biomolecules Article A Marine Diterpenoid Modulates the Proteasome Activity in Murine Macrophages Stimulated with LPS Yisett González 1, Deborah Doens 1,Héctor Cruz 1,2, Ricardo Santamaría 3, Marcelino Gutiérrez 3 , Alejandro Llanes 1,* and Patricia L. Fernández 1,* 1 Centro de Biología Celular y Molecular de Enfermedades, Instituto de Investigaciones Científicas y Servicios de Alta Tecnología (INDICASAT AIP),Edificio 219, Ciudad del Saber, 0801 Panamá, Panamá; [email protected] (Y.G.); [email protected] (D.D.); [email protected] (H.C.) 2 Facultad de Ciencias de la Salud Dr. William C. Gorgas, Universidad Latina de Panamá, 0801 Panamá, Panamá 3 Centro de Biodiversidad y Descubrimiento de Drogas, INDICASAT AIP, Edificio 219, Ciudad del Saber, 0801 Panamá, Panamá; [email protected] (R.S.); [email protected] (M.G.) * Correspondence: [email protected] (A.L.); [email protected] (P.L.F.), Tel.: +507-517-0739 Received: 13 August 2018; Accepted: 1 October 2018; Published: 5 October 2018 Abstract: The proteasome is an intracellular complex that degrades damaged or unfolded proteins and participates in the regulation of several processes. The immunoproteasome is a specialized form that is expressed in response to proinflammatory signals and is particularly abundant in immune cells. In a previous work, we found an anti-inflammatory effect in a diterpenoid extracted from the octocoral Pseudopterogorgia acerosa, here called compound 1. This compound prevented the degradation of inhibitor κB α (IκBα) and the subsequent activation of nuclear factor κB (NFκB), suggesting that this effect might be due to inhibition of the ubiquitin-proteasome system. Here we show that compound 1 inhibits the proteasomal chymotrypsin-like activity (CTL) of murine macrophages in the presence of lipopolysaccharide (LPS) but not in its absence. This effect might be due to the capacity of this compound to inhibit the activity of purified immunoproteasome. The compound inhibits the cell surface expression of major histocompatibility complex (MHC)-I molecules and the production of proinflammatory cytokines induced by LPS in vitro and in vivo, respectively. Molecular docking simulations predicted that compound 1 selectively binds to the catalytic site of immunoproteasome subunits β1i and β5i, which are responsible for the CTL activity. Taken together these findings suggest that the compound could be a selective inhibitor of the immunoproteasome, and hence could pave the way for its future evaluation as a candidate for the treatment of inflammatory disorders and autoimmune diseases. Keywords: marine diterpenoid; proteasome inhibitors; immunoproteasome 1. Introduction The proteasome is an enzymatic complex found in the nucleus and cytoplasm of eukaryotic cells, archaea and certain bacteria. This complex is responsible for the degradation of intracellular proteins that are damaged or misfolded. It works in collaboration with the ubiquitin system, which tags proteins for proteasome processing. The proteasome plays an important role in the regulation of many cellular processes, such as the cell cycle, the defense against oxidative stress and inflammatory responses. The proteasome is composed of two types of domains: a core particle and one or two regulatory domains. The core particle is formed by four stacking rings, each of them consisting of seven α or β subunits. Central rings have three catalytic subunits, namely β1, β2 and β5, which have caspase-like, trypsin-like and chymotrypsin-like (CTL) activity, respectively. An alternative form Biomolecules 2018, 8, 109; doi:10.3390/biom8040109 www.mdpi.com/journal/biomolecules Biomolecules 2018, 8, 109 2 of 17 of the proteasome, called immunoproteasome, is present in most animal cells but it is abundantly expressed in immune cells, where its primary role is to process proteins for antigen presentation by major histocompatibility complex (MHC) class I molecules [1,2]. Expression of the immunoproteasome is induced by interferon-γ (IFN-γ), tumor necrosis factor (TNF) and bacterial lipopolysaccharide (LPS) under inflammatory conditions, such as infections or autoimmune diseases [3–5]. In the presence of such stimuli, catalytic subunits of the constitutive form are respectively substituted by inducible subunits β1i (LPM2), β2i (MECL-1) and β5i (LMP7) to form the immunoproteasome. Unlike its constitutive counterparts, which have caspase-like activity, the β1i subunit also has CTL activity [6,7]. The proteasome has been implicated as a modulator of inflammatory responses by participating in the activation of nuclear factor κB (NFκB), a transcription factor that regulates the expression of many genes involved in inflammation [8]. Five NFκB family members have been described, namely RelA (p65), RelB, cRel, p50 and p52, respectively encoded by genes rela, relb, crel, nfkb1 and nfkb2. After the stimulus, NFκB proteins form dimers, which bind to κB sites on target genes either as homodimers or heterodimers. In resting cells, NFκB is sequestered in the cytoplasm by inhibitor κB (IκB) proteins. Activation of NFκB is triggered by phosphorylation of IκB, followed by its ubiquitination and proteasomal degradation, thus releasing NFκB and promoting its translocation into the nucleus [9]. It has been demonstrated that the immunoproteasome subunit β1i is involved in the proteolytic processing of NFκB precursor proteins (p100/p105), as well as in the degradation of inhibitor κB α (IκBα)[10–12]. Later, it was observed that β1i-deficient retinal pigment epithelial cells exhibited diminished activation of NFκB in response to TNF [13]. However, the role of the immunoproteasome in NFκB activation and in the degradation of IκB proteins is still under debate [10,14–17]. Other studies have demonstrated that immunoproteasome subunits are not essential in the activation of NFκB either in cancer cell lines or in peritoneal macrophages stimulated with TNF [17,18]. Due to the role of the proteasome in many physiological processes, it has become a major target for the design of new drugs as a therapeutic for several diseases. Many proteasome inhibitors have been identified from natural and synthetic sources. Two of them, bortezomib and carfilzomib, are currently approved for the treatment of multiple myeloma. Although a number of second-generation proteasome inhibitors are in clinical trials [19], undesirable side effects have been associated to these molecules. The immunoproteasome has emerged as a therapeutic target and as a strategy to reduce the toxicity associated with the inhibition of the constitutive proteasome in cells [20,21]. These molecules are not only valuable as potential therapeutics but would also allow a better understanding of the physiological roles attributed to the immunoproteasome. Several highly selective immunoproteasome inhibitors have been recently described, including both peptidic [22] and nonpeptidic inhibitors [23]. In previous studies, we have shown a marked anti-inflammatory activity for a pseudopterane diterpene (compound 1) isolated from the octocoral Pseudopterogorgia acerosa [16]. Compound 1 inhibited the production and expression of proinflammatory mediators in macrophages stimulated with LPS, TNF and other toll-like receptor ligands. Our results showed that this anti-inflammatory effect is due to the inhibition of IκBα degradation and the subsequent activation of NFκB. We then analyzed if the effect of compound 1 might be influenced by a modulation of the ubiquitin-proteasome system, affecting the proteasomal degradation of phosphorylated IκBα. We show herein that compound 1 inhibits the CTL activity of the proteasome induced by LPS in vitro and reduces the expression of MHC class I in macrophages. This inhibitory effect might occur by a mechanism that involved the modulation of immunoproteasome activity, since a reduction in the CTL activity of the purified immunoproteasome was observed. In vivo, compound 1 reduces the production of proinflammatory mediators in the lung of animals treated by intranasal inoculation of LPS. Molecular docking simulations predicted that compound 1 preferentially interacts with the catalytic site of subunits β1i and β5i, suggesting that the effect of this compound might be dependent on immunoproteasome activity. Biomolecules 2018, 8, 109 3 of 17 2. Materials and Methods 2.1. Mice In vivo studies were carried out by using female C57Bl/6 mice with an age of eight weeks, obtained from Instituto de Investigaciones Científicas y Servicios de Alta Tecnología (INDICASAT)’s mouse facility. Mice were kept at 25 ◦C under a light/dark cycle of 12 h and had free access to food and water. All experiments were performed in accordance with guidelines from the Institutional Animal Welfare Committee and the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was also approved by the Institutional Animal Care and Use Committee of INDICASAT AIP (IACUC-15-004). 2.2. Acute Pulmonary Inflammation C57BL/6 mice (n = 5) were anesthetized with Ketamine/Xylazine (93/6 mg/Kg) and then treated by intranasal inoculation with lipopolysaccharide (LPS) from Escherichia coli 0111:B4 (Sigma Aldrich, Saint Louis, MO, USA) (0.5 mg/Kg) or saline for control group. Compound 1 (5 mg/Kg) was administered by intraperitoneal (i.p.) injection 2 h before and 10 h after LPS administration. The control group was not treated with compound 1. Mice were euthanized 24 h after the challenge with LPS and the concentrations of tumor necrosis factor (TNF) and interleukin