The fundamental importance of the 26S proteasome in health and disease suggests that its function must be finely controlled, and yet our knowledge about proteasome regulation remains limited. Posttranslational modifications, especially phosphorylation, of proteasome subunits have been shown to impact proteasome function through different mechanisms, although the vast majority of proteasome phosphorylation events have not been studied. Here, we have characterized 1 of the most frequently detected proteasome phosphosites, namely Ser361 of Rpn1, a base subunit of the 19S regulatory particle. Using a variety of approaches including CRISPR/Cas9-mediated gene editing and quantitative mass spectrometry, we found that loss of Rpn1-S361 phosphorylation reduces proteasome activity, impairs cell proliferation, and causes oxidative stress as well as mitochondrial dysfunction. A screen of the human kinome identified several kinases including PIM1/2/3 that catalyze S361 phosphorylation, while its level is reversibly controlled by the proteasome-resident phosphatase, UBLCP1. Mechanistically, Rpn1-S361 phosphorylation is required for proper assembly of the 26S proteasome, and we have utilized a genetic code expansion system to directly demonstrate that S361-phosphorylated Rpn1 more readily forms a precursor complex with Rpt2, 1 of the first steps of 19S base assembly. These findings have revealed a prevalent and biologically important mechanism governing proteasome formation and function.

Hepatocellular carcinoma (HCC) is the most common form of hepatic malignancies. The diagnosis of HCC remains challenging due to the low sensitivity and specificity of the diagnostic method. Exosomes, which are abundant in various proteins from parent cells, play pivotal roles in intercellular communication and have been confirmed as promising sources of disease biomarkers. Herein, we performed a simple but robust proteomic profiling on exosomes derived from 1 μL of serum using a data-independent acquisition (DIA) method for the first time, to screen potential biomarkers for the diagnosis of HCC. Ten pivotal differentially expressed proteins (DEPs) (von Willebrand factor (VWF), LGALS3BP, TGFB1, SERPINC1, HPX, HP, HBA1, FGA, FGG, and FGB) were screened as a potential candidate biomarker panel, which could completely discriminate patients with HCC from normal control (NC). Interestingly, Gene Expression Profiling Interactive Analysis (GEPIA) revealed that the expression levels of four genes increased and those of six genes decreased in HCC tissues compared with normal tissues, which were in concordance with protein expression levels. In conclusion, we screened 10 exosomal proteins holding promise for acting as a potential candidate biomarker panel for detection of HCC through a simple but robust proteomic profiling.

Runt-related transcription factor 1 (Runx1) plays a key role in cartilage formation, but its function in articular cartilage formation is unclear. We generated non-inducible and inducible Runx1-deficient mice (Runx1f/fCol2α1-Cre and Runx1f/fCol2α1-CreER mice) and found that chondrocyte-specific Runx1-deficient mice developed a spontaneous osteoarthritis (OA)-like phenotype and showed exacerbated articular cartilage destruction under OA, characterized by articular cartilage degradation and cartilage ossification, with decreased Col2α1 expression and increased Mmp13 and Adamts5 expression. RNA-sequencing analysis of hip articular cartilage from the Runx1f/fCol2α1-Cre mice compared to that from wild-type mice and subsequent validation analyses demonstrated that Runx1 is a central regulator in multiple signaling pathways, converging signals of the Hippo/Yap, TGFβ/Smad, and Wnt/β-catenin pathways into a complex network to regulate the expression of downstream genes, thereby controlling a series of osteoarthritic pathological processes. RNA-sequencing analysis of mutant knee joints showed that Runx1’s role in signaling pathways in articular cartilage is different from that in whole knee joints, indicating that Runx1 regulation is tissue-specific. Histopathologic analysis confirmed that Runx1 deficiency decreased the levels of YAP and p-Smad2/3 and increased the levels of active β-catenin. Overexpression of Runx1 dramatically increased YAP expression in chondrocytes. Adeno-associated virus-mediated Runx1 overexpression in the knee joints of osteoarthritic mice showed the protective effect of Runx1 on articular cartilage damaged in OA. Our results notably showed that Runx1 is a central regulator of articular cartilage homeostasis by orchestrating the YAP, TGFβ, and Wnt signaling pathways in the formation of articular cartilage and OA, and targeting Runx1 and its downstream genes may facilitate the design of novel therapeutic approaches for OA.