Recombinant human hair keratin proteins for halting bleeding

Keratins derived from human hair have been widely used for tissue engineering. However, some drawbacks relative to the traditional keratins extracts have been found: (a): difficultly controlling the amino acid composition; (b): batch to batch inconsistent quality; and (c): producing complex keratin and keratin-associated proteins (KAPs), which problems have made some studies concerning human hair keratins stagnant, especially in the mechanism studies related to hemostasis of keratins. Herein, a type-I human hair keratin of K37 and a type-II human hair keratin of K81 were heterologously expressed and firstly used for haemostatic application. SDS-PAGE analysis shows that the recombinant keratins had higher purity compared to the extracted keratins. The circular dichroism (CD) spectra of K37 and K81 suggested that the secondary structures were rich in α-helix. In addition, the recombinant keratin proteins could enhance fibrin colt formation at the site of injury and decrease the bleeding time and blood loss in liver puncture and femoral artery injury rat models. This study provides a new strategy for future works involving design and mechanism studies of keratin biomaterials

A Novel NADP(H)-Dependent 7alpha-HSDH: Discovery and Construction of Substrate Selectivity Mutant by C-Terminal Truncation

7α-Hydroxysteroid dehydrogenase (7α-HSDH) plays an important role in the biosynthesis of tauroursodeoxycholic acid (TUDCA) using complex substrate chicken bile powder as raw material. However, chicken bile powder contains 4.74% taurocholic acid (TCA), and a new by-product tauroursocholic acid (TUCA) will be produced, having the risk of causing colorectal cancer. Here, we obtained a novel NADP(H)-dependent 7α-HSDH with good thermostability from Ursus thibetanus gut microbiota (named St-2-2). St-2-2 could catalyze taurochenodeoxycholic acid (TCDCA) and TCA with the catalytic activity of 128.13 and 269.39 U/mg, respectively. Interestingly, by a structure-based C-terminal truncation strategy, St-2-2△C10 only remained catalytic activity on TCDCA (14.19 U/mg) and had no activity on TCA. As a result, it can selectively catalyze TCDCA in waste chicken bile powder. MD simulation and structural analysis indicated that enhanced surface hydrophilicity and improved C-terminal rigidity affected the entry and exit of substrates. Hydrogen bond interactions between different subunits and interaction changes in Phe249 of the C-terminal loop inverted the substrate catalytic activity. This is the first report on substrate selectivity of 7α-HSDH by C-terminal truncation strategy and it can be extended to other 7α-HSDHs (J-1-1, S1-a-1)

iRhom2 loss alleviates renal injury in long-term PM2.5-exposed mice by suppression of inflammation and oxidative stress

Particulate matter (PM2.5) is a risk factor for organ injury and disease progression, such as lung, brain and liver. However, its effects on renal injury and the underlying molecular mechanism have not been understood. The inactive rhomboid protein 2 (iRhom2), also known as rhomboid family member 2 (Rhbdf2), is a necessary modulator for shedding of tumor necrosis factor-α (TNF-α) in immune cells, and has been explored in the pathogenesis of chronic renal diseases. In the present study, we found that compared to the wild type (iRhom2+/+) mice, iRhom2 knockout (iRhom2-/-) protected PM2.5-exposed mice from developing severe renal injury, accompanied with improved renal pathological changes and functions. iRhom2-/- mice exhibited reduced inflammatory response, as evidenced by the reduction of interleukin 1β (IL-1β), IL-6, tumor necrosis factor-α (TNF-α) and IL-18 in kidney samples, which might be, at least partly, through inactivating TNF-α converting enzyme/TNF-α receptors (TACE/TNFRs) and inhibitor of α/nuclear factor κ B (IκBα/NF-κB) signaling pathways. In addition, oxidative stress was also restrained by iRhom2-/- in kidney of PM2.5-exposed mice by enhancing heme oxygenase/nuclear factor erythroid 2-related factor 2 (HO-1/Nrf-2) expressions, and reducing phosphorylated c-Jun N-terminal kinase (JNK). In vitro, blockage of HO-1 or Nrf-2 rescued the inflammatory response and oxidative stress that were reduced by iRhom2 knockdown in PM2.5-incubated RAW264.7 cells. Similar results were observed in JNK activator-treated cells. Taken together, our findings indicated that iRhom2 played an essential role in regulating PM2.5-induced chronic renal damage, thus revealing a potential target for preventing chronic kidney diseases development. Keywords: PM2.5, Renal injury, iRhom2, Inflammation, Oxidative stress, JN

The deubiquitinating enzyme 13 retards non-alcoholic steatohepatitis via blocking inactive rhomboid protein 2-dependent pathway

Nowadays potential preclinical drugs for the treatment of nonalcoholic steatohepatitis (NASH) have failed to achieve expected therapeutic efficacy because the pathogenic mechanisms are underestimated. Inactive rhomboid protein 2 (IRHOM2), a promising target for treatment of inflammation-related diseases, contributes to deregulated hepatocyte metabolism-associated nonalcoholic steatohepatitis (NASH) progression. However, the molecular mechanism underlying Irhom2 regulation is still not completely understood. In this work, we identify the ubiquitin-specific protease 13 (USP13) as a critical and novel endogenous blocker of IRHOM2, and we also indicate that USP13 is an IRHOM2-interacting protein that catalyzes deubiquitination of Irhom2 in hepatocytes. Hepatocyte-specific loss of the Usp13 disrupts liver metabolic homeostasis, followed by glycometabolic disorder, lipid deposition, increased inflammation, and markedly promotes NASH development. Conversely, transgenic mice with Usp13 overexpression, lentivirus (LV)- or adeno-associated virus (AAV)-driven Usp13 gene therapeutics mitigates NASH in 3 models of rodent. Mechanistically, in response to metabolic stresses, USP13 directly interacts with IRHOM2 and removes its K63-linked ubiquitination induced by ubiquitin-conjugating enzyme E2N (UBC13), a ubiquitin E2 conjugating enzyme, and thus prevents its activation of downstream cascade pathway. USP13 is a potential treatment target for NASH therapy by targeting the Irhom2 signaling pathway