The Janus-like role of proline metabolism in cancer.

The metabolism of the non-essential amino acid L-proline is emerging as a key pathway in the metabolic rewiring that sustains cancer cells proliferation, survival and metastatic spread. Pyrroline-5-carboxylate reductase (PYCR) and proline dehydrogenase (PRODH) enzymes, which catalyze the last step in proline biosynthesis and the first step of its catabolism, respectively, have been extensively associated with the progression of several malignancies, and have been exposed as potential targets for anticancer drug development. As investigations into the links between proline metabolism and cancer accumulate, the complexity, and sometimes contradictory nature of this interaction emerge. It is clear that the role of proline metabolism enzymes in cancer depends on tumor type, with different cancers and cancer-related phenotypes displaying different dependencies on these enzymes. Unexpectedly, the outcome of rewiring proline metabolism also differs between conditions of nutrient and oxygen limitation. Here, we provide a comprehensive review of proline metabolism in cancer; we collate the experimental evidence that links proline metabolism with the different aspects of cancer progression and critically discuss the potential mechanisms involved.

Structural and functional characterization of C0021158, a high-affinity monoclonal antibody that inhibits Arginase 2 function via a novel non-competitive mechanism of action.

Arginase 2 (ARG2) is a binuclear manganese metalloenzyme that catalyzes the hydrolysis of L-arginine. The dysregulated expression of ARG2 within specific tumor microenvironments generates an immunosuppressive niche that effectively renders the tumor 'invisible' to the host's immune system. Increased ARG2 expression leads to a concomitant depletion of local L-arginine levels, which in turn leads to suppression of anti-tumor T-cell-mediated immune responses. Here we describe the isolation and characterization of a high affinity antibody (C0021158) that inhibits ARG2 enzymatic function completely, effectively restoring T-cell proliferation in vitro. Enzyme kinetic studies confirmed that C0021158 exhibits a noncompetitive mechanism of action, inhibiting ARG2 independently of L-arginine concentrations. To elucidate C0021158's inhibitory mechanism at a structural level, the co-crystal structure of the Fab in complex with trimeric ARG2 was solved. C0021158's epitope was consequently mapped to an area some distance from the enzyme's substrate binding cleft, indicating an allosteric mechanism was being employed. Following C0021158 binding, distinct regions of ARG2 undergo major conformational changes. Notably, the backbone structure of a surface-exposed loop is completely rearranged, leading to the formation of a new short helix structure at the Fab-ARG2 interface. Moreover, this large-scale structural remodeling at ARG2's epitope translates into more subtle changes within the enzyme's active site. An arginine residue at position 39 is reoriented inwards, sterically impeding the binding of L-arginine. Arg39 is also predicted to alter the pKA of a key catalytic histidine residue at position 160, further attenuating ARG2's enzymatic function. In silico molecular docking simulations predict that L-arginine is unable to bind effectively when antibody is bound, a prediction supported by isothermal calorimetry experiments using an L-arginine mimetic. Specifically, targeting ARG2 in the tumor microenvironment through the application of C0021158, potentially in combination with standard chemotherapy regimens or alternate immunotherapies, represents a potential new strategy to target immune cold tumors.

Extensive sequence and structural evolution of Arginase 2 inhibitory antibodies enabled by an unbiased approach to affinity maturation.

Affinity maturation is a powerful technique in antibody engineering for the in vitro evolution of antigen binding interactions. Key to the success of this process is the expansion of sequence and combinatorial diversity to increase the structural repertoire from which superior binding variants may be selected. However, conventional strategies are often restrictive and only focus on small regions of the antibody at a time. In this study, we used a method that combined antibody chain shuffling and a staggered-extension process to produce unbiased libraries, which recombined beneficial mutations from all six complementarity-determining regions (CDRs) in the affinity maturation of an inhibitory antibody to Arginase 2 (ARG2). We made use of the vast display capacity of ribosome display to accommodate the sequence space required for the diverse library builds. Further diversity was introduced through pool maturation to optimize seven leads of interest simultaneously. This resulted in antibodies with substantial improvements in binding properties and inhibition potency. The extensive sequence changes resulting from this approach were translated into striking structural changes for parent and affinity-matured antibodies bound to ARG2, with a large reorientation of the binding paratope facilitating increases in contact surface and shape complementarity to the antigen. The considerable gains in therapeutic properties seen from extensive sequence and structural evolution of the parent ARG2 inhibitory antibody clearly illustrate the advantages of the unbiased approach developed, which was key to the identification of high-affinity antibodies with the desired inhibitory potency and specificity.

Discovery and Optimization of wt-RET/KDR-Selective Inhibitors of RETV804M Kinase.

A combination of focused library and virtual screening, hit expansion, and rational design has resulted in the development of a series of inhibitors of RETV804M kinase, the anticipated drug-resistant mutant of RET kinase. These agents do not inhibit the wild type (wt) isoforms of RET or KDR and therefore offer a potential adjunct to RET inhibitors currently undergoing clinical evaluation.

