Dysregulated interactions between lamin A and SUN1 induce abnormalities in the nuclear envelope and endoplasmic reticulum in progeric laminopathies.

Hutchinson-Gilford progeria syndrome (HGPS) is a human progeroid disease caused by a point mutation on the LMNA gene. We reported previously that the accumulation of the nuclear envelope protein SUN1 contributes to HGPS nuclear aberrancies. However, the mechanism by which interactions between mutant lamin A (also known as progerin or LAΔ50) and SUN1 produce HGPS cellular phenotypes requires further elucidation. Using light and electron microscopy, this study demonstrated that SUN1 contributes to progerin-elicited structural changes in the nuclear envelope and the endoplasmic reticulum (ER) network. We further identified two domains through which full-length lamin A associates with SUN1, and determined that the farnesylated cysteine within the CaaX motif of lamin A has a stronger affinity for SUN1 than does the lamin A region containing amino acids 607 to 656. Farnesylation of progerin enhanced its interaction with SUN1 and reduced SUN1 mobility, thereby promoting the aberrant recruitment of progerin to the ER membrane during postmitotic assembly of the nuclear envelope, resulting in the accumulation of SUN1 over consecutive cellular divisions. These results indicate that the dysregulated interaction of SUN1 and progerin in the ER during nuclear envelope reformation determines the progression of HGPS.

Double homeobox gene, Duxbl, promotes myoblast proliferation and abolishes myoblast differentiation by blocking MyoD transactivation.

Homeobox genes encode transcription factors that regulate embryonic development programs including organogenesis, axis formation and limb development. Previously, we identified and cloned a mouse double homeobox gene, Duxbl, whose homeodomain exhibits the highest identity (67 %) to human DUX4, a candidate gene of facioscapulohumeral muscular dystrophy (FSHD). Duxbl proteins have been shown to be expressed in elongated myocytes and myotubes of trunk and limb muscles during embryogenesis. In this study, we found that Duxbl maintained low expression levels in various adult muscles. Duxbl proteins were induced to express in activated satellite cells and colocalized with MyoG, a myogenic differentiating marker. Furthermore, Duxbl proteins were not detected in quiescent satellite cells but detected in regenerated myocytes and colocalized with MyoD and MyoG following cardiotoxin-induced muscle injury. Ectopic Duxbl overexpressions in C2C12 myoblast cells promoted cell proliferation through mainly enhancing cyclin D1 and hyper-phosphorylated retinoblastoma protein but reducing p21 expression. However, Duxbl overexpression in C2C12 cells inhibited myogenic differentiation by decreasing MyoD downstream gene expressions, including M-cadherin, MyoG, p21 and cyclin D3 but not MyoD itself. Duxbl overexpressions also promoted cell proliferation but blocked MyoD-induced myogenic conversion in multipotent mesenchymal C3H10T1/2 cells. In addition, results of a luciferase reporter assay suggest that Duxbl negatively regulated MyoG promoter activity through the proximal two E boxes. In conclusion, these results indicate that Duxbl may play a crucial role in myogenesis and postnatal muscle regeneration by activating and proliferating satellite and myoblast cells.

Mitochondrial injury induced by a Salmonella genotoxin triggers the proinflammatory senescence-associated secretory phenotype.

Bacterial genotoxins damage host cells by targeting their chromosomal DNA. In the present study, we demonstrate that a genotoxin of Salmonella Typhi, typhoid toxin, triggers the senescence-associated secretory phenotype (SASP) by damaging mitochondrial DNA. The actions of typhoid toxin disrupt mitochondrial DNA integrity, leading to mitochondrial dysfunction and disturbance of redox homeostasis. Consequently, it facilitates the release of damaged mitochondrial DNA into the cytosol, activating type I interferon via the cGAS-STING pathway. We also reveal that the GCN2-mediated integrated stress response plays a role in the upregulation of inflammatory components depending on the STING signaling axis. These SASP factors can propagate the senescence effect on T cells, leading to senescence in these cells. These findings provide insights into how a bacterial genotoxin targets mitochondria to trigger a proinflammatory SASP, highlighting a potential therapeutic target for an anti-toxin intervention.

