Molecular mechanism for activation of the 26S proteasome by ZFAND5.

Various hormones, kinases, and stressors (fasting, heat shock) stimulate 26S proteasome activity. To understand how its capacity to degrade ubiquitylated proteins can increase, we studied mouse ZFAND5, which promotes protein degradation during muscle atrophy. Cryo-electron microscopy showed that ZFAND5 induces large conformational changes in the 19S regulatory particle. ZFAND5's AN1 Zn-finger domain interacts with the Rpt5 ATPase and its C terminus with Rpt1 ATPase and Rpn1, a ubiquitin-binding subunit. Upon proteasome binding, ZFAND5 widens the entrance of the substrate translocation channel, yet it associates only transiently with the proteasome. Dissociation of ZFAND5 then stimulates opening of the 20S proteasome gate. Using single-molecule microscopy, we showed that ZFAND5 binds ubiquitylated substrates, prolongs their association with proteasomes, and increases the likelihood that bound substrates undergo degradation, even though ZFAND5 dissociates before substrate deubiquitylation. These changes in proteasome conformation and reaction cycle can explain the accelerated degradation and suggest how other proteasome activators may stimulate proteolysis.

The effector PHYL1JWB from Candidatus Phytoplasma ziziphi induces abnormal floral development by destabilising flower development proteins.

Phytoplasmas can induce complex and substantial phenotypic changes in their hosts in ways that favour their colonisation, but the mechanisms underlying these changes remain largely unknown. Jujube witches' broom (JWB) disease is a typical phytoplasma disease causing great economic loss in Chinese jujube (Ziziphus jujuba Mill.). Here, we reported an effector, PHYL1JWB from Candidatus Phytoplasma ziziphi, which implicated in inducing abnormal floral organogenesis. Utilising a combination of in vivo and in vitro methods, we investigated the influence of PHYL1JWB on the proteins associated with floral development. Our findings reveal that PHYL1JWB facilitates the proteasome-mediated degradation of essential flower morphogenetic regulators, including AP1, SEP1, SEP2, SEP3, SEP4, CAL, and AGL6, through a distinctive pathway that is dependent on the activity of the 26S proteasome, thus obviating the requirement for lysine ubiquitination of the substrates. Further, the Y2H analysis showed that the leucine at position 75th in second α helix of PHYL1JWB is fundamental for the interactions of PHYL1JWB with AP1 and SEP1-4 in jujube and Arabidopsis. Our research carry profound implications for elucidating the contribution of PHYL1JWB to the aberrant floral development in diseased jujube, and help to establish a robust theoretical underpinning for the prophylaxis and therapy of JWB disease.

Lhs1 dependent ERAD is determined by transmembrane domain context.

Transmembrane proteins have unique requirements to fold and integrate into the endoplasmic reticulum (ER) membrane. Most notably, transmembrane proteins must fold in three separate environments: extracellular domains fold in the oxidizing environment of the ER lumen, transmembrane domains (TMDs) fold within the lipid bilayer, and cytosolic domains fold in the reducing environment of the cytosol. Moreover, each region is acted upon by a unique set of chaperones and monitored by components of the ER associated quality control machinery that identify misfolded domains in each compartment. One factor is the ER lumenal Hsp70-like chaperone, Lhs1. Our previous work established that Lhs1 is required for the degradation of the unassembled α-subunit of the epithelial sodium channel (αENaC), but not the homologous ß- and γENaC subunits. However, assembly of the ENaC heterotrimer blocked the Lhs1-dependent ER associated degradation (ERAD) of the α-subunit, yet the characteristics that dictate the specificity of Lhs1-dependent ERAD substrates remained unclear. We now report that Lhs1-dependent substrates share a unique set of features. First, all Lhs1 substrates appear to be unglycosylated, and second they contain two TMDs. Each substrate also contains orphaned or unassembled TMDs. Additionally, interfering with inter-subunit assembly of the ENaC trimer results in Lhs1-dependent degradation of the entire complex. Finally, our work suggests that Lhs1 is required for a subset of ERAD substrates that also require the Hrd1 ubiquitin ligase. Together, these data provide hints as to the identities of as-yet unconfirmed substrates of Lhs1 and potentially of the Lhs1 homolog in mammals, GRP170.

Proteasomal degradation within endocytic organelles mediates antigen cross-presentation.

