Safety and efficacy of an anti-human APC antibody for prophylaxis of congenital factor deficiencies in preclinical models.

Rebalance of coagulation and anticoagulation to achieve a hemostatic effect has recently gained attention as an alternative therapeutic strategy for hemophilia. We engineered a humanized chimeric antibody, SR604, based on a previously published murine antibody, HAPC1573, which selectively blocks the anticoagulant activity of human activated protein C (APC). SR604 effectively blocked the anticoagulation activities of APC in human plasma deficient in various coagulation factors in vitro with affinities ∼60 times greater than that of HAPC1573. SR604 exhibited prophylactic and therapeutic efficacy in the tail-bleeding and knee-injury models of hemophilia A and B mice expressing human APC (humanized hemophilic mice). SR604 did not interfere with the cytoprotection and endothelial barrier function of APC, nor were there obvious toxicity effects in humanized hemophilic mice. Pharmacokinetic study showed a high bioavailability (106%) of subcutaneously injected SR604 in cynomolgus monkeys. These results demonstrate that SR604 is expected to be a safe and effective therapeutic and/or prophylactic agent with a prolonged half-life for patients with congenital factor deficiencies including hemophilia A and B.

Developing high-affinity decoy receptors to treat multiple myeloma and diffuse large B cell lymphoma.

Disease relapse and treatment-induced immunotoxicity pose significant clinical challenges for patients with hematological cancers. Here, we reveal distinctive requirements for neutralizing TNF receptor ligands APRIL and BAFF and their receptor activity in MM and DLBCL, impacting protein translation and production in MM cells and modulating the translation efficiency of the ATM interactor (ATMIN/ACSIZ). Therapeutically, we investigated the use of BCMA decoy receptor (sBCMA-Fc) as an inhibitor of APRIL and BAFF. While wild-type sBCMA-Fc effectively blocked APRIL signaling in MM, it lacked activity in DLBCL due to its weak BAFF binding. To expand the therapeutic utility of sBCMA-Fc, we engineered an affinity-enhanced mutant sBCMA-Fc fusion molecule (sBCMA-Fc V3) 4- and 500-fold stronger in binding to APRIL and BAFF, respectively. The mutant sBCMA-Fc V3 clone significantly enhanced antitumor activity against both MM and DLBCL. Importantly, we also demonstrated an adequate toxicity profile and on-target mechanism of action in nonhuman primate studies.

Structural insights into catalytic mechanism and product delivery of cyanobacterial acyl-acyl carrier protein reductase.

Long-chain alk(a/e)nes represent the major constituents of conventional transportation fuels. Biosynthesis of alkanes is ubiquitous in many kinds of organisms. Cyanobacteria possess two enzymes, acyl-acyl carrier protein (acyl-ACP) reductase (AAR) and aldehyde-deformylating oxygenase (ADO), which function in a two-step alkane biosynthesis pathway. These two enzymes act in series and possibly form a complex that efficiently converts long chain fatty acyl-ACP/fatty acyl-CoA into hydrocarbon. While the structure of ADO has been previously described, structures of both AAR and AAR-ADO complex have not been solved, preventing deeper understanding of this pathway. Here, we report a ligand-free AAR structure, and three AAR-ADO complex structures in which AARs bind various ligands. Our results reveal the binding pattern of AAR with its substrate/cofactor, and suggest a potential aldehyde-transferring channel from AAR to ADO. Based on our structural and biochemical data, we proposed a model for the complete catalytic cycle of AAR.

Identification of residues important for the activity of aldehyde-deformylating oxygenase through investigation into the structure-activity relationship.

