Background: Copper/zinc superoxide dismutase (SOD1) genetic mutants are associated with familial amyotrophic lateral sclerosis (ALS). Mutant proteins form abnormal aggregates. Results: We used imaging of live cells to observe SOD1 proteins harboring mutations associated with ALS. Conclusion: SOD1 mutations impair its dimerization, leading to subsequent aggregation. Significance: Analysis of the SOD1 quaternary structure in living human cells correlates with previous biochemical data.

Human phosphoserine phosphatase (HPSP) regulates the levels of glycine and D-serine, the putative co-agonists for the glycine site of the NMDA receptor in the brain. Here, we describe the first crystal structures of the HPSP in complexes with the competitive inhibitor 2-amino-3-phosphonopropionic acid (AP3) at 2.5 Å, and the phosphate ion (Pi) and the product uncompetitive inhibitor L-serine (HPSP⅐L-Ser⅐Pi) at 2.8 Å. The complex structures reveal that the open-closed environmental change of the active site, generated by local rearrangement of the ␣-helical bundle domain, is important to substrate recognition and hydrolysis. The maximal extent of this structural rearrangement is shown to be about 13 Å at the L4 loop and about 25°at the helix ␣3. Both the structural change and mutagenesis data suggest that Arg-65 and Glu-29 play an important role in the binding of the substrate. Interestingly, the AP3 binding mode turns out to be significantly different from that of the natural substrate, phospho-L-serine, and the HPSP⅐L-Ser⅐Pi structure provides a structural basis for the feedback control mechanism of serine. These analyses allow us to provide a clear model for the mechanism of HPSP and a framework for structure-based drug development. Phosphoserine phosphatase (PSP)1 is an important enzyme in the phosphorylated pathway of serine biosynthesis, which contributes a major portion of the endogenous L-serine (1, 2). In the mammalian nervous system, D-serine is converted from L-serine by serine racemase (3, 4) and acts as a co-agonist of the N-methyl-D-aspartate (NMDA) subtype of glutamate receptors, a major neurotransmitter receptor family (5). NMDA receptors require coactivation at a glycine site where D-serine, present in high levels in the mammalian brain, is three times more potent than glycine (6). Recently, a subset of NMDA receptors has been found to be activated by glycine or D-serine in the absence of glutamate (7). The enzymatic reaction of PSP is Mg 2ϩ -dependent and results in the dephosphorylation of phospho-Lserine with the formation of a phosphoenzyme intermediate, which is subsequently autodephosphorylated. The resulting product, L-serine, is not only a precursor for the biosynthesis of glycine but also an uncompetitive inhibitor for the enzymatic reaction of PSP (8). It may be possible to regulate NMDA activity by using selective inhibitors against serine racemase and/or PSP.The PSP family and P-type ATPases are members of the haloacid dehalogenase-like hydrolase family (9 -11). The crystal structures have been elucidated for several members of the haloacid dehalogenase superfamily: the L-2-haloacid dehalogenases, CheY, P-type ATPase and, recently, Methanococcus jannaschii (MJ) PSP (12-16). Three conserved motifs have been observed in amino acid sequences in members of the haloacid dehalogenase superfamily, and these residues are located in the active pocket. The significance of these motifs was confirmed by an assay of mutants for substrate hydrolysis (17). Other phosphoesters, including phosphothreo...

Lung alveolar macrophages (AMs) are in the first line of immune defense against respiratory pathogens and play key roles in the pathogenesis of Mycobacterium tuberculosis (Mtb) in humans. Nevertheless, AMs are available only in limited amounts for in vitro studies, which hamper the detailed molecular understanding of host-Mtb interactions in these macrophages. The recent establishment of the self-renewing and primary Max Planck Institute (MPI) cells, functionally very close to lung AMs, opens unique opportunities for in vitro studies of host-pathogen interactions in respiratory diseases. Here, we investigated the suitability of MPI cells as a host cell system for Mtb infection. Bacterial, cellular, and innate immune features of MPI cells infected with Mtb were characterized. Live bacteria were readily internalized and efficiently replicated in MPI cells, similarly to primary murine macrophages and other cell lines. MPI cells were also suitable for the determination of anti-tuberculosis (TB) drug activity. The primary innate immune response of MPI cells to live Mtb showed significantly higher and earlier induction of the pro-inflammatory cytokines TNFα, interleukin 6 (IL-6), IL-1α, and IL-1β, as compared to stimulation with heat-killed (HK) bacteria. MPI cells previously showed a lack of induction of the anti-inflammatory cytokine IL-10 to a wide range of stimuli, including HK Mtb. By contrast, we show here that live Mtb is able to induce significant amounts of IL-10 in MPI cells. Autophagy experiments using light chain 3B immunostaining, as well as LysoTracker labeling of acidic vacuoles, demonstrated that MPI cells efficiently control killed Mtb by elimination through phagolysosomes. MPI cells were also able to accumulate lipid droplets in their cytoplasm following exposure to lipoproteins. Collectively, this study establishes the MPI cells as a relevant, versatile host cell model for TB research, allowing a deeper understanding of AMs functions in this pathology.

The generation of compact quantum dots (QDs) probes is of critical importance for visualizing molecular interaction occurring in biological context, particularly when using the Förster resonance energy transfer (FRET) approach. This Article reports novel water-soluble compact CdSe/ZnS QDs prepared by ligand exchange reaction using thiolated nitrilotriacetate (NTA). The resulting NTA-QDs revealed higher stability and remarkable conjugation efficiency compared to the other QDs prepared with different ligands by using the ligand exchange method. The Ni-NTA group is a well-known binding moiety for the detection and purification of oligohistidine-tagged recombinant proteins. We demonstrated that NiNTA-QDs prepared by Ni(2+) complexation exhibited highly specific binding ability toward 6-histidine (His)-tagged peptides present in various experimental conditions (buffer solution, agarose beads, and HEK cells). Importantly, the compact NiNTA-QDs serve as an efficient FRET donor. These results suggest that the stable and highly selective multifunctional NTA-QDs can be useful for labeling and tracking molecular interactions within biological context.

The use of Cyan Fluorescent Proteins, with a distinctive lifetime signature, opens up new alternatives to track and semi-quantify the relative expression of proteins in vivo using a single excitation source and emission channel.