Genomic Landscape Reveals Chromosomally-Mediated Antimicrobial Resistome and Virulome of a High-Risk International Clone II Acinetobacter baumannii AB073 from Thailand.

This study utilized integrative bioinformatics' tools together with phenotypic assays to understand the whole-genome features of a carbapenem-resistant international clone II Acinetobacter baumannii AB073. Overall, we found the isolate to be resistant to seven antibiotic classes, penicillins, ß-lactam/ß-lactamase inhibitor combinations, cephalosporins, carbapenems, aminoglycosides, fluoroquinolones, and folate pathway antagonists. These resistance phenotypes are related to various chromosomal-located antibiotic resistance determinants involved in different mechanisms such as reduced permeability, antibiotic target protection, antibiotic target alteration, antibiotic inactivation, and antibiotic efflux. IC2 A. baumannii AB073 could not transfer antibiotic resistance by conjugation experiments. Likewise, mobilome analysis found that AB073 did not carry genetic determinants involving horizontal gene transfer. Moreover, this isolate also carried multiple genes associated with the ability of iron uptake, biofilm formation, immune invasion, virulence regulations, and serum resistance. In addition, the genomic epidemiological study showed that AB073-like strains were successful pathogens widespread in various geographic locations and clinical sources. In conclusion, the comprehensive analysis demonstrated that AB073 contained multiple genomic determinants which were important characteristics to classify this isolate as a successful international clone II obtained from Thailand.

A family of carboxypeptidases catalyzing α- and ß-tubulin tail processing and deglutamylation.

Tubulin posttranslational modifications represent an important mechanism involved in the regulation of microtubule functions. The most widespread among them are detyrosination, α∆2-tubulin, and polyglutamylation. Here, we describe a family of tubulin-modifying enzymes composed of two closely related proteins, KIAA0895L and KIAA0895, which have tubulin metallocarboxypeptidase activity and thus were termed TMCP1 and TMCP2, respectively. We show that TMCP1 (also known as MATCAP) acts as α-tubulin detyrosinase that also catalyzes α∆2-tubulin. In contrast, TMCP2 preferentially modifies ßI-tubulin by removing three amino acids from its C terminus, generating previously unknown ßI∆3 modification. We show that ßI∆3-tubulin is mostly found on centrioles and mitotic spindles and in cilia. Moreover, we demonstrate that TMCPs also remove posttranslational polyglutamylation and thus act as tubulin deglutamylases. Together, our study describes the identification and comprehensive biochemical analysis of a previously unknown type of tubulin-modifying enzymes involved in the processing of α- and ß-tubulin C-terminal tails and deglutamylation.

NALCN-mediated sodium influx confers metastatic prostate cancer cell invasiveness.

There is growing evidence that ion channels are critically involved in cancer cell invasiveness and metastasis. However, the molecular mechanisms of ion signaling promoting cancer behavior are poorly understood and the complexity of the underlying remodeling during metastasis remains to be explored. Here, using a variety of in vitro and in vivo techniques, we show that metastatic prostate cancer cells acquire a specific Na+ /Ca2+ signature required for persistent invasion. We identify the Na+ leak channel, NALCN, which is overexpressed in metastatic prostate cancer, as a major initiator and regulator of Ca2+ oscillations required for invadopodia formation. Indeed, NALCN-mediated Na+ influx into cancer cells maintains intracellular Ca2+ oscillations via a specific chain of ion transport proteins including plasmalemmal and mitochondrial Na+ /Ca2+ exchangers, SERCA and store-operated channels. This signaling cascade promotes activity of the NACLN-colocalized proto-oncogene Src kinase, actin remodeling and secretion of proteolytic enzymes, thus increasing cancer cell invasive potential and metastatic lesions in vivo. Overall, our findings provide new insights into an ion signaling pathway specific for metastatic cells where NALCN acts as persistent invasion controller.

The sodium leak channel NALCN regulates cell excitability of pituitary endocrine cells.

Anterior pituitary endocrine cells that release hormones such as growth hormone and prolactin are excitable and fire action potentials. In these cells, several studies previously showed that extracellular sodium (Na+ ) removal resulted in a negative shift of the resting membrane potential (RMP) and a subsequent inhibition of the spontaneous firing of action potentials, suggesting the contribution of a Na+ background conductance. Here, we show that the Na+ leak channel NALCN conducts a Ca2+ - Gd3+ -sensitive and TTX-resistant Na+ background conductance in the GH3 cell line, a cell model of pituitary endocrine cells. NALCN knockdown hyperpolarized the RMP, altered GH3 cell electrical properties and inhibited prolactin secretion. Conversely, the overexpression of NALCN depolarized the RMP, also reshaping the electrical properties of GH3 cells. Overall, our results indicate that NALCN is functional in GH3 cells and involved in endocrine cell excitability as well as in hormone secretion. Indeed, the GH3 cell line suitably models native pituitary cells that display a similar Na+ background conductance and appears as a proper cellular model to study the role of NALCN in cellular excitability.

Functional expression of CLIFAHDD and IHPRF pathogenic variants of the NALCN channel in neuronal cells reveals both gain- and loss-of-function properties.

