Polyamines are organic polycations essential for cell growth and differentiation; their aberrant accumulation is often associated with diseases, including many types of cancer. To maintain polyamine homeostasis, the catalytic activity and protein abundance of ornithine decarboxylase (ODC), the committed enzyme for polyamine biosynthesis, are reciprocally controlled by the regulatory proteins antizyme isoform 1 (Az₁) and antizyme inhibitor (AzIN). Az₁ suppresses polyamine production by inhibiting the assembly of the functional ODC homodimer and, most uniquely, by targeting ODC for ubiquitin-independent proteolytic destruction by the 26S proteasome. In contrast, AzIN positively regulates polyamine levels by competing with ODC for Az₁ binding. The structural basis of the Az₁-mediated regulation of polyamine homeostasis has remained elusive. Here we report crystal structures of human Az₁ complexed with either ODC or AzIN. Structural analysis revealed that Az₁ sterically blocks ODC homodimerization. Moreover, Az₁ binding triggers ODC degradation by inducing the exposure of a cryptic proteasome-interacting surface of ODC, which illustrates how a substrate protein may be primed upon association with Az₁ for ubiquitin-independent proteasome recognition. Dynamic and functional analyses further indicated that the Az₁-induced binding and degradation of ODC by proteasome can be decoupled, with the intrinsically disordered C-terminal tail fragment of ODC being required only for degradation but not binding. Finally, the AzIN–Az₁ structure suggests how AzIN may effectively compete with ODC for Az₁ to restore polyamine production. Taken together, our findings offer structural insights into the Az-mediated regulation of polyamine homeostasis and proteasomal degradation.

Malic enzymes are widely distributed in nature, and have important biological functions. They catalyze the oxidative decarboxylation of malate to produce pyruvate and CO₂ in the presence of divalent cations (Mg²⁺, Mn²⁺). Most malic enzymes have a clear selectivity for the dinucleotide cofactor, being able to use either NAD⁺ or NADP⁺, but not both. Structural studies of the human mitochondrial NAD⁺‐dependent malic enzyme established that malic enzymes belong to a new class of oxidative decarboxylases. Here we report the crystal structure of the pigeon cytosolic NADP⁺‐dependent malic enzyme, in a closed form, in a quaternary complex with NADP⁺, Mn²⁺, and oxalate. This represents the first structural information on an NADP⁺‐dependent malic enzyme. Despite the sequence conservation, there are large differences in several regions of the pigeon enzyme structure compared to the human enzyme. One region of such differences is at the binding site for the 2′‐phosphate group of the NADP⁺ cofactor, which helps define the cofactor selectivity of the enzymes. Specifically, the structural information suggests Lys362 may have an important role in the NADP⁺ selectivity of the pigeon enzyme, confirming our earlier kinetic observations on the K362A mutant. Our structural studies also revealed differences in the organization of the tetramer between the pigeon and the human enzymes, although the pigeon enzyme still obeys 222 symmetry.

A unique feature of rheumatoid arthritis (RA) is the presence of anti-citrullinated protein antibodies (ACPA). Several risk factors for RA are known to increase the expression or activity of peptidyl arginine deiminases (PADs), which catalyze citrullination and, when dysregulated, can result in hypercitrullination. However, the consequence of hypercitrullination is unknown and the function of each PAD has yet to be defined. Th cells of RA patients are hypoglycolytic and hyperproliferative due to impaired expression of PFKFB3 and ATM, respectively. Here, we report that these features are also observed in peripheral blood mononuclear cells (PBMCs) from healthy at-risk individuals (ARIs). PBMCs of ARIs are also hypercitrullinated and produce more IL-2 and Th17 cytokines but fewer Th2 cytokines. These abnormal features are due to impaired induction of PTPN22, a phosphatase that also suppresses citrullination independently of its phosphatase activity. Attenuated phosphatase activity of PTPN22 results in aberrant expression of IL-2, ATM, and PFKFB3, whereas diminished nonphosphatase activity of PTPN22 leads to hypercitrullination mediated by PADs. PAD2- or PAD4-mediated hypercitrullination reduces the expression of Th2 cytokines. By contrast, only PAD2-mediated hypercitrullination can increase the expression of Th17 cytokines. Taken together, our data depict a molecular signature of preclinical RA that is triggered by impaired induction of PTPN22.

Peptidylarginine deiminase 4 (PAD4) is a homodimeric enzyme that catalyzes Ca²⁺-dependent protein citrullination, which results in the conversion of arginine to citrulline. This paper demonstrates the functional role of dimerization in the regulation of PAD4 activity. To address this question, we created a series of dimer interface mutants of PAD4. The residues Arg8, Tyr237, Asp273, Glu281, Tyr435, Arg544 and Asp547, which are located at the dimer interface, were mutated to disturb the dimer organization of PAD4. Sedimentation velocity experiments were performed to investigate the changes in the quaternary structures and the dissociation constants (K_d) between wild-type and mutant PAD4 monomers and dimers. The kinetic data indicated that disrupting the dimer interface of the enzyme decreases its enzymatic activity and calcium-binding cooperativity. The K_d values of some PAD4 mutants were much higher than that of the wild-type (WT) protein (0.45 µM) and were concomitant with lower k_cat values than that of WT (13.4 s⁻¹). The K_d values of the monomeric PAD4 mutants ranged from 16.8 to 45.6 µM, and the k_cat values of the monomeric mutants ranged from 3.3 to 7.3 s⁻¹. The k_cat values of these interface mutants decreased as the K_d values increased, which suggests that the dissociation of dimers to monomers considerably influences the activity of the enzyme. Although dissociation of the enzyme reduces the activity of the enzyme, monomeric PAD4 is still active but does not display cooperative calcium binding. The ionic interaction between Arg8 and Asp547 and the Tyr435-mediated hydrophobic interaction are determinants of PAD4 dimer formation.