During MHC-I-restricted antigen processing, peptides generated by cytosolic proteasomes are translocated by the transporter associated with antigen processing (TAP) into the endoplasmic reticulum, where they bind to newly synthesized MHC-I molecules. Dendritic cells and other cell types can also generate MHC-I complexes with peptides derived from internalized proteins, a process called cross-presentation. Here, we show that active proteasomes within cross-presenting cell phagosomes can generate these peptides. Active proteasomes are detectable within endocytic compartments in mouse bone marrow-derived dendritic cells. In TAP-deficient mouse dendritic cells, cross-presentation is enhanced by the introduction of human ß2 -microglobulin, which increases surface expression of MHC-I and suggests a role for recycling MHC-I molecules. In addition, surface MHC-I can be reduced by proteasome inhibition and stabilized by MHC-I-restricted peptides. This is consistent with constitutive proteasome-dependent but TAP-independent peptide loading in the endocytic pathway. Rab-GTPase mutants that restrain phagosome maturation increase proteasome recruitment and enhance TAP-independent cross-presentation. Thus, phagosomal/endosomal binding of peptides locally generated by proteasomes allows cross-presentation to generate MHC-I-peptide complexes identical to those produced by conventional antigen processing.

Interplay between proteasome function and inflammatory responses in e-cig vapor condensate-challenged lung epithelial cells.

Exposure to cigarettes and other nicotine-based products results in persistent inflammation in the lung. In recent years, electronic cigarettes (e-cigs) have become extremely popular among adults and youth alike. E-cigarette vapor-induced oxidative stress promotes protein breakdown, DNA damage and cell death, culminating in a variety of respiratory diseases. The proteasome, a multi-catalytic protease, superintends protein degradation within the cell. When cells are stimulated with inflammatory cytokines such as IFN-γ and TNF-α, the constitutive catalytic proteasome subunits are replaced by the inducible subunits-low-molecular mass polypeptide (LMP)2 (ß1i), multi-catalytic endopeptidase complex-like (MECL)1 (ß2i), and LMP7 (ß5i), which are required for the production of certain MHC class I-restricted T-cell epitopes. In this study, we used human alveolar epithelial cells (A549) and exposed them to filtered air or (1%) tobacco-flavored (TF) electronic cigarette vapor condensate (ECVC) ± nicotine (6 mg/ml) (TF-ECVC ± N) for 24 h. We observed an increase in the levels of IFN-γ, TNF-α, and inducible proteasome subunits (LMP7/PSMB8, LMP2/PSMB9, MECL1/PSMB10), and a reduced expression of constitutive proteasome subunits (ß1/PSMB6 and ß2/PSMB7) in challenged A549 cells. Interestingly, knockdown of the inducible proteasome subunit LMP7 reversed ECVC-induced expression of NADPH oxidase and immunoproteasome subunits in A549 cells. In addition, pre-exposure to an LMP7 inhibitor (ONX-0914) abrogated the mRNA expression of several NOX subunits and rescued the excessive production/release of inflammatory cytokines/chemokines (IL-6, IL-8, CCL2, and CCL5) in ECVC-challenged cells. Our findings suggest an important role of LMP7 in regulating the expression of inflammatory mediators during ECVC exposure. Overall, our results provide evidence for proteasome-dependent ROS-mediated inflammation in ECVC-challenged cells.

Proteasome Interactome and Its Role in the Mechanisms of Brain Plasticity.

Proteasomes are highly conserved multienzyme complexes responsible for proteolytic degradation of the short-lived, regulatory, misfolded, and damaged proteins. They play an important role in the processes of brain plasticity, and decrease in their function is accompanied by the development of neurodegenerative pathology. Studies performed in different laboratories both on cultured mammalian and human cells and on preparations of the rat and rabbit brain cortex revealed a large number of proteasome-associated proteins. Since the identified proteins belong to certain metabolic pathways, multiple enrichment of the proteasome fraction with these proteins indicates their important role in proteasome functioning. Extrapolation of the experimental data, obtained on various biological objects, to the human brain suggests that the proteasome-associated proteins account for at least 28% of the human brain proteome. The proteasome interactome of the brain contains a large number of proteins involved in the assembly of these supramolecular complexes, regulation of their functioning, and intracellular localization, which could be changed under different conditions (for example, during oxidative stress) or in different phases of the cell cycle. In the context of molecular functions of the Gene Ontology (GO) Pathways, the proteins of the proteasome interactome mediate cross-talk between components of more than 30 metabolic pathways annotated in terms of GO. The main result of these interactions is binding of adenine and guanine nucleotides, crucial for realization of the nucleotide-dependent functions of the 26S and 20S proteasomes. Since the development of neurodegenerative pathology is often associated with regioselective decrease in the functional activity of proteasomes, a positive therapeutic effect would be obviously provided by the factors increasing proteasomal activity. In any case, pharmacological regulation of the brain proteasomes seems to be realized through the changes in composition and/or activity of the proteins associated with proteasomes (deubiquitinase, PKA, CaMKIIα, etc.).

Commentary: Are There Indeed Spliced Peptides in the Immunopeptidome?

Proteasome-generated spliced epitopes presented by HLA class I complexes are emerging targets for T cell targeted immunotherapies. Their identification by mass spectrometry triggered heated debates, which find a representative opinion in one of the two fronts in the recent perspective article by Arie Admon. Briefly, he suggests that proteasomes cannot efficiently catalyze such a reaction, and, thus, that all spliced peptides identified in HLA class I immunopeptidomes and other specimens are artifacts. This hypothesis is in contrast with in vitro, in cellula, and in vivo results published since the discovery of proteasome-catalyzed peptide splicing in 2004.

