Hongfei Jiang ¹ Qiuyu Gong ² Renshuai Zhang ^1*

• ¹ The Affiliated Hospital of Qingdao University, Qingdao, China
• ² The First Affiliated Hospital of Xi'an Jiaotong University, Xi'an, Shaanxi, China

Inflammation is the body's protective response to detrimental irritants (such as pathogens or irritants), which can clear out necrotic cells and initiate tissue repair. However, when inflammation becomes chronic or lasts too long, it can be harmful and may lead to a wide variety of diseases, including cardiovascular diseases, cancer, diabetes, arthritis, Alzheimer's disease, pulmonary diseases, and autoimmune diseases [1]. The COVID-19 that swept the world has further increased scientists' attentions to inflammation research. Many signaling pathways have been reported to be involved in the initiation and progression of inflammation, and some diagnosis markers for inflammation have been widely used in clinic, such as Leukocyte count, procalcitonin, C-reactive protein and IL-6. However, clinically available targets for inflammation diagnosis, especially treatment, are still relatively few. The discovery of novel inflammatory biomarkers has important implications for the diagnosis and treatment of inflammation and inflammation-related diseases.In recent years, the connection between pyroglutamyl aminopeptidase 1 (PGP-1, EC 3.4.19.3) and inflammation has received increasing attention from scientists. This viewpoint provides our opinions on PGP-1 as a promising novel target of inflammation. PGP-1 is a kind of pyroglutamyl peptidase, which is a typical cysteine peptidase catalyzing the removal of L-pyroglutamate (pGlu) from the N-terminus of some peptides and proteins (Figure 1A, 1B) [2]. PGP-1 has been found in almost all the domains of life including mammals, avian, fish, plants, protists and fungi. In addition, PGP-1 is highly conserved and ubiquitously distributed in human tissues, and its novel physiological roles are constantly being proposed in recent decades.In previous study, PGP-1 was thought to be involved in the absorption of peptides and proteins from the mammalian alimentary tract due to its presence in both the small intestine and duodenum PAGE \* Arabic \* MERGEFORMAT 3 [3]. In addition, also based on the widespread distribution of PGP-1 in functionally dissimilar tissues, researchers proposed that PGP-1 may participate in the intracellular catabolism and resynthesis of peptides [4]. PGP-1 was also believed to affect the certain disease states (e.g., memory impairment) through regulating the levels of free pGlu, given the proven pharmacological properties of pGlu [5]. Furthermore, PGP-1 exerted physiological roles by regulating physiologically important peptide hormones with pGlu at N-terminus (e.g., thyrotropin-releasing hormone and gonadotropin-releasing hormone) [6]. Our group reported the first PGP-1 probe in 2016, and with help of the probe, we found that the cellular inflammation was accompanied by the increase of PGP-1 [7]. Subsequently, we utilized a near-infrared PGP-1 fluorescent probe to further reveal the up-regulation of PGP-1 in inflammatory mouse model [8]. In addition, our study also suggested for the first time that knocking down of PGP-1 led to the weakness of inflammatory process (down-regulation of TNF-α expression) in RAW264.7 cells. Based on these studies, we proposed that PGP-1 was a new potential inflammatory cytokine.In the following years, the relationship between PGP-1 and many inflammatory models were demonstrated utilizing various fluorescent probes. These inflammatory models included freund's incomplete adjuvant or lipopolysaccharide induced macrophagocyte (RAW264.7), human normal hepatocyte cell (LO-2), hepatocellular carcinoma cell (HepG 2), human ovarian cancer cell (ES-2) and mouse model. For example, Hu et al. designed a bioluminescent probe for quantitating PGP-1 activity by attaching a pGlu group onto the aminoluciferin using an amide bond [9]. The imaging study of living cells demonstrated the up-regulation of PGP-1 in human ovarian cancer cell line ES-2 that was preincubated with a common immunopotentiator, Freund's incomplete adjuvant. In vivo imaging also revealed that PGP-1 was highly expressed in mouse model of inflammatory liver disease. It is worth noted that inflammation has been considered to involve in the development and progression of tumor [10]. The relationship between PGP-1 and inflammation-related diseases, especially tumor, has also been demonstrated in various tumor cell models (HepG2, Huh-7, A549, and HCT116) and tumor-bearing mice model (HepG2 tumorbearing mice). Targeting inflammatory factors has been regarded as an important strategy for hepatocellular carcinoma (HCC) diagnosis and treatment considering chronic inflammation is strongly associated with HCC. For example, the study from Guo et al. found that PGP-1 was aberrant expressed in HCC cell lines (HepG 2 and HuH-7) and HepG2 tumor-bearing mice [11].The overexpression of PGP-1 in HCC cells promoted tumor