New insight into transglutaminase 2 and link to neurodegenerative diseases.

Formation of toxic protein aggregates is a common feature and mainly contributes to the pathogenesis of neurodegenerative diseases (NDDs), which include amyotrophic lateral sclerosis (ALS), Alzheimer's, Parkinson's, Huntington's, and prion diseases. The transglutaminase 2 (TG2) gene encodes a multifunctional enzyme, displaying four types of activity, such as transamidation, GTPase, protein disulfide isomerase, and protein kinase activities. Many studies demonstrated that the calcium-dependent transamidation activity of TG2 affects the formation of insoluble and toxic amyloid aggregates that mainly consisted of NDD-related proteins. So far, many important and NDD-related substrates of TG2 have been identified, including amlyoid-ß, tau, α-synuclein, mutant huntingtin, and ALS-linked trans-activation response (TAR) DNA-binding protein 43. Recently, the formation of toxic inclusions mediated by several TG2 substrates were efficiently inhibited by TG2 inhibitors. Therefore, the development of highly specific TG2 inhibitors would be an important tool in alleviating the progression of TG2-related brain disorders. In this review, the authors discuss recent advances in TG2 biochemistry, several mechanisms of molecular regulation and pleotropic signaling functions, and the presumed role of TG2 in the progression of many NDDs. [BMB Reports 2018; 51(1): 5-13].

Isocitrate protects DJ-1 null dopaminergic cells from oxidative stress through NADP+-dependent isocitrate dehydrogenase (IDH).

DJ-1 is one of the causative genes for early onset familiar Parkinson's disease (PD) and is also considered to influence the pathogenesis of sporadic PD. DJ-1 has various physiological functions which converge on controlling intracellular reactive oxygen species (ROS) levels. In RNA-sequencing analyses searching for novel anti-oxidant genes downstream of DJ-1, a gene encoding NADP+-dependent isocitrate dehydrogenase (IDH), which converts isocitrate into α-ketoglutarate, was detected. Loss of IDH induced hyper-sensitivity to oxidative stress accompanying age-dependent mitochondrial defects and dopaminergic (DA) neuron degeneration in Drosophila, indicating its critical roles in maintaining mitochondrial integrity and DA neuron survival. Further genetic analysis suggested that DJ-1 controls IDH gene expression through nuclear factor-E2-related factor2 (Nrf2). Using Drosophila and mammalian DA models, we found that IDH suppresses intracellular and mitochondrial ROS level and subsequent DA neuron loss downstream of DJ-1. Consistently, trimethyl isocitrate (TIC), a cell permeable isocitrate, protected mammalian DJ-1 null DA cells from oxidative stress in an IDH-dependent manner. These results suggest that isocitrate and its derivatives are novel treatments for PD associated with DJ-1 dysfunction.

PINK1 phosphorylates transglutaminase 2 and blocks its proteasomal degradation.

Parkinson's disease (PD) is characterized by progressive dopaminergic neuronal loss and the formation of abnormal protein aggregates, referred to as Lewy bodies (LBs). PINK1 is a serine/threonine protein kinase that protects cells from stress-induced mitochondrial dysfunction. PINK1 gene mutations cause one form of autosomal recessive early-onset PD. Transglutaminase 2 (TG2) is an intracellular protein cross-linking enzyme that has an important role in LB formation during PD pathogenesis. This study identifies PINK1 as a novel TG2 binding partner and shows that PINK1 stabilizes the half-life of TG2 via inhibition of TG2 ubiquitination and subsequent proteasomal degradation. PINK1 affects TG2 stability in a kinase-dependent manner. In addition, PINK1 directly phosphorylates TG2 in carbonyl cyanide m-chlorophenyl hydrazine-induced mitochondrial damaged states, thereby enhancing TG2 accumulation and intracellular protein cross-linking products. This study further confirms the functional link between upstream PINK1 and downstream TG2 in Drosophila melanogaster. These data suggest that PINK1 positively regulates TG2 activity, which may be closely associated with aggresome formation in neuronal cells.

Osteoconduction capacity of human deciduous and permanent teeth ash in a rat calvarial bone defect model.

The aim of this study was to confirm the osteoconduction capacities and determine the potential of permanent teeth ash (PTA), and deciduous teeth ash (DTA) as bone substitutes. Rats (n = 71) were divided randomly into four groups: sham, micro macroporous biphasic calcium phosphate (MBCP), PTA, and DTA. A sample of the each group was transplanted into preformed 8-mm calvarial defects (one per rat). The density of new bone was calculated and the crystallinities of the PTA and DTA were analyzed by X-ray diffraction. The degree of new bone formation was high in the MBCP and DTA groups but low in the PTA groups. The DTA was highly crystalline, whereas the PTA was not. The percentages of ß-tricalcium phosphate in the DTA and PTA were 10.7 and 3.7%, respectively. DTA has a high osteoconduction capacity, suggesting that it is a useful bone substitute.

Multiple teeth fractures in dentinogenesis imperfecta: a case report.