Crystal structure of an L chain optimised 14F7 anti-ganglioside Fv suggests a unique tumour-specificity through an unusual H-chain CDR3 architecture.

Targeted cancer immunotherapy offers increased efficacy concomitantly with reduced side effects. One antibody with promising clinical potential is 14F7, which specifically recognises the NeuGc GM3 ganglioside. This antigen is found in the plasma membrane of a range of tumours, but is essentially absent from healthy human cells. 14F7 can discriminate NeuGc GM3 from the very similar NeuAc GM3, a common component of cell membranes. The molecular basis for this unique specificity is poorly understood. Here we designed and expressed 14F7-derived single-chain Fvs (scFvs), which retained the specificity of the parent antibody. Detailed expression and purification protocols are described as well as the synthesis of the NeuGc GM3 trisaccharide. The most successful scFv construct, which comprises an alternative variable light chain (VLA), allowed structure determination to 2.2 Å resolution. The structure gives insights into the conformation of the important CDR H3 loop and the suspected antigen binding site. Furthermore, the presence of VLA instead of the original VL elucidates how this subdomain indirectly stabilises the CDR H3 loop. The current work may serve as a guideline for the efficient production of scFvs for structure determination.

Inter-Enzyme Allosteric Regulation of Chorismate Mutase in Corynebacterium glutamicum: Structural Basis of Feedback Activation by Trp.

Corynebacterium glutamicum is widely used for the industrial production of amino acids, nucleotides, and vitamins. The shikimate pathway enzymes DAHP synthase (CgDS, Cg2391) and chorismate mutase (CgCM, Cgl0853) play a key role in the biosynthesis of aromatic compounds. Here we show that CgCM requires the formation of a complex with CgDS to achieve full activity, and that both CgCM and CgDS are feedback regulated by aromatic amino acids binding to CgDS. Kinetic analysis showed that Phe and Tyr inhibit CgCM activity by inter-enzyme allostery, whereas binding of Trp to CgDS strongly activates CgCM. Mechanistic insights were gained from crystal structures of the CgCM homodimer, tetrameric CgDS, and the heterooctameric CgCM-CgDS complex, refined to 1.1, 2.5, and 2.2 Å resolution, respectively. Structural details from the allosteric binding sites reveal that DAHP synthase is recruited as the dominant regulatory platform to control the shikimate pathway, similar to the corresponding enzyme complex from Mycobacterium tuberculosis.

Quantum chemical modeling of the reaction path of chorismate mutase based on the experimental substrate/product complex.

Chorismate mutase is a well-known model enzyme, catalyzing the Claisen rearrangement of chorismate to prephenate. Recent high-resolution crystal structures along the reaction coordinate of this enzyme enabled computational analyses at unprecedented detail. Using quantum chemical simulations, we investigated how the catalytic reaction mechanism is affected by electrostatic and hydrogen-bond interactions. Our calculations showed that the transition state (TS) was mainly stabilized electrostatically, with Arg90 playing the leading role. The effect was augmented by selective hydrogen-bond formation to the TS in the wild-type enzyme, facilitated by a small-scale local induced fit. We further identified a previously underappreciated water molecule, which separates the negative charges during the reaction. The analysis includes the wild-type enzyme and a non-natural enzyme variant, where the catalytic arginine was replaced with an isosteric citrulline residue.

1H, 13C, 15N backbone assignment of the human heat-labile enterotoxin B-pentamer and chemical shift mapping of neolactotetraose binding.

The major virulence factor of enterotoxigenic Escherichia coli is the heat-labile enterotoxin (LT), an AB5 toxin closely related to the cholera toxin. LT consists of six subunits, the catalytically active A-subunit and five B-subunits arranged as a pentameric ring (LTB), which enable the toxin to bind to the epithelial cells in the intestinal lumen. LTB has two recognized binding sites; the primary binding site is responsible for anchoring the toxin to its main receptor, the GM1-ganglioside, while the secondary binding site recognizes blood group antigens. Herein, we report the 1H, 13C, 15N main chain assignment of LTB from human isolates (hLTB; 103 a.a. per subunit, with a total molecular mass of 58.5 kDa). The secondary structure was predicted based on 13C', 13Cα, 13Cß, 1HN and 15N chemical shifts and compared to a published crystal structure of LTB. Neolactotetraose (NEO) was titrated to hLTB and chemical shift perturbations were measured. The chemical shift perturbations were mapped onto the crystal structure, confirming that NEO binds to the primary binding site of hLTB and competes with GM1-binding. Our new data further lend support to the hypothesis that binding at the primary binding site is transmitted to the secondary binding site of the toxin, where it may influence the binding to blood group antigens.

High-Resolution Crystal Structures Elucidate the Molecular Basis of Cholera Blood Group Dependence.