The excitability of neurons is tightly dependent on their ion channel repertoire. Among these channels, the leak sodium channel NALCN plays a crucial role in the maintenance of the resting membrane potential. Importantly, NALCN mutations lead to complex neurodevelopmental syndromes, including infantile hypotonia with psychomotor retardation and characteristic facies (IHPRF) and congenital contractures of limbs and face, hypotonia and developmental delay (CLIFAHDD), which are recessively and dominantly inherited, respectively. Unfortunately, the biophysical properties of NALCN are still largely unknown to date, as well as the functional consequences of both IHPRF and CLIFAHDD mutations on NALCN current. Here we have set-up the heterologous expression of NALCN in the neuronal cell line NG108-15 to investigate the electrophysiological properties of NALCN carrying representative IHPRF and CLIFAHDD mutations. Several original properties of the wild-type (wt) NALCN current were retrieved: mainly carried by external Na+, blocked by Gd3+, insensitive to TTX and potentiated by low external Ca2+ concentration. However, we found that this current displays a time-dependent inactivation in the -80/-40 mV range of membrane potential, and a non linear current-voltage relationship indicative of voltage sensitivity. Importantly, no detectable current was recorded with the IHPRF missense mutation p.Trp1287Leu (W1287L), while the CLIFAHDD mutants, p.Leu509Ser (L509S) and p.Tyr578Ser (Y578S), showed higher current densities and slower inactivation, compared to wt NALCN current. This study reveals that heterologous expression of NALCN channel can be achieved in the neuronal cell line NG108-15 to study the electrophysiological properties of wt and mutants. From our results, we conclude that IHPRF and CLIFAHDD missense mutations are loss- and gain-of-function variants, respectively.

[6]-Gingerol inhibits de novo fatty acid synthesis and carnitine palmitoyltransferase-1 activity which triggers apoptosis in HepG2.

The de novo fatty acid synthesis catalyzed by key lipogenic enzymes, including fatty acid synthase (FASN) has emerged as one of the novel targets of anti-cancer approaches. The present study explored the possible inhibitory efficacy of [6]-gingerol on de novo fatty acid synthesis associated with mitochondrial-dependent apoptotic induction in HepG2 cells. We observed a dissipation of mitochondrial membrane potential accompanied by a reduction of fatty acid levels. [6]-gingerol administration manifested inhibition of FASN expression, indicating FASN is a major target of [6]-gingerol inducing apoptosis in HepG2 cells. Indeed, we found that increased ROS generation could likely be a mediator of the anti-cancer effect of [6]-gingerol. A reduction of fatty acid levels and induction of apoptosis were restored by inhibition of acetyl-CoA carboxylase (ACC) activity, suggesting an accumulation of malonyl-CoA level could be the major cause of apoptotic induction of [6]-gingerol in HepG2 cells. The present study also showed that depletion of fatty acid following [6]-gingerol treatment caused an inhibitory effect on carnitine palmitoyltransferase-1 activity (CPT-1), whereas C75 augmented CPT-1 activity, indicating that [6]-gingerol exhibits the therapeutic benefit on suppression of fatty acid ß-oxidation.

The selective target of capsaicin on FASN expression and de novo fatty acid synthesis mediated through ROS generation triggers apoptosis in HepG2 cells.

The inhibition of the mammalian de novo synthesis of long-chain saturated fatty acids (LCFAs) by blocking the fatty acid synthase (FASN) enzyme activity in tumor cells that overexpress FASN can promote apoptosis, without apparent cytotoxic to non-tumor cells. The present study aimed to focus on the potent inhibitory effect of capsaicin on the fatty acid synthesis pathway inducing apoptosis of capsaicin in HepG2 cells. The use of capsaicin as a source for a new FASN inhibitor will provide new insight into its possible application as a selective anti-cancer therapy. The present findings showed that capsaicin promoted apoptosis as well as cell cycle arrest in the G0/G1 phase. The onset of apoptosis was correlated with a dissipation of mitochondrial membrane potential (ΔΨm). Apoptotic induction by capsaicin was mediated by inhibition of FASN protein expression which was accompanied by decreasing its activity on the de novo fatty acid synthesis. The expression of FASN was higher in HepG2 cells than in normal hepatocytes that were resistant to undergoing apoptosis following capsaicin administration. Moreover, the inhibitory effect of capsaicin on FASN expression and activity was found to be mediated by an increase of intracellular reactive oxygen species (ROS) generation. Treatment of HepG2 cells with capsaicin failed to alter ACC and ACLY protein expression, suggesting ACC and ACLY might not be the specific targets of capsaicin to induce apoptosis. An accumulation of malonyl-CoA level following FASN inhibition represented a major cause of mitochondrial-dependent apoptotic induction instead of deprivation of fatty acid per se. Here, we also obtained similar results with C75 that exhibited apoptosis induction by reducing the levels of fatty acid without any change in the abundance of FASN expression along with increasing ROS production. Collectively, our results provide novel evidence that capsaicin exhibits a potent anti-cancer property by targeting FASN protein in HepG2 cells.