progression, whereas knockdown ofPAGE \* Arabic \* MERGEFORMAT 3PGP-1 significantly suppressed tumor cell growth and migration. These data suggested that PGP-1 was involved in the development of HCC and can be regarded as a potential HCC biomarker and therapeutic target. In addition, Liu group also explored the expression of PGP-1 in HepG2, RAW264 cells, inflammation model mice pre-cultured with lipopolysaccharide and HepG2 tumorbearing mice by constructing ratiometric fluorescent sensors [12,13]. The imaging results of the inflammation model mice pre-cultured with lipopolysaccharide showed that the content of PGP-1 did increase after lipopolysaccharide induction. Furthermore, the in vivo imaging of HepG2 tumorbearing mice also suggested that the expression level of PGP-1 in the living body was closely related to inflammation-related tumor. Moreover, the up-regulation of PGP-1 in burnt skin tissues and serum samples from burns patients during the occurrence of inflammation resulting from burns has also been demonstrated [14]. These findings together suggest that PGP-1 is indeed involved in inflammatory response in vitro and in vivo. PGP-1, as a novel inflammatory cytokine, has been demonstrated in cellular level, animal models and patient samples. However, more efforts are needed to identify GP-1 as a clinical indicator and therapeutic target for inflammation and inflammation-related diseases. Several problems deserve the attention of scientists: Firstly, the direct mechanism that PGP-1 involves inflammation progress is complicated and unclear. Although the classical IL-6/STAT3 pathway is thought to play key roles in PGP-1 promoting the progression of hepatocellular tumor associated with chronic inflammation, how PGP-1 is involved in the occurrence and progression of inflammation still needs more direct evidences [11]. Elucidating the reaction between PGP-1 and inflammation-related proteins may provide a solution to this issue, since the dissociation of N-terminal peptide bonds is the typical functional feature of PGP-1.Secondly, the feasibility of PGP-1 as a therapeutic target for inflammation has not yet been demonstrated in vivo. The development of PGP-1 inhibitors is the key to solve this issue. As early as 1982, Fujiwara et al. reported a specific inhibitor for PGP-1, N-carbobenzoxypyroglutamyl diazomethyl ketone, which covalently bond to the activity site with Ki of 0.12 mM [15]. In addition, Ma group reported seven natural inhibitors against PGP-1, which showed inhibition activity PAGE \* Arabic \* MERGEFORMAT 3 against PGP-1 at micromolar level [16,17]. It is worth noting that, up to now, only a few PGP-1 inhibitors have been reported, and studies on their activity in vivo are lacking. Therefore, it would be extremely useful to develop effective PGP-1 inhibitors for exploring the role of PGP-1 as a therapeutic target for inflammation. On the one hand, the analysis and summary of the characteristics of PGP-1 substrates can provide reference for the design of inhibitors. PGP-1 shows broad substrate specificity, however, it is highly specific for amino terminal pGlu residues. Minor alterations to the pGlu residue may significantly affect the ability of PGP-1 to cleave the adjacent peptide bond. For example, increasing the size of the pyroglutamyl ring from 5 to 6 members or introduction of a second ureido nitrogen into the ring almost completely eliminate the ability of PGP-1 cleaving the adjacent peptide bond [18]. In addition, if the pGlu residues in amino terminal are attached to proline (Pro), the pGlu-Pro bonds are not normally hydrolyzed by mammalian PGP-1 [19]. On the other hand, virtual screening is also an ideal strategy to obtain PGP-1 inhibitors. It is worth noting that the human PGP-1 enzyme has been purified and enzymatically characterized, however, its crystal structure is not known [20]. Virtual screening based on non-human species PGP-1 has limited accuracy. It is believed that with the elucidation of human PGP-1 structure, virtual screening will greatly improve the probability of PGP-1 inhibitor discovery.Thirdly, it has been widely recognized that chronic inflammation might result in carcinogenesis, and this induced process usually goes on silently for a long time [10]. PGP-1 inhibitors have the potential to prevent the tumorigenesis and progression by inhibiting chronic inflammation. If PGP-1 inhibitors used in the anti-chronic inflammation are derived from food materials, they may be easier to incorporate into the daily diet and easier to use for cancer prevention. Thus, inhibitors of PGP-1 derived from food materials, such as edible plants, should be greater concerned in the prevention of inflammation-related tumor.In conclusion, PGP-1 shows great potential in the diagnosis and treatment of inflammation and inflammation-related diseases, however, more investigation to elucidate the detailed mechanism of PGP-1's involvement in inflammation progression and further validate the effectiveness of PGP-