Cholera is the prime example of blood-group-dependent diseases, with individuals of blood group O experiencing the most severe symptoms. The cholera toxin is the main suspect to cause this relationship. We report the high-resolution crystal structures (1.1-1.6 Å) of the native cholera toxin B-pentamer for both classical and El Tor biotypes, in complexes with relevant blood group determinants and a fragment of its primary receptor, the GM1 ganglioside. The blood group A determinant binds in the opposite orientation compared to previously published structures of the cholera toxin, whereas the blood group H determinant, characteristic of blood group O, binds in both orientations. H-determinants bind with higher affinity than A-determinants, as shown by surface plasmon resonance. Together, these findings suggest why blood group O is a risk factor for severe cholera.

Electrostatic transition state stabilization rather than reactant destabilization provides the chemical basis for efficient chorismate mutase catalysis.

For more than half a century, transition state theory has provided a useful framework for understanding the origins of enzyme catalysis. As proposed by Pauling, enzymes accelerate chemical reactions by binding transition states tighter than substrates, thereby lowering the activation energy compared with that of the corresponding uncatalyzed process. This paradigm has been challenged for chorismate mutase (CM), a well-characterized metabolic enzyme that catalyzes the rearrangement of chorismate to prephenate. Calculations have predicted the decisive factor in CM catalysis to be ground state destabilization rather than transition state stabilization. Using X-ray crystallography, we show, in contrast, that a sluggish variant of Bacillus subtilis CM, in which a cationic active-site arginine was replaced by a neutral citrulline, is a poor catalyst even though it effectively preorganizes chorismate for the reaction. A series of high-resolution molecular snapshots of the reaction coordinate, including the apo enzyme, and complexes with substrate, transition state analog and product, demonstrate that an active site, which is only complementary in shape to a reactive substrate conformer, is insufficient for effective catalysis. Instead, as with other enzymes, electrostatic stabilization of the CM transition state appears to be crucial for achieving high reaction rates.

Surface assembly of poly(I:C) on PEGylated microspheres to shield from adverse interactions with fibroblasts.

By expressing an array of pattern recognition receptors (PRRs), fibroblasts play an important role in stimulating and modulating the response of the innate immune system. The TLR3 ligand polyriboinosinic acid-polyribocytidylic acid, poly(I:C), a mimic of viral dsRNA, is a vaccine adjuvant candidate to activate professional antigen presenting cells (APCs). However, owing to its ligation with extracellular TLR3 on fibroblasts, subcutaneously administered poly(I:C) bears danger towards autoimmunity. It is thus in the interest of its clinical safety to deliver poly(I:C) in such a way that its activation of professional APCs is as efficacious as possible, whereas its interference with non-immune cells such as fibroblasts is controlled or even avoided. Complementary to our previous work with monocyte-derived dendritic cells (MoDCs), here we sought to control the delivery of poly(I:C) surface-assembled on microspheres to human foreskin fibroblasts (HFFs). Negatively charged polystyrene (PS) microspheres were equipped with a poly(ethylene glycol) (PEG) corona through electrostatically driven coatings with a series of polycationic poly(L-lysine)-graft-poly(ethylene glycol) copolymers, PLL-g-PEG, of varying grafting ratios g from 2.2 up to 22.7. Stable surface assembly of poly(I:C) was achieved by incubation of polymer-coated microspheres with aqueous poly(I:C) solutions. Notably, recognition of both surface-assembled and free poly(I:C) by extracellular TLR3 on HFFs halted their phagocytic activity. Ligation of surface-assembled poly(I:C) with extracellular TLR3 on HFFs could be controlled by tuning the grafting ratio g and thus the chain density of the PEG corona. When assembled on PLL-5.7-PEG-coated microspheres, poly(I:C) was blocked from triggering class I MHC molecule expression on HFFs. Secretion of interleukin (IL)-6 by HFFs after exposure to surface-assembled poly(I:C) was distinctly lower as compared to free poly(I:C). Overall, surface assembly of poly(I:C) may have potential to contribute to the clinical safety of this vaccine adjuvant candidate.

Structural analysis of the conserved ubiquitin-binding motifs (UBMs) of the translesion polymerase iota in complex with ubiquitin.

Ubiquitin-binding domains (UBDs) provide specificity to the ubiquitin system, which is also involved in translesion synthesis (TLS) in eukaryotic cells. Upon DNA damage, the UBDs (UBM domains) of polymerase iota (Pol ι) interact with ubiquitinated proliferating cell nuclear antigen to regulate the interchange between processive DNA polymerases and TLS. We report a biophysical analysis and solution structures of the two conserved UBM domains located in the C-terminal tail of murine Pol ι in complex with ubiquitin. The 35-amino acid core folds into a helix-turn-helix motif, which belongs to a novel domain fold. Similar to other UBDs, UBMs bind to ubiquitin on the hydrophobic surface delineated by Leu-8, Ile-44, and Val-70, however, slightly shifted toward the C terminus. In addition, UBMs also use electrostatic interactions to stabilize binding. NMR and fluorescence spectroscopy measurements revealed that UBMs bind monoubiquitin, and Lys-63- but not Lys-48-linked chains. Importantly, these biophysical data are supported by functional studies. Indeed, yeast cells expressing ubiquitin mutants specifically defective for UBM binding are viable but sensitive to DNA damaging conditions that require TLS for repair.