Nuclear translocation of PKM2 mediates keratinocyte metabolic reprogramming in psoriasis.

PKM2 mediates the Warburg effects and is crucial for tumorigenesis, but its role in hyperplastic skin disorders remains elusive. In this study, we investigated the function of PKM2 in psoriatic keratinocytes. We found that PKM2 expression and its nuclear translocation were induced in the epidermis of psoriasis patients, contributing to aerobic glycolysis and cell growth. Moreover, mass spectrometry combined with immunoprecipitation analysis revealed that PKM2 could interact with TRIM33, an E3 ubiquitin ligase in the nucleus, and this interaction is critical for the nuclear retention of PKM2. As a result of TRIM33-mediated ubiquitination, PKM2 nuclear protein kinase function is promoted, thus leading to the phosphorylation of STAT3. In addition, blocking PKM2 nuclear translocation abrogated TRIM33-triggered glycolysis and cell proliferation in keratinocytes. Taken together, our experiments demonstrate that ubiquitination regulates the nuclear retention of PKM2 in keratinocytes. Moreover, our results highlight a novel mechanism accounting for the metabolic reprogramming of keratinocytes in psoriasis patients.

Serotonin/5-HT7 receptor provides an adaptive signal to enhance pigmentation response to environmental stressors through cAMP-PKA-MAPK, Rab27a/RhoA, and PI3K/AKT signaling pathways.

Serotonin (5-HT), a neurotransmitter, is essential for normal and pathological pigmentation processing, and its receptors may be therapeutical targets. The effect and behavior of the 5-HT7 receptor (5-HT7R) in melanogenesis in high vertebrates remain unknown. Herein, we examine the role and molecular mechanism of 5-HT7R in the pigmentation of human skin cells, human tissue, mice, and zebrafish models. Firstly, 5-HT7R protein expression decreased significantly in stress-induced depigmentation skin and vitiligo epidermis. Stressed mice received transdermal serotonin 5-HT7R selective agonists (LP-12, 0.01%) for 12 or 60 days. Mice might recover from persistent stress-induced depigmentation. The downregulation of tyrosinase (Tyr), microphthalmia-associated transcription factor (Mitf) expression, and 5-HT7R was consistently restored in stressed skin. High-throughput RNA sequencing showed that structural organization (dendrite growth and migration) and associated pathways were activated in the dorsal skin of LP-12-treated animals. 5-HT7R selective agonist, LP-12, had been demonstrated to enhance melanin production, dendrite growth, and chemotactic motility in B16F10 cells, normal human melanocytes (NHMCs), and zebrafish. Mechanistically, the melanogenic, dendritic, and migratory functions of 5-HT7R were dependent on the downstream signaling of cAMP-PKA-ERK1/2, JNK MAPK, RhoA/Rab27a, and PI3K/AKT pathway activation. Importantly, pharmacological inhibition and genetic siRNA of 5-HT7R by antagonist SB269970 partially/completely abolished these functional properties and the related activated pathways in both NHMCs and B16F10 cells. Consistently, htr7a/7b genetic knockdown in zebrafish could blockade melanogenic effects and abrogate 5-HT-induced melanin accumulation. Collectively, we have first identified that 5-HT7R regulates melanogenesis, which may be a targeted therapy for pigmentation disorders, especially those worsened by stress.

Kaempferol, the melanogenic component of Sanguisorba officinalis, enhances dendricity and melanosome maturation/transport in melanocytes.

Kaempferol, a representative flavonoid constituent of Sanguisorba officinalis, promotes melanogenesis, but the underlying mechanisms remain unknown. Here, we evaluated the effects of kaempferol on melanocytes morphology and behavior and determined the mechanisms regulating kaempferol-induced pigmentation. We observed that kaempferol increased melanin contents and dendritic length and stimulated melanocyte migration both in vitro and vivo. It significantly enhanced the expression of microphthalmia-associated transcription factor (MITF) and downstream enzymes of melanin biosynthesis-tyrosinase (TYR), tyrosinase-related protein (TRP-1), and dopachrome tautomerase (DCT). It also induced melanosome maturation (increased stage III and IV melanosomes) and melanin transfer to dendritic tips; this was evidenced as follows: kaempferol-treated melanocytes exhibited the perimembranous accumulation of HMB45-positive melanosomes and increased the expression of Rab27A, RhoA, and Cdc42, which improved melanosome transport to perimembranous actin filaments. These results jointly indicated that kaempferol promotes melanogenesis and melanocyte growth. Additionally, kaempferol stimulated the phosphorylation of P38/ERK MAPK and downregulated p-PI3K, p-AKT, and p-P70s6K expression. Pre-incubation with P38 (SB203580) and ERK (PD98059) signaling inhibitors reversed the melanogenic and dendritic effects and MITF expression. PI3K/AKT inhibitor augmented kaempferol-induced melanin content and dendrite length. In summary, kaempferol regulated melanocytes' dendritic growth and melanosome quantity, maturation, and transport via P38/ERK MAPK and PI3K/AKT signaling pathways.

