The methionine salvage pathway is widely distributed among some eubacteria, yeast, plants and animals and recycles the sulfur-containing metabolite 5-methylthioadenosine (MTA) to methionine. In eukaryotic cells, the methionine salvage pathway takes place in the cytosol and usually involves six enzymatic activities: MTA phosphorylase (MTAP, EC 2.4.2.28), 5′-methylthioribose-1-phosphate isomerase (mtnA, EC 5.3.1.23), 5′-methylthioribulose-1-phosphate dehydratase (mtnB, EC: 4.2.1.109), 2,3-dioxomethiopentane-1-phosphate enolase/phosphatase (mtnC, EC 3.1.3.77), aci-reductone dioxygenase (mtnD, EC 1.13.11.54) and 4-methylthio-2-oxo-butanoate (MTOB) transaminase (EC 2.6.1.-). The aim of this study was to complete the available information on the methionine salvage pathway in human by identifying the enzyme responsible for the dehydratase step. Using a bioinformatics approach, we propose that a protein called APIP could perform this role. The involvement of this protein in the methionine salvage pathway was investigated directly in HeLa cells by transient and stable short hairpin RNA interference. We show that APIP depletion specifically impaired the capacity of cells to grow in media where methionine is replaced by MTA. Using a Shigella mutant auxotroph for methionine, we confirm that the knockdown of APIP specifically affects the recycling of methionine. We also show that mutation of three potential phosphorylation sites does not affect APIP activity whereas mutation of the potential zinc binding site completely abrogates it. Finally, we show that the N-terminal region of APIP that is missing in the short isoform is required for activity. Together, these results confirm the involvement of APIP in the methionine salvage pathway, which plays a key role in many biological functions like cancer, apoptosis, microbial proliferation and inflammation.

Deoxyribose-phosphate aldolase (EC 4.1.2.4), which converts 2-deoxy-d-ribose-5-phosphate into glyceraldehyde-3-phosphate and acetaldehyde, belongs to the core metabolism of living organisms. It was previously shown that human cells harbor deoxyribose phosphate aldolase activity but the protein responsible of this activity has never been formally identified. This study provides the first experimental evidence that DERA, which is mainly expressed in lung, liver and colon, is the human deoxyribose phosphate aldolase. Among human cell lines, the highest DERA mRNA level and deoxyribose phosphate aldolase activity were observed in liver-derived Huh-7 cells. DERA was shown to interact with the known stress granule component YBX1 and to be recruited to stress granules after oxidative or mitochondrial stress. In addition, cells in which DERA expression was down-regulated using shRNA formed fewer stress granules and were more prone to apoptosis after clotrimazole stress, suggesting the importance of DERA for stress granule formation. Furthermore, the expression of DERA was shown to permit cells in which mitochondrial ATP production was abolished to make use of extracellular deoxyinosine to maintain ATP levels. This study unraveled a previously undescribed pathway which may allow cells with high deoxyribose-phosphate aldolase activity, such as liver cells, to minimize or delay stress-induced damage by producing energy through deoxynucleoside degradation.

The sodium-iodide symporter (NIS) is an integral membrane protein that plays a crucial role in iodide accumulation, especially in the thyroid. As for many other membrane proteins, its intracellular sorting and distribution have a tremendous effect on its function, and constitute an important aspect of its regulation. Many short sequences have been shown to contribute to protein trafficking along the sorting or endocytic pathways. Using bioinformatics tools, we identified such potential sites on human NIS [tyrosine-based motifs, SH2-(Src homology 2), SH3- and PDZ (post-synaptic density-95/discs large tumour suppressor/zonula occludens-1)-binding motifs, and diacidic, dibasic and dileucine motifs] and analysed their roles using mutagenesis. We found that several of these sites play a role in protein stability and/or targeting to the membrane. Aside from the mutation at position 178 (SH2 plus tyrosine-based motif) that affects iodide uptake, the most drastic effect is associated with the mutation of an internal PDZ-binding motif at position 121 that completely abolishes NIS expression at the plasma membrane. Mutating the sites located on the C-terminal domain of the protein has no effect except for the creation of a diacidic motif that decreases the total NIS protein level without affecting its expression at the plasma membrane.

The sodium/iodide symporter (NIS) expresses at the basolateral plasma membrane of the thyroid follicular cell and mediates iodide accumulation required for normal thyroid hormonogenesis. Loss-of-function NIS variants cause congenital hypothyroidism due to impaired iodide accumulation in thyroid follicular cells underscoring the significance of NIS for thyroid physiology. Here we report novel findings derived from the thorough characterization of the nonsense NIS mutant p.R636* NIS-leading to a truncated protein missing the last eight amino acids-identified in twins with congenital hypothyroidism. R636* NIS is severely mislocalized into intracellular vesicular compartments due to the lack of a conserved carboxy-terminal type 1 PDZ-binding motif. As a result, R636* NIS is barely targeted to the plasma membrane and therefore iodide transport is reduced. Deletion of the PDZ-binding motif causes NIS accumulation into late endosomes and lysosomes. Using PDZ domain arrays, we revealed that the PDZ-domain containing protein SCRIB binds to the carboxy-terminus of NIS by a PDZ-PDZ interaction. Furthermore, in CRISPR/Cas9based SCRIB deficient cells, NIS expression at the basolateral plasma membrane is compromised, leading to NIS localization into intracellular vesicular compartments.We conclude that the PDZ-binding motif is a plasma membrane retention signal that participates in the polarized expression of NIS by selectively interacting with the PDZ-domain containing protein SCRIB, thus retaining the transporter at the basolateral plasma membrane. Our data provide insights into the molecular mechanisms that regulate NIS expression at the plasma membrane, a topic of great interest in the 2 of 13 | MARTÍN eT Al.