development of next-generation cancer therapies. Growing evidence suggests that calpain-induced intracellular proteolysis is responsible for cancer growth and stromal regulation. Calpain is a family of stress-responsive intracellular proteases and is inducible in cancer and stromal cells during carcinogenesis. Objective: Here, we shed light on the recent advances that have been made in understanding how calpain contributes to stromal regulation in cancer. Conclusions: Calpains are activated in stromal cells, including pancreatic stellate cells and mesenchymal cells, thereby inducing fibrogenic responses in cancer stroma. Moreover, these molecules contribute to epithelial-mesenchymal transition and endothelial-mesenchymal transition to provide mesenchymal stromal cells in the microenvironment, and concomitantly participate in cancer angiogenesis. In addition to the conventional calpains, the unconventional calpain-9 is associated with epithelial-mesenchymal transition. Animal experiments showed that targeting calpain systems antagonizes cancer development; thus, this approach is promising for cancer control.

Targeted Protein Degradation: An Emerging Therapeutic Strategy in Cancer

: Drug discovery in the scope of cancer therapy has been focused on conventional agents that nonselectively induce DNA damage or selectively inhibit the activity of key oncogenic molecules without affecting their protein levels. An emerging therapeutic strategy that garnered attention in recent years is the induction of Targeted Protein Degradation (TPD) of cellular targets by hijacking the intracellular proteolysis machinery. This novel approach offers several advantages over conventional inhibitors and introduces a paradigm shift in several pharmacological aspects of drug therapy. While TPD has been found to be the major mode of action of clinically approved anticancer agents such as fulvestrant and thalidomide, recent years have witnessed systematic endeavors to expand the repertoire of proteins amenable to therapeutic ablation by TPD. Such endeavors have led to three major classes of agents that induce protein degradation, including molecular glues, Proteolysis Targeting Chimeras (PROTACs) and Hydrophobic Tag (HyT)-based degraders. Here, we briefly highlight agents in these classes and key advances made in the field with a focus on clinical translation in cancer therapy.

Topical issues concerning treatment of intracellular bacterial pathogens in children

The article considers the issues of pharmacodynamics, clinical efficacy and safety of macrolide antibiotics. It also discusses the development of microorganisms resistance to macrolides. A number of studies in vivo and in vitro on various cellular and animal models have shown that macrolides have anti-inflammatory and immunomodulatory effects. The presence of these effects in macrolides may be especially relevant during the period of increasing incidence of influenza and the new COVID-19 coronavirus infection. It is known that the severity of viral infections is associated with the release of proinflammatory cytokines. Macrolides change the entire inflammatory response cascade by weakening the excess production of cytokines. Besides, macrolides can affect the activity of phagocytes, changing their function, including chemotaxis, phagocytosis, destroying bacteria and viruses. Earlier it was reported that macrolides (in particular, clarithromycin) can interfere with the replication cycle of the influenza virus, thus leading to its production inhibition by intracellular proteolysis of hemagglutinin. One of the commonly prescribed drugs in this group is clarithromycin. The article summarizes the literature data and presents the materials of our own clinical observations on the clarithromycin treatment of children with intracellular bacterial pathogens, the causative agents of which are Mycoplasma pneumoniae and Chlamydia pneumoniae. KEYWORDS: macrolides, clarithromycin, intracellular bacterial pathogens, children, antibacterial therapy. FOR CITATION: Savenkova M.S., Savenkov M.P. Topical issues concerning treatment of intracellular bacterial pathogens in children. Russian Journal of Woman and Child Health. 2020;3(4):326–335. DOI: 10.32364/2618-8430-2020-3-4-326-335.

Signature activities of 20S proteasome include degradation of the ubiquitin-tag with the protein under hypoxia

Careful removal of unwanted proteins is necessary for cell survival. The primary constitutive intracellular protease is the 26S proteasome complex, often found in equilibrium with its free catalytic subcomplex– the 20S core particle. Protein degradation by 26S is tightly regulated by prior ubiquitination of substrates, whereas 20S is amenable to substrates with an unstructured segment. Differentiating their contributions to intracellular proteolysis is challenging due to their common catalytic sites. Here, by chemically synthesizing a synoptic set of homogenous ubiquitinated proteins, we ascribe signature features to 20S function and demonstrate a unique property: degrading the ubiquitin-tag along with the target protein. Cryo-EM confirms that a ubiquitinated substrate can induce asymmetric conformational changes to 20S. Mass-spectrometry of intracellular peptidome under hypoxia and in human failing heart identifies the signature properties of 20S in cells. Moreover, the ability of 20S proteasome to clear toxic proteins rapidly, contributes to better survival under these conditions.