Ekaterina Kuzina

Ubiquitin-independent proteosomal degradation of myelin basic protein contributes to development of neurodegenerative autoimmunity

Recent findings indicate that the ubiquitin‐proteasome system is involved in the pathogenesis of cancer as well as autoimmune and several neurodegenerative diseases, and is thus a target for novel therapeutics. One disease that is related to aberrant protein degradation is multiple sclerosis, an autoimmune disorder involving the processing and presentation of myelin autoantigens that leads to the destruction of axons. Here, we show that brain‐derived proteasomes from SJL mice with experimental autoimmune encephalomyelitis (EAE) in an ubiquitin‐independent manner generate significantly increased amounts of myelin basic protein peptides that induces cytotoxic lymphocytes to target mature oligodendrocytes ex vivo. Ten times enhanced release of immunogenic peptides by cerebral proteasomes from EAE‐SJL mice is caused by a dramatic shift in the balance between constitutive and β1ihigh immunoproteasomes in the CNS of SJL mice with EAE. We found that during EAE, β1i is increased in resident CNS cells, whereas β5i is imported by infiltrating lymphocytes through the blood‐brain barrier. Peptidyl epoxyketone specifically inhibits brain‐derived β1ihigh immunoproteasomes in vitro (kobs/[I] = 240 M‐1s‐1), and at a dose of 0.5 mg/kg, it ameliorates ongoing EAE in vivo. Therefore, our findings provide novel insights into myelin metabolism in pathophysiologic conditions and reveal that the β1i subunit of the immunoproteasome is a potential target to treat autoimmune neurologic diseases.—Belogurov Jr., A., Kuzina, E., Kudriaeva, A., Kononikhin, A., Kovalchuk, S., Surina, Y., Smirnov, I., Lomakin, Y., Bacheva, A., Stepanov, A., Karpova, Y., Lyupina, Y., Kharybin, O., Melamed, D., Ponomarenko, N., Sharova, N., Nikolaev, E., Gabibov, A. Ubiquitin‐independent proteosomal degradation of myelin basic protein contributes to development of neurodegenerative autoimmunity. FASEB J. 29, 1901‐1913 (2015). www.fasebj.org

Multiple sclerosis autoantigen myelin basic protein escapes control by ubiquitination during proteasomal degradation.

Background: Most proteins must be ubiquitinated prior to proteasomal degradation. Results: Myelin basic protein (MBP) is hydrolyzed by the 26S proteasome without ubiquitination in vitro and in mammalian cells. Conclusion: Proteasome-mediated hydrolysis of the multiple sclerosis autoantigen MBP is uncontrolled by the ubiquitination system. Significance: Results reveal the first example of an autoantigen degraded by the proteasome without ubiquitin. The vast majority of cellular proteins are degraded by the 26S proteasome after their ubiquitination. Here, we report that the major component of the myelin multilayered membrane sheath, myelin basic protein (MBP), is hydrolyzed by the 26S proteasome in a ubiquitin-independent manner both in vitro and in mammalian cells. As a proteasomal substrate, MBP reveals a distinct and physiologically relevant concentration range for ubiquitin-independent proteolysis. Enzymatic deimination prevents hydrolysis of MBP by the proteasome, suggesting that an abnormally basic charge contributes to its susceptibility toward proteasome-mediated degradation. To our knowledge, our data reveal the first case of a pathophysiologically important autoantigen as a ubiquitin-independent substrate of the 26S proteasome.

Immunoproteasome enhances intracellular proteolysis of myelin basic protein

300 Proteasome is a multisubunit protein complex that exhibits proteolytic activity and is present in the nuclei and cytoplasm of cells. The 20S proteasome, which has a molecular weight of 700 kDa and a sedimenta tion coefficient of 20S, is present as a proteolytic core in a more complex particle, the 26S proteasome [1]. The degradation of proteins in the cell is regulated by the ubiquitinylation system, which marks the old or defective protein molecules for their recognition by the proteasome and subsequent proteolysis [2]. One of the key biological functions of the proteasome is the hydrolysis of intracellular proteins to the antigenic peptides, which are then presented on the cell surface on the major histocompatibility complex class I mole cules [3]. Recent studies indicate the existence of a molecular mechanism by which the peptides gener ated by the proteasome can also be presented on the major histocompatibility complex class II molecules [4]. The catalytic activity of a constitutive proteasome is mediated by three subunits, β1, β2, and β5, which are constitutively expressed in cells. The proteasome, which contains corresponding immunosubunits β1i, β2i, and β5i the catalytic center, is called the immuno proteasome and is significantly different from the con stitutive proteasome in its activity and substrate speci ficity. The set of antigenic peptides produced by the immunoproteasome differs from the set of peptides produced by the constitutive proteasome [5, 6]. It was recently shown that immunoproteasome not only changes the degradation spectrum of antigenic pro teins but also ensures the maintenance of protein homeostasis under conditions of oxidative stress caused by the action of interferons on the cell [7]. The amount of immunoproteasome in cells increases in various diseases (hematologic malignancies [8], rheu matoid arthritis [9], autoimmune colitis [10], Alzhe imer’s disease [11], and Huntington disease [12]). One of the most common and socially significant autoimmune diseases is multiple sclerosis (MS), which is characterized by the destruction of the myelin sheath of nerve fibers. Myelin basic protein (MBP) is a major autoantigen in multiple sclerosis. At present, the molecular mechanisms underlying the develop ment of multiple sclerosis are being actively studied. Recent studies have demonstrated an important role of both the constitutive proteasome and the immuno proteasome in the development of this disease [13]. Earlier, we studied in vitro the proteolysis of MBP by the proteasome isolated from normal mice and mice with experimental autoimmune encephalomy elitis (EAE), an experimental model of MS [14]. Dur ing further development of this research, we created a model to study the intracellular proteolysis of MBP in mammalian cells. Here we show that the intracellular hydrolysis of MBP is significantly accelerated when the proteasome–immunoproteasome balance is shifted toward the latter. It is known that the expression of the myelin basic protein in mammals is detected solely in the central and peripheral nervous systems. This protein is local ized in the membrane of specialized cells, oligoden drocytes and Schwann cells, forming the myelin sheath of axons. Unfortunately, work with primary human neuronal cultures is associated with numerous experimental difficulties (first of all, the lack of mate rial and ethical concerns). In view of this, at the first step of this work, to study the proteolysis of MBP ex vivo we created a genetic construct that made it possible to express a recombinant human MBP in mammalian cells. For this purpose, a DNA fragment 546 bp long, encoding the full length MBP, was amplificated by PCR using specific overlapping oligo nucleotides and then cloned into the pBudCE4.1/EF Immunoproteasome Enhances Intracellular Proteolysis of Myelin Basic Protein