Dentinogenesis imperfecta (DGI) is a hereditary defect consisting of opalescent teeth composed of irregularly formed and hypomineralized dentin. This paper presents the multiple fractures of DGI-affected teeth and suggests the reason of low fracture resistance by observing the dentin microstructures directly using scanning electron microscope (SEM) and by measuring its surface hardness using the Vickers hardness test. SEM revealed that while the enamel microstructure was similar in the DGI-affected and normal teeth, the microstructure of the DGI-affected dentin was poorly woven and more loosely packed than that of the normal dentin. The Vickers hardness of the DGI-affected dentin was 4.89 times softer than the normal dentin. The low fracture resistance of DGI-affected teeth can be attributed to the poorly woven microstructure of their dentin, which leads to a reduction in hardness.

Covalent NEDD8 conjugation increases RCAN1 protein stability and potentiates its inhibitory action on calcineurin.

Similar to ubiquitin, regulatory roles for NEDD8 (neural precursor cell-expressed developmentally down-regulated 8) are being clarified during cell growth, signal transduction, immune response, and development. However, NEDD8 targets and their functional alterations are not well known. Regulator of calcineurin 1 (RCAN1/DSCR1P1) is located near the Down syndrome critical region on the distal part of chromosome 21, and its gene product is an endogenous inhibitor of calcineurin signaling. RCAN1 is modified by ubiquitin and consequently undergoes proteasomal degradation. Here we report that NEDD8 is conjugated to RCAN1 (RCAN1-1S) via three lysine residues, K96, K104, and K107. Neddylation enhances RCAN1 protein stability without affecting its cellular location. In addition, we found that neddylation significantly inhibits proteasomal degradation of RCAN1, which may underlie the ability of NEDD8 to enhance RCAN1 stability. Furthermore, neddylation increases RCAN1 binding to calcineurin, which potentiates its inhibitory activity toward downstream NFAT signaling. The present study provides a new regulatory mechanism of RCAN1 function and highlights an important role for diverse RCAN1-involved cellular physiology.

Identification of RUNX3 as a component of the MST/Hpo signaling pathway.

Recent genetic screens of fly mutants and molecular analysis have revealed that the Hippo (Hpo) pathway controls both cell proliferation and cell death. Deregulation of its human counterpart (the MST pathway) has been implicated in human cancers. However, how this pathway is linked with the known tumor suppressor network remains to be established. RUNX3 functions as a tumor suppressor of gastric cancer, lung cancer, bladder cancer, and colon cancer. Here, we show that RUNX3 is a principal and evolutionarily conserved component of the MST pathway. SAV1/WW45 facilitates the close association between MST2 and RUNX3. MST2, in turn, stimulates the SAV1-RUNX3 interaction. In addition, we show that siRNA-mediated RUNX3 knockdown abolishes MST/Hpo-mediated cell death. By establishing that RUNX3 is an endpoint effector of the MST pathway and that RUNX3 is capable of inducing cell death in cooperation with MST and SAV1, we define an evolutionarily conserved novel regulatory mechanism loop for tumor suppression in human cancers.

ASK1 negatively regulates the 26 S proteasome.

The 26 S proteasome, composed of the 20 S core and 19 S regulatory particle, plays a central role in ubiquitin-dependent proteolysis. Disruption of this process contributes to the pathogenesis of the various diseases; however, the mechanisms underlying the regulation of 26 S proteasome activity remain elusive. Here, cell culture experiments and in vitro assays demonstrated that apoptosis signal-regulating kinase 1 (ASK1), a member of the MAPK kinase kinase family, negatively regulated 26 S proteasome activity. Immunoprecipitation/Western blot analyses revealed that ASK1 did not interact with 20 S catalytic core but did interact with ATPases making up the 19 S particle, which is responsible for recognizing polyubiquitinated proteins, unfolding them, and translocating them into the 20 S catalytic core in an ATP-dependent process. Importantly, ASK1 phosphorylated Rpt5, an AAA ATPase of the 19 S proteasome, and inhibited its ATPase activity, an effect that may underlie the ability of ASK1 to inhibit 26 S proteasome activity. The current findings point to a novel role for ASK1 in the regulation of 26 S proteasome and offer new strategies for treating human diseases caused by proteasome malfunction.

Parkin directly modulates 26S proteasome activity.

Parkinson's disease (PD) is a common neurodegenerative disease that involves the deterioration of dopaminergic neurons in the substantia nigra pars compacta. Although the etiology of PD remains poorly understood, recent genetic, postmortem, and experimental evidence shows that abnormal protein accumulation and subsequent aggregate formation are prominent features of both sporadic and familial PD. While proteasome dysfunction is observed in PD, diverse mutations in the parkin gene are linked to early-onset autosomal-recessive forms of familial PD. We demonstrate that parkin, an E3 ubiquitin ligase, activates the 26S proteasome in an E3 ligase activity-independent manner. Furthermore, an N-terminal ubiquitin-like domain within parkin is critical for the activation of the 26S proteasome through enhancing the interaction between 19S proteasomal subunits, whereas the PD-linked R42P mutant abolishes this action. The current findings point to a novel role for parkin for 26S proteasome assembly and suggest that parkin mutations contribute to the proteasomal dysfunction in PD.