Pex5p is the receptor for the peroxisomal targeting signal 1 (PTS1) that consists of a C-terminal tripeptide (consensus (S/A/C)(K/R/H)(L/M)). Hexadecapeptides recognized by Pex5p from Homo sapiens and Saccharomyces cerevisiae were identified by screening a two-hybrid peptide library, and the targeting ability of the peptides was demonstrated using the green fluorescent protein as reporter. The PTS1 receptors recognized in a speciesspecific manner a broad range of C-terminal tripeptides, and these are reported herein. In addition, residues upstream of the tripeptide influenced the strength of the interaction in the two-hybrid system as well as in an in vitro competition assay. In peptides interacting with the human protein, hydrophobic residues were found with high frequency especially at positions ؊2 and ؊5, whereas peptides interacting with S. cerevisiae Pex5p were more hydrophilic and frequently contained arginine at position ؊2. In instances where the terminal tripeptide deviated from the consensus, upstream residues exerted a greater influence on the ability of the hexadecapeptides to bind Pex5p.

Genetic regulators and environmental stimuli modulate T-cell activation in autoimmunity and cancer. The enzyme co-factor tetrahydrobiopterin (BH4) is involved in the production of monoamine neurotransmitters, the generation of nitric oxide, and pain1,2. Here we uncover a link between these processes, identifying a fundamental role for BH4 in T-cell biology. We find that genetic inactivation of GTP cyclohydrolase 1 (GCH1, the rate-limiting enzyme in the synthesis of BH4) and inhibition of sepiapterin reductase (SPR, the terminal enzyme in its synthetic pathway) severely impair the proliferation of mature mouse and human T cells. BH4 production in activated T cells is linked to alterations in iron metabolism and mitochondrial bioenergetics. In vivo blockade of BH4 synthesis abrogates T-cell-mediated autoimmunity and allergic inflammation, while enhancing BH4 levels through GCH1 overexpression augments responses by CD4- and CD8-expressing T cells, increasing their antitumour activity in vivo. Administration of BH4 to mice markedly reduces tumour growth and expands the population of intratumoral effector T cells. Kynurenine—a tryptophan metabolite that blocks antitumour immunity—inhibits T-cell proliferation in a manner that can be rescued by BH4. Finally, we report the development of a potent SPR antagonist for possible clinical use. Our data uncover GCH1, SPR and their downstream metabolite BH4 as critical regulators of T-cell biology that can be readily manipulated to either block autoimmunity or enhance anticancer immunity.

To identify members of the translocation machinery for peroxisomal proteins, we made use of the two‐hybrid system to establish a protein linkage map centered around Pex5p from Saccharomyces cerevisiae, the receptor for the C‐terminal peroxisomal targeting signal (PTS1). Among the five interaction partners identified, Pex14p was found to be induced under conditions allowing peroxisome proliferation. Deletion of the corresponding gene resulted in the inability of yeast cells to grow on oleate as well as the absence of peroxisomal structures. The PEX14 gene product of ∼38 kDa was biochemically and ultrastructurally demonstrated to be a peroxisomal membrane protein, despite the lack of a membrane‐spanning domain. This protein was shown to interact with itself, with Pex13p and with both PTS receptors, Pex5p and Pex7p, indicating a central function for the import of peroxisomal matrix proteins, either as a docking protein or as a releasing factor at the organellar membrane.

No abstract