Functional degradation of myelin basic protein. The proteomic approach

Proteolytic degradation of autoantigens is of prime importance in current biochemistry and immunology. The fundamental issue is the functional role of peptides produced in the process of change of the hydrolysis specificity during the transition from the normal to a pathologic state. In some cases identification of specific peptide fragments can be a diagnostic and prognostic criterion of the pathology progress. The subject of this work is the comparative study of degradation peculiarities of one of the major neuroantigens, myelin basic protein, by proteases activated upon the development of a pathological demyelinating process, and by proteasomes of different origin. Comparison of the specificity of the tested biocatalysts in some cases demonstrated critical changes in the set of myelin basic protein fragments capable of being presented on the major histocompatibility complex class I upon neurodegeneration, which may promote the development of autoimmune pathological processes.

Glatiramer Acetate and Nanny Proteins Restrict Access of the Multiple Sclerosis Autoantigen Myelin Basic Protein to the 26S Proteasome

We recently showed that myelin basic protein (MBP) is hydrolyzed by 26S proteasome without ubiquitination. The previously suggested concept of charge-mediated interaction between MBP and the proteasome led us to attempt to compensate or mimic its positive charge to inhibit proteasomal degradation. We demonstrated that negatively charged actin and calmodulin (CaM), as well as basic histone H1.3, inhibit MBP hydrolysis by competing with the proteasome and MBP, respectively, for binding their counterpart. Interestingly, glatiramer acetate (GA), which is used to treat multiple sclerosis (MS) and is structurally similar to MBP, inhibits intracellular and in vitro proteasome-mediated MBP degradation. Therefore, the data reported in this study may be important for myelin biogenesis in both the normal state and pathophysiological conditions.

The Transcriptome of Type I Murine Astrocytes under Interferon-Gamma Exposure and Remyelination Stimulus

Astrocytes are considered to be an important contributor to central nervous system (CNS) disorders, particularly multiple sclerosis. The transcriptome of these cells is greatly affected by cytokines released by lymphocytes, penetrating the blood–brain barrier—in particular, the classical pro-inflammatory cytokine interferon-gamma (IFNγ). We report here the transcriptomal profiling of astrocytes treated using IFNγ and benztropine, a putative remyelinization agent. Our findings indicate that the expression of genes involved in antigen processing and presentation in astrocytes are significantly upregulated upon IFNγ exposure, emphasizing the critical role of this cytokine in the redirection of immune response towards self-antigens. Data reported herein support previous observations that the IFNγ-induced JAK-STAT signaling pathway may be regarded as a valuable target for pharmaceutical interventions.

Erratum to: “Therapeutic effect of MBP immunodominant peptides encapsulated in nanovehicles in the development of experimental autoimmune encephalomyelitis in DA rats”

Multiple sclerosis (MS) is a severe autoimmune neurodegenerative disease. It attacks mainly young people. The development of new approaches to MS treatment is a challenge to modern immunology and pharmacology. In the present study, a high therapeutic efficacy of immunodominant peptides of myelin basic protein (MBP) incorporated into unilamellar mannosylated liposomes in the development of experimental autoimmune encephalomyelitis (EAE) is demonstrated in DA rats. MBP is a component of the oligodendrocyte membrane, which forms the axonal sheath. This protein is among the major autoantigens in MS. We have analyzed the binding pattern of anti-MBP autoantibodies from MS patients using a previously designed MBP epitope library. Utilizing the same approach, we have investigated the pool of anti-MBP antibodies from SJL/J and C57BL/6mice and DA rats with EAE. According to the autoantibody binding patterns, the rodent model most closely mimicking MS is EAE in DA rats. We have chosen three immunodominant MBP fragments encapsulated in unilamellar mannosylated liposomes for the treatment of the verified DA rodent model. MBP fragment 46–62 is the most efficient in mitigating the first EAE attack, whereas MBP 124–139 and 147–160 inhibit the development of pathology at the regression stage. Simultaneous administration of these peptides in liposomes significantly reduces the level of antibodies against MBP. The synergistic therapeutic effect of MBP fragments reduces the integral disease score by inhibiting the first EAE attack and mitigating the subsequent relapse. Thus, our findings offer new opportunities for the efficient treatment of multiple sclerosis.