Hypoxia-Inducible Factor Prolyl Hydroxylase Inhibitor Prevents Steroid-Associated Osteonecrosis of the Femoral Head in Rabbits by Promoting Angiogenesis and Inhibiting Apoptosis

The purpose of this study was to investigate the preventive effect of ethyl 3,4-dihydroxybenzoate(EDHB) on steroid-associated femoral head osteonecrosis(ONFH) in a rabbit model. New Zealand white rabbits were randomly divided into two groups (prevention group and model group), each containing 24 rabbits. Osteonecrosis was induced by lipopolysaccharide(LPS) combined with methylprednisolone(MPS). The prevention group received an intraperitoneal injection of EDHB at 50 mg/kg body weight every other day starting three days before establishing rabbit models of osteonecrosis, for a total of nine doses. Osteonecrosis was verified by haematoxylin-eosin (HE) staining. The expression of HIF-1α and VEGF was analyzed by immunohistochemistry. Angiogenesis, apoptosis and microstructural parameters were also analyzed. The rabbit models of osteonecrosis were successfully established and observed by HE staining. Histopathological observations indicated that EDHB reduced the rate of empty lacunae and the incidence of osteonecrosis. Immunohistochemical staining for HIF-1α and VEGF suggested that EDHB therapy inhibited degradation of HIF-1α and promoted expression of VEGF. Ink artery infusion angiography and microvessel density analysis revealed that there were more microvessels in the prevention group than in the model group. The TUNEL apoptosis assay suggested that EDHB intervention could reduce the number of apoptotic cells in avascular osteonecrosis of the femoral head. Micro-CT scanning indicated that the treatment group had better microstructural parameters than the model group. EDHB prevents steroid-associated osteonecrosis of the femoral head in rabbits by promoting angiogenesis and inhibiting apoptosis of bone cells and hematopoietic tissue.     

RNF2 is recruited by WASH to ubiquitinate AMBRA1 leading to downregulation of autophagy

WASH (Wiskott-Aldrich syndrome protein (WASP) and SCAR homolog) was identified to function in endosomal sorting via Arp2/3 activation. We previously demonstrated that WASH is a new interactor of BECN1 and present in the BECN1-PIK3C3 complex with AMBRA1. The AMBRA1-DDB1-CUL4A complex is an E3 ligase for K63-linked ubiquitination of BECN1, which is required for starvation-induced autophagy. WASH suppresses autophagy by inhibition of BECN1 ubiquitination. However, how AMBRA1 is regulated during autophagy remains elusive. Here, we found that RNF2 associates with AMBRA1 to act as an E3 ligase to ubiquitinate AMBRA1 via K48 linkage. RNF2 mediates ubiquitination of AMBRA1 at lysine 45. Notably, RNF2 deficiency enhances autophagy induction. Upon autophagy induction, RNF2 potentiates AMBRA1 degradation with the help of WASH. WASH deficiency impairs the association of RNF2 with AMBRA1 to impede AMBRA1 degradation. Our findings reveal another novel layer of regulation of autophagy through WASH recruitment of RNF2 for AMBRA1 degradation leading to downregulation of autophagy.     

Dystrophin Involved in the Susceptibility of Slow Muscles to Hindlimb Unloading via Concomitant Activation of TGF-β1/Smad3 Signaling and Ubiquitin–Proteasome Degradation in Mice

While it is well known that the slow-twitch muscles are vulnerable to microgravity conditions, the molecular and cellular mechanisms underlying this phenomenon remain unknown. Dystrophin, which constitutes an important link between the cytoskeleton and the extracellular matrix, is hypothesized to be involved in force generation and mechanical stabilization of the skeletal muscle. Here we have shown that after a 14-day hindlimb unloading (HU) of the C57BL/10 mice, the expression of dystrophin was significantly down-regulated in the fast-twitch myofibers, while in the slow-twitch myofibers, it was up-regulated. In order to investigate the role of dystrophin in HU-induced susceptibility to muscle atrophy, we compared the degradation signaling mechanisms of slow-twitch soleus muscle in dystrophin-deficient (mdx) and the wild-type (WT) mice. We found that mdx mice manifest less reduction of muscle mass and myofiber cross-sectional area than the control animals. Also, the expression of two ubiquitin ligases (MuRF1, Atrogin-1), which plays a crucial role in the ubiquitin–proteasome-mediated muscular degradation, was significantly down-regulated in soleus muscle of the hindlimb-unloaded mdx mice. In comparison, in the soleus muscle of unloaded WT mice, these ligases were significantly up-regulated. Whereas the hindlimb unloading reduced the expression of transforming growth factor β (TGF-β1)/Smad3 in mdx mice, in WT mice, the expression of this growth factor was augmented in response to unloading. Correspondingly, as a result of HU of the mdx mice, the expression of four subtypes of the myosin heavy chain and troponin I was reduced or it exhibited a delayed slow-to-fast transition. In summary, our results suggest that dystrophin exerts an intermediary and positive role in the disuse atrophy of the slow-twitch muscles. This effect is mediated through the activation of TGF-β1/Smad3 signaling and downstream ubiquitin–proteasome pathway.     