Mutations in spike protein and allele variations in ACE2 impact targeted therapy strategies against SARS-CoV-2.

Coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has spread rapidly worldwide with high rates of transmission and substantial mortality. To date, however, no effective treatments or enough vaccines for COVID-19 are available. The roles of angiotensin converting enzyme 2 (ACE2) and spike protein in the treatment of COVID-19 are major areas of research. In this study, we explored the potential of ACE2 and spike protein as targets for the development of antiviral agents against SARS-CoV-2. We analyzed clinical data, genetic data, and receptor binding capability. Clinical data revealed that COVID-19 patients with comorbidities related to an abnormal renin-angiotensin system exhibited more early symptoms and poorer prognoses. However, the relationship between ACE2 expression and COVID-19 progression is still not clear. Furthermore, if ACE2 is not a good targetable protein, it would not be applicable across a wide range of populations. The spike-S1 receptor-binding domain that interacts with ACE2 showed various amino acid mutations based on sequence analysis. We identified two spike-S1 point mutations (V354F and V470A) by receptor-ligand docking and binding enzyme-linked immunosorbent assays. These variants enhanced the binding of the spike protein to ACE2 receptors and were potentially associated with increased infectivity. Importantly, the number of patients infected with the V354F and V470A mutants has increased with the development of the SARS-CoV-2 pandemic. These results suggest that ACE2 and spike-S1 are likely not ideal targets for the design of peptide drugs to treat COVID-19 in different populations.

Adaptive evolution of GPR39 in diverse directions in vertebrates.

G protein-coupled receptors (GPCRs) play an important role in physiology and disease and represent productive drug targets. Orphan GPCRs, which have unknown endogenous ligands, are considered drug targets and consequently have attracted great interest in identifying their endogenous cognate ligands for deorphanization. However, additional studies have shown that GPCRs, including many orphan GPCRs, can constitutively activate G protein signaling in a ligand-independent manner. GPR39 is such an orphan GPCR with constitutive activity. Here, we performed a phylogenetic and selection analysis of GPR39 in vertebrates, and we found that GPR39 underwent positive selection in different branches of vertebrates. Using luciferase reporter assays, we demonstrated that human, frog and chicken GPR39 can constitutively activate Gq and G12 signaling pathways in a ligand-independent manner. Zebrafish GPR39 can constitutively activate Gs, Gq and G12 signaling pathways in a ligand-independent manner. We further found that the zebrafish-H2967.35 site is crucial for the activity of the Gs signaling pathway. In addition, our mutagenesis studies indicated that the positive selection sites of GPR39 from different species had important effects on the constitutive activity of the receptor. Our results revealed the adaptive evolution of GPR39 in diverse directions, which led to differences in constitutive activity.

Constitutively active BRS3 is a genuinely orphan GPCR in placental mammals.

G protein-coupled receptors (GPCRs) play an important role in physiology and disease and represent the most productive drug targets. Orphan GPCRs, with their endogenous ligands unknown, were considered a source of drug targets and consequently attract great interest to identify their endogenous cognate ligands for deorphanization. However, a contrary view to the ubiquitous existence of endogenous ligands for every GPCR is that there might be a significant overlooked fraction of orphan GPCRs that function constitutively in a ligand-independent manner only. Here, we investigated the evolution of the bombesin receptor-ligand family in vertebrates in which one member-bombesin receptor subtype-3 (BRS3)-is a potential orphan GPCR. With analysis of 17 vertebrate BRS3 structures and 10 vertebrate BRS3 functional data, our results demonstrated that nonplacental vertebrate BRS3 still connects to the original ligands-neuromedin B (NMB) and gastrin-releasing peptide (GRP)-because of adaptive evolution, with significantly changed protein structure, especially in three altered key residues (Q127R, P205S, and R294H) originally involved in ligand binding/activation, whereas the placental mammalian BRS3 lost the binding affinity to NMB/GRP and constitutively activates Gs/Gq/G12 signaling in a ligand-independent manner. Moreover, the N terminus of placental mammalian BRS3 underwent positive selection, exhibiting significant structural differences compared to nonplacental vertebrate BRS3, and this domain plays an important role in constitutive activity of placental mammalian BRS3. In conclusion, constitutively active BRS3 is a genuinely orphan GPCR in placental mammals, including human. To our knowledge, this study identified the first example that might represent a new group of genuinely orphan GPCRs that will never be deorphanized by the discovery of a natural ligand and provided new perspectives in addition to the current ligand-driven GPCR deorphanization.