Abi plays an opposing role to Abl in Drosophila axonogenesis and synaptogenesis.

Abl tyrosine kinase (Abl) regulates axon guidance by modulating actin dynamics. Abelson interacting protein (Abi), originally identified as a kinase substrate of Abl, also plays a key role in actin dynamics, yet its role with respect to Abl in the developing nervous system remains unclear. Here we show that mutations in abi disrupt axonal patterning in the developing Drosophila central nervous system (CNS). However, reducing abi gene dosage by half substantially rescues Abl mutant phenotypes in pupal lethality, axonal guidance defects and locomotion deficits. Moreover, we show that mutations in Abl increase synaptic growth and spontaneous synaptic transmission frequency at the neuromuscular junction. Double heterozygosity for abi and enabled (ena) also suppresses the synaptic overgrowth phenotypes of Abl mutants, suggesting that Abi acts cooperatively with Ena to antagonize Abl function in synaptogenesis. Intriguingly, overexpressing Abi or Ena alone in cultured cells dramatically redistributed peripheral F-actin to the cytoplasm, with aggregates colocalizing with Abi and/or Ena, and resulted in a reduction in neurite extension. However, co-expressing Abl with Abi or Ena redistributed cytoplasmic F-actin back to the cell periphery and restored bipolar cell morphology. These data suggest that abi and Abl have an antagonistic interaction in Drosophila axonogenesis and synaptogenesis, which possibly occurs through the modulation of F-actin reorganization.

Kaempferol protects against propacetamol-induced acute liver injury through CYP2E1 inactivation, UGT1A1 activation, and attenuation of oxidative stress, inflammation and apoptosis in mice.

Acetaminophen (APAP) overdose can induce acute liver injury (ALI) with significant morbidity and mortality. Propacetamol is an APAP prodrug, which is clinically bioequivalent to APAP. Kaempferol, a dietary flavonoid, has antioxidant, anti-inflammatory, and anti-apoptotic effects. In this study, we investigated the protective effect of kaempferol on propacetamol-induced ALI and its underlying mechanism in mice. Kaempferol pretreatment (125 mg/kg) before propacetamol injection significantly decreased propacetamol-induced serum ALT and AST activities, and DNA fragmentation. Kaempferol administration also reduced propacetamol-induced oxidative stress by inhibiting thiobarbituric acid reactive substances (TBARS) and 3-nitrotyrosine (3-NT) formation partly through downregulation of cytochrome P450 2E1 (CYP2E1) expression, upregulation of UDP glucuronosyltransferase family 1 member A1 (UGT1A1) expression, restoration of the activities of antioxidant enzymes including SOD, GPx and catalase toward normal, recovery of propacetamol-suppressed Nrf2 and GCLC expressions, and maintenance of normal glutathione level. Furthermore, kaempferol markedly attenuated APAP-induced serum TNF-α and IL-6 productions, downregulated APAP-induced phosphorylations of JNK and ERK, and decreased early hepatic apoptosis via decreasing Bax/Bcl-2 ratio and caspase 3 activation. Furthermore, administration of N-acetylcysteine (NAC) and kaempferol significantly rescued more mice than a low dose of NAC only did when a lethal dose of propacetamol injected and therapized at a delayed time point. These data suggested that kaempferol protects the liver against propacetamol-induced injury through anti-oxidative, anti-inflammatory and anti-apoptotic activities.

Cardiac fibrosis in mouse expressing DsRed tetramers involves chronic autophagy and proteasome degradation insufficiency.

Proteinopathy in the heart which often manifests excessive misfolded/aggregated proteins in cardiac myocytes can result in severe fibrosis and heart failure. Here we developed a mouse model, which transgenically express tetrameric DsRed, a red fluorescent protein (RFP), in an attempt to mimic the pathological mechanisms ofcardiac fibrosis. Whilst DsRed is expressed and forms aggregation in most mouse organs, certain pathological defects are specifically recapitulated in cardiac muscle cells including mitochondria damages, aggresome-like residual bodies, excessive ubiquitinated proteins, and the induction of autophagy. The proteinopathy and cellular injuries caused by DsRed aggregates may be due to impaired or overburdened ubiquitin-proteasome system and autophagy-lysosome systems. We further identified that DsRed can be ubiquitinated and associated with MuRF1, a muscle-specific E3 ligase. Concomitantly, an activation of NF-κB signaling and a strong TIMP1 induction were noted, suggesting that RFP-induced fibrosis was augmented by a skewed balance between TIMP1 and MMPs. Taken together, our study highlights the molecular consequences of uncontrolled protein aggregation leading to congestive heart failure, and provides novel insights into fibrosis formation that can be exploited for improved therapy.