The effect of albumin fusion structure on the production and bioactivity of the somatostatin-28 fusion protein in Pichia pastoris

Somatostatin, a natural inhibitor of growth hormone (GH), and its analogs have been used in clinical settings for the treatment of acromegaly, gigantism, thyrotropinoma, and other carcinoid syndromes. However, natural somatostatin is limited for clinical usage because of its short half-life in vivo. Albumin fusion technology was used to construct long-acting fusion proteins and Pichia pastoris was used as an expression system. Three fusion proteins (SS28)₂-HSA, (SS28)₃-HSA, and HSA-(SS28)₂, were constructed with different fusion copies of somatostatin-28 and fusion orientations. The expression level of (SS28)₃-HSA was much lower than (SS28)₂-HSA and HSA-(SS28)₂ due to the additional fusion of the somatostatin-28 molecule. MALDI-TOF mass spectrometry revealed that severe degradation occurred in the fermentation process. Similar to the standard, somatostatin-14, all three fusion proteins were able to inhibit GH secretion in blood, with (SS28)₂-HSA being the most effective one. A pharmacokinetics study showed that (SS28)₂-HSA had a prolonged half-life of 2 h. These results showed that increasing the number of small protein copies fused to HSA may not be a suitable method for improving protein bioactivity.     

Tyr-301 Phosphorylation Inhibits Pyruvate Dehydrogenase by Blocking Substrate Binding and Promotes the Warburg Effect

The mitochondrial pyruvate dehydrogenase complex (PDC) plays a crucial role in regulation of glucose homoeostasis in mammalian cells. PDC flux depends on catalytic activity of the most important enzyme component pyruvate dehydrogenase (PDH). PDH kinase inactivates PDC by phosphorylating PDH at specific serine residues, including Ser-293, whereas dephosphorylation of PDH by PDH phosphatase restores PDC activity. The current understanding suggests that Ser-293 phosphorylation of PDH impedes active site accessibility to its substrate pyruvate. Here, we report that phosphorylation of a tyrosine residue Tyr-301 also inhibits PDH α 1 (PDHA1) by blocking pyruvate binding through a novel mechanism in addition to Ser-293 phosphorylation. In addition, we found that multiple oncogenic tyrosine kinases directly phosphorylate PDHA1 at Tyr-301, and Tyr-301 phosphorylation of PDHA1 is common in EGF-stimulated cells as well as diverse human cancer cells and primary leukemia cells from human patients. Moreover, expression of a phosphorylation-deficient PDHA1 Y301F mutant in cancer cells resulted in increased oxidative phosphorylation, decreased cell proliferation under hypoxia, and reduced tumor growth in mice. Together, our findings suggest that phosphorylation at distinct serine and tyrosine residues inhibits PDHA1 through distinct mechanisms to impact active site accessibility, which act in concert to regulate PDC activity and promote the Warburg effect.     

Divalent cations activate TRPV1 through promoting conformational change of the extracellular region

Divalent cations Mg²⁺ and Ba²⁺ selectively and directly potentiate transient receptor potential vanilloid type 1 heat activation by lowering the activation threshold into the room temperature range. We found that Mg²⁺ potentiates channel activation only from the extracellular side; on the intracellular side, Mg²⁺ inhibits channel current. By dividing the extracellularly accessible region of the channel protein into small segments and perturbing the structure of each segment with sequence replacement mutations, we observed that the S1–S2 linker, the S3–S4 linker, and the pore turret are all required for Mg²⁺ potentiation. Sequence replacements at these regions substantially reduced or eliminated Mg²⁺-induced activation at room temperature while sparing capsaicin activation. Heat activation was affected by many, but not all, of these structural alternations. These observations indicate that extracellular linkers and the turret may interact with each other. Site-directed fluorescence resonance energy transfer measurements further revealed that, like heat, Mg²⁺ also induces structural changes in the pore turret. Interestingly, turret movement induced by Mg²⁺ precedes channel activation, suggesting that Mg²⁺-induced conformational change in the extracellular region most likely serves as the cause of channel activation instead of a coincidental or accommodating structural adjustment.