SUMOylation controls Hu antigen R posttranscriptional activity in liver cancer.

The posttranslational modification of proteins critically influences many biological processes and is a key mechanism that regulates the function of the RNA-binding protein Hu antigen R (HuR), a hub in liver cancer. Here, we show that HuR is SUMOylated in the tumor sections of patients with hepatocellular carcinoma in contrast to the surrounding tissue, as well as in human cell line and mouse models of the disease. SUMOylation of HuR promotes major cancer hallmarks, namely proliferation and invasion, whereas the absence of HuR SUMOylation results in a senescent phenotype with dysfunctional mitochondria and endoplasmic reticulum. Mechanistically, SUMOylation induces a structural rearrangement of the RNA recognition motifs that modulates HuR binding affinity to its target RNAs, further modifying the transcriptomic profile toward hepatic tumor progression. Overall, SUMOylation constitutes a mechanism of HuR regulation that could be potentially exploited as a therapeutic strategy for liver cancer.

Ratiometric Fluorescent Sensors Illuminate Cellular Magnesium Imbalance in a Model of Acetaminophen-Induced Liver Injury.

Magnesium(II) plays catalytic, structural, regulatory, and signaling roles in living organisms. Abnormal levels of this metal have been associated with numerous pathologies, including cardiovascular disease, diabetes, metabolic syndrome, immunodeficiency, cancer, and, most recently, liver pathologies affecting humans. The role of Mg2+ in the pathophysiology of liver disease, however, has been occluded by concomitant changes in concentration of interfering divalent cations, such as Ca2+, which complicates the interpretation of experiments conducted with existing molecular Mg2+ indicators. Herein, we introduce a new quinoline-based fluorescent sensor, MagZet1, that displays a shift in its excitation and emission wavelengths, affording ratiometric detection of cellular Mg2+ by both fluorescence microscopy and flow cytometry. The new sensor binds the target metal with a submillimolar dissociation constant─well suited for detection of changes in free Mg2+ in cells─and displays a 10-fold selectivity against Ca2+. Furthermore, the fluorescence ratio is insensitive to changes in pH in the physiological range, providing an overall superior performance over existing indicators. We provide insights into the metal selectivity profile of the new sensor based on computational modeling, and we apply it to shed light on a decrease in cytosolic free Mg2+ and altered expression of metal transporters in cellular models of drug-induced liver injury caused by acetaminophen overdose.

Methionine Cycle Rewiring by Targeting miR-873-5p Modulates Ammonia Metabolism to Protect the Liver from Acetaminophen.

Drug-induced liver injury (DILI) development is commonly associated with acetaminophen (APAP) overdose, where glutathione scavenging leads to mitochondrial dysfunction and hepatocyte death. DILI is a severe disorder without effective late-stage treatment, since N-acetyl cysteine must be administered 8 h after overdose to be efficient. Ammonia homeostasis is altered during liver diseases and, during DILI, it is accompanied by decreased glycine N-methyltransferase (GNMT) expression and S-adenosylmethionine (AdoMet) levels that suggest a reduced methionine cycle. Anti-miR-873-5p treatment prevents cell death in primary hepatocytes and the appearance of necrotic areas in liver from APAP-administered mice. In our study, we demonstrate a GNMT and methionine cycle activity restoration by the anti-miR-873-5p that reduces mitochondrial dysfunction and oxidative stress. The lack of hyperammoniemia caused by the therapy results in a decreased urea cycle, enhancing the synthesis of polyamines from ornithine and AdoMet and thus impacting the observed recovery of mitochondria and hepatocyte proliferation for regeneration. In summary, anti-miR-873-5p appears to be an effective therapy against APAP-induced liver injury, where the restoration of GNMT and the methionine cycle may prevent mitochondrial dysfunction while activating hepatocyte proliferative response.

Structural insight into the unique conformation of cystathionine ß-synthase from Toxoplasma gondii.