The inhibitory effect of the hepatitis B virus singly-spliced RNA-encoded p21.5 protein on HBV nucleocapsid formation.

Hepatitis B virus (HBV) is the smallest DNA virus and the major cause of acute and chronic hepatitis. The 3.2 kb HBV viral genome generates four major species of unspliced viral transcript as well as several alternatively spliced RNAs. A 2.2 kb singly-spliced RNA is the most abundant spliced RNA and is widely expressed among all HBV genotypes. The expression of the singly-spliced RNA, as well as that of its encoded protein HBSP, is strongly associated with hepatopathology during HBV infection. Here, we report a novel inhibitory role of a p21.5 protein, which is encoded by a 2.2 kb singly-spliced RNA, in the modulation of HBV replication. We show that overexpression of the singly-spliced RNA is able to efficiently inhibit HBV replication. Furthermore, a mutation in the ATG start codon of the precore region completely abolishes the inhibitory effect of the singly-spliced RNA, indicating that a viral protein (p21.5) derived from the singly-spliced RNA is the mediator of the inhibition. Furthermore, p21.5 is able to form a homodimer that interacts with core dimers forming hybrid viral assembly components. Sucrose gradient fractionation revealed that co-expression of p21.5 resulted in a spread distribution pattern of core proteins ranging from low to high sucrose densities. When compared with p22, p21.5 is almost ten times more efficient at destabilizing HBV nucleocapsid assembly in Huh7 cells overexpressing either p21.5 or p22 protein. Moreover, in vivo expression of p21.5 protein by tail vein injection was found to decrease the amount of nucleocapsid in the livers of HBV-expressing BALB/c mice. In conclusion, our study reveals that the HBV 2.2 kb singly-spliced RNA encodes a 21.5 kDa viral protein that significantly interferes with the assembly of nucleocapsids during HBV nucleocapsid formation. These findings provide a possible strategy for elimination of HBV particles inside cells.

Targeting MCT-1 oncogene inhibits Shc pathway and xenograft tumorigenicity.

Overexpression of Shc adaptor proteins is associated with mitogenesis, carcinogenesis and metastasis. Multiple copies in T-cell malignancy 1 (MCT-1) oncoprotein promotes cell proliferation, survival and tumorigenic effects. Our current data show that MCT-1 is a novel regulator of Shc-Ras-MEK-ERK signaling and MCT-1 is significantly co-activated with Shc gene in human carcinomas. The knockdown of MCT-1 enhances apoptotic cell death accompanied with the activation of caspases and cleavage of caspase substrates under environmental stress. The cancer cell proliferation, chemo-resistance and tumorigenic capacity are proved to be effectively suppressed by targeting MCT-1. Accordingly, an important linkage between MCT-1 oncogenicity and Shc pathway in tumor development has now been established. Promoting MCT-1 expression by gene hyperactivation may be recognized as a tumor marker and MCT-1 may serve as a molecular target of cancer therapy.

Role of a propeller loop in the quaternary structure and enzymatic activity of prolyl dipeptidases DPP-IV and DPP9.

The dipeptidyl peptidase (DPP) family members, including DPP-IV, DPP8, DPP9 and others, cleave the peptide bond after the penultimate proline residue and are drug target rich. The dimerization of DPP-IV is required for its activity. A propeller loop located at the dimer interface is highly conserved within the family. Here we carried out site-directed mutagenesis on the loop of DPPIV and identified several residues important for dimer formation and enzymatic activity. Interestingly, the corresponding residues on DPP9 have a different impact whereby the mutations decrease activity without changing dimerization. Thus the propeller loop seems to play a varying role in different DPPs.