Proteasomes Are Critical for Maintenance of CD133+CD24+ Kidney Progenitor Cells.

Kidney progenitor cells, although rare and dispersed, play a key role in the repair of renal tubules after acute kidney damage. However, understanding these cells has been challenging due to the limited access to primary renal tissues and the absence of immortalized cells to model kidney progenitors. Previously, our laboratory utilized the renal proximal tubular epithelial cell line, RPTEC/TERT1, and the flow cytometry technique to sort and establish a kidney progenitor cell model called Human Renal Tubular Precursor TERT (HRTPT) which expresses CD133 and CD24 and exhibits the characteristics of kidney progenitors, such as self-renewal capacity and multi-potential differentiation. In addition, a separate cell line was established, named Human Renal Epithelial Cell 24 TERT (HREC24T), which lacks CD133 expression and shows no progenitor features. To further characterize HRTPT CD133+CD24+ progenitor cells, we performed proteomic profiling which showed high proteasomal expression in HRTPT kidney progenitor cells. RT-qPCR, Western blot, and flow cytometry analysis showed that HRTPT cells possess higher proteasomal expression and activity compared to HREC24T non-progenitor cells. Importantly, inhibition of the proteasomes with bortezomib reduced the expression of progenitor markers and obliterated the potential for self-renewal and differentiation of HRTPT progenitor cells. In conclusion, proteasomes are critical in preserving progenitor markers expression and self-renewal capacity in HRTPT kidney progenitors.

Chronic Administration of Non-Constitutive Proteasome Inhibitor Modulates Long-Term Potentiation and Glutamate Signaling-Related Gene Expression in Murine Hippocampus.

Proteasomes degrade most intracellular proteins. Several different forms of proteasomes are known. Little is known about the role of specific proteasome forms in the central nervous system (CNS). Inhibitors targeting different proteasome forms are used in clinical practice and were shown to modulate long-term potentiation (LTP) in hippocampal slices of untreated animals. Here, to address the role of non-constitutive proteasomes in hippocampal synaptic plasticity and reveal the consequences of their continuous inhibition, we studied the effect of chronic administration of the non-constitutive proteasome inhibitor ONX-0914 on the LTP induced by two different protocols: tetanic stimulation and theta-burst stimulation (TBS). Both the tetanus- and TBS-evoked potentiation contribute to the different forms of hippocampal-dependent memory and learning. Field-excitatory postsynaptic potentials (fEPSPs) in hippocampal slices from control animals and animals treated with DMSO or ONX-0914 were compared. LTP induced by the TBS was not affected by ONX-0914 administration; however, chronic injections of ONX-0914 led to a decrease in fEPSP slopes after tetanic stimulation. The observed effects correlated with differential expression of genes involved in synaptic plasticity, glutaminergic synapse, and synaptic signaling. Obtained results indicate that non-constitutive proteasomes are likely involved in the tetanus-evoked LTP, but not the LTP occurring after TBS, supporting the relevance and complexity of the role of specific proteasomes in synaptic plasticity, memory, and learning.

[Changes in the Activities and Contents of Individual Forms of Proteasomes in Samples of the Cerebral Cortex during Pathology Development in 5xFAD Mice].

The ubiquitin-proteasome system (UPS) provides hydrolysis of most intracellular proteins in proteasomes. There are various forms of proteasomes that differ, among other things, in the set of proteolytic subunits and the presence of activators. Alzheimer's disease (AD) is characterized by disturbances in the functional state of the UPS. At the same time, an increase in the expression of certain forms of proteasomes, in particular, proteasomes containing immune subunits (nonconstitutive proteasomes), has been shown. Here, we studied dynamic changes in the expression of catalytic proteasome subunit genes and corresponding proteins in the cerebral cortex of animals using a mouse model of AD (5xFAD transgenic mice). Increases by 4 and 6 folds in transcripts of the PSMB9 and PSMB8 genes encoding immune proteasome subunits were detected, as well as a significant increase in the content of immune ß-subunits (by 2.8 folds, ß1i; 2.2 folds, ß2i) in samples from 5xFAD mice at the age of 380 days, compared with samples from mice at 60 days of age. Moreover, the activation of both 20S and 26S proteasomes containing immune subunits were revealed in samples from 380 days old 5xFAD mice by electrophoresis in native conditions. This indicates activated synthesis of the immune subunits and assembly of nonconstitutive proteasomes at the terminal stage of pathology development. The obtained data, in combination with the available literature, indicate that the activation of nonconstitutive proteasomes is a universal phenomenon characteristic of various animal models of AD, which may reflect both the development of neuroinflammation and adaptive processes in tissues induced by the accumulation of toxic protein aggegates.