Cysteine plays a major role in the redox homeostasis and antioxidative defense mechanisms of many parasites of the phylum Apicomplexa. Of relevance to human health is Toxoplasma gondii, the causative agent of toxoplasmosis. A major route of cysteine biosynthesis in this parasite is the reverse transsulfuration pathway involving two key enzymes cystathionine ß-synthase (CBS) and cystathionine γ-lyase (CGL). CBS from T. gondii (TgCBS) catalyzes the pyridoxal-5́-phosphate-dependent condensation of homocysteine with either serine or O-acetylserine to produce cystathionine. The enzyme can perform alternative reactions that use homocysteine and cysteine as substrates leading to the endogenous biosynthesis of hydrogen sulfide, another key element in maintaining the intracellular redox equilibrium. In contrast with human CBS, TgCBS lacks the N-terminal heme binding domain and is not responsive to S-adenosylmethionine. Herein, we describe the structure of a TgCBS construct that lacks amino acid residues 466-491 and shows the same activity of the native protein. TgCBS Δ466-491 was determined alone and in complex with reaction intermediates. A complementary molecular dynamics analysis revealed a unique domain organization, similar to the pathogenic mutant D444N of human CBS. Our data provides one missing piece in the structural diversity of CBSs by revealing the so far unknown three-dimensional arrangement of the CBS-type of Apicomplexa. This domain distribution is also detected in yeast and bacteria like Pseudomonas aeruginosa. These results pave the way for understanding the mechanisms by which TgCBS regulates the intracellular redox of the parasite, and have far-reaching consequences for the functional understanding of CBSs with similar domain distribution.

ARL15 modulates magnesium homeostasis through N-glycosylation of CNNMs.

Cyclin M (CNNM1-4) proteins maintain cellular and body magnesium (Mg2+) homeostasis. Using various biochemical approaches, we have identified members of the CNNM family as direct interacting partners of ADP-ribosylation factor-like GTPase 15 (ARL15), a small GTP-binding protein. ARL15 interacts with CNNMs at their carboxyl-terminal conserved cystathionine-ß-synthase (CBS) domains. In silico modeling of the interaction between CNNM2 and ARL15 supports that the small GTPase specifically binds the CBS1 and CNBH domains. Immunocytochemical experiments demonstrate that CNNM2 and ARL15 co-localize in the kidney, with both proteins showing subcellular localization in the endoplasmic reticulum, Golgi apparatus and the plasma membrane. Most importantly, we found that ARL15 is required for forming complex N-glycosylation of CNNMs. Overexpression of ARL15 promotes complex N-glycosylation of CNNM3. Mg2+ uptake experiments with a stable isotope demonstrate that there is a significant increase of 25Mg2+ uptake upon knockdown of ARL15 in multiple kidney cancer cell lines. Altogether, our results establish ARL15 as a novel negative regulator of Mg2+ transport by promoting the complex N-glycosylation of CNNMs.

Structural Insights into the Intracellular Region of the Human Magnesium Transport Mediator CNNM4.

The four member family of "Cyclin and Cystathionine ß-synthase (CBS) domain divalent metal cation transport mediators", CNNMs, are the least-studied mammalian magnesium transport mediators. CNNM4 is abundant in the brain and the intestinal tract, and its abnormal activity causes Jalili Syndrome. Recent findings show that suppression of CNNM4 in mice promotes malignant progression of intestinal polyps and is linked to infertility. The association of CNNM4 with phosphatases of the regenerating liver, PRLs, abrogates its Mg2+-efflux capacity, thus resulting in an increased intracellular Mg2+ concentration that favors tumor growth. Here we present the crystal structures of the two independent intracellular domains of human CNNM4, i.e., the Bateman module and the cyclic nucleotide binding-like domain (cNMP). We also derive a model structure for the full intracellular region in the absence and presence of MgATP and the oncogenic interacting partner, PRL-1. We find that only the Bateman module interacts with ATP and Mg2+, at non-overlapping sites facilitating their positive cooperativity. Furthermore, both domains dimerize autonomously, where the cNMP domain dimer forms a rigid cleft to restrict the Mg2+ induced sliding of the inserting CBS1 motives of the Bateman module, from a twisted to a flat disk shaped dimer.

Current Structural Knowledge on the CNNM Family of Magnesium Transport Mediators.

The cyclin and cystathionine ß-synthase (CBS) domain magnesium transport mediators, CNNMs, are key players in maintaining the homeostasis of magnesium in different organs. The human family includes four members, whose impaired activity causes diseases such as Jalili Syndrome or Familial Hypomagnesemia, but is also linked to neuropathologic disorders, altered blood pressure, and infertility. Recent findings demonstrated that CNNMs are associated with the highly oncogenic phosphatases of the regenerating liver to promote tumor growth and metastasis, which has attracted renewed focus on their potential exploitation as targets for cancer treatment. However, the exact function of CNNMs remains unclear and is subject to debate, proposed as either direct transporters, sensors, or homeostatic factors. This review gathers the current structural knowledge on the CNNM family, highlighting similarities and differences with the closely related structural partners such as the bacterial Mg2+/Co2+ efflux protein CorC and the Mg2+ channel MgtE.