BACKGROUND: Aldehyde-deformylating oxygenase (ADO) is a key enzyme involved in the biosynthetic pathway of fatty alk(a/e)nes in cyanobacteria. However, cADO (cyanobacterial ADO) showed extreme low activity with the k cat value below 1 min-1, which would limit its application in biofuel production. To identify the activity related key residues of cADO is urgently required. RESULTS: The amino acid residues which might affect cADO activity were identified based on the crystal structures and sequence alignment of cADOs, including the residues close to the di-iron center (Tyr39, Arg62, Gln110, Tyr122, Asp143 of cADO-1593), the protein surface (Trp 178 of cADO-1593), and those involved in two important hydrogen bonds (Gln49, Asn123 of cADO-1593, and Asp49, Asn123 of cADO-sll0208) and in the oligopeptide whose conformation changed in the absence of the di-iron center (Leu146, Asn149, Phe150 of cADO-1593, and Thr146, Leu148, Tyr150 of cADO-sll0208). The variants of cADO-1593 from Synechococcus elongatus PCC7942 and cADO-sll0208 from Synechocystis sp. PCC6803 were constructed, overexpressed, purified and kinetically characterized. The k cat values of L146T, Q49H/N123H/F150Y and W178R of cADO-1593 and L148R of cADO-sll0208 were increased by more than two-fold, whereas that of R62A dropped by 91.1%. N123H, Y39F and D143A of cADO-1593, and Y150F of cADO-sll0208 reduced activities by ≤ 20%. CONCLUSIONS: Some important amino acids, which exerted some effects on cADO activity, were identified. Several enzyme variants exhibited greatly reduced activity, while the k cat values of several mutants are more than two-fold higher than the wild type. This study presents the report on the relationship between amino acid residues and enzyme activity of cADOs, and the information will provide a guide for enhancement of cADO activity through protein engineering.

Structure-oriented substrate specificity engineering of aldehyde-deformylating oxygenase towards aldehydes carbon chain length.

BACKGROUND: Aldehyde-deformylating oxygenase (ADO) is an important enzyme involved in the biosynthetic pathway of fatty alk(a/e)nes in cyanobacteria. However, ADO exhibits quite low chain-length specificity with respect to the substrates ranging from C4 to C18 aldehydes, which is not suitable for producing fuels with different properties or different chain lengths. RESULTS: Based on the crystal structures of cADOs (cyanobacterial ADO) with substrate analogs bound, some amino acids affecting the substrate specificity of cADO were identified, including the amino acids close to the aldehyde group and the hydrophobic tail of the substrate and those along the substrate channel. Using site-directed mutagenesis, selected amino acids were replaced with bulky ones introducing steric hindrance to the binding pocket via large functional groups. All mutants were overexpressed, purified and kinetically characterized. All mutants, except F87Y, displayed dramatically reduced activity towards C14,16,18 aldehydes. Notably, the substrate preferences of some mutants towards different chain-length substrates were enhanced: I24Y for n-heptanal, I27F for n-decanal and n-dodecanal, V28F for n-dodecanal, F87Y for n-decanal, C70F for n-hexanal, A118F for n-butanal, A121F for C4,6,7 aldehydes, V184F for n-dodecanal and n-decanal, M193Y for C6-10 aldehydes and L198F for C7-10 aldehydes. The impact of the engineered cADO mutants on the change of the hydrocarbon profile was demonstrated by co-expressing acyl-ACP thioesterase BTE, fadD and V184F in E. coli, showing that n-undecane was the main fatty alkane. CONCLUSIONS: Some amino acids, which can control the chain-length selectivity of substrates of cADO, were identified. The substrate specificities of cADO were successfully changed through structure-guided protein engineering, and some mutants displayed different chain-length preference. The in vivo experiments of V184F in genetically engineered E. coli proved the importance of engineered cADOs on the distribution of the fatty alkane profile. The results would be helpful for the production of fatty alk(a/e)nes in cyanobacteria with different properties.

Structural insights into the catalytic mechanism of aldehyde-deformylating oxygenases.

The fatty alk(a/e)ne biosynthesis pathway found in cyanobacteria gained tremendous attention in recent years as a promising alternative approach for biofuel production. Cyanobacterial aldehyde-deformylating oxygenase (cADO), which catalyzes the conversion of Cn fatty aldehyde to its corresponding Cn-1 alk(a/e)ne, is a key enzyme in that pathway. Due to its low activity, alk(a/e)ne production by cADO is an inefficient process. Previous biochemical and structural investigations of cADO have provided some information on its catalytic reaction. However, the details of its catalytic processes remain unclear. Here we report five crystal structures of cADO from the Synechococcus elongates strain PCC7942 in both its iron-free and iron-bound forms, representing different states during its catalytic process. Structural comparisons and functional enzyme assays indicate that Glu144, one of the iron-coordinating residues, plays a vital role in the catalytic reaction of cADO. Moreover, the helix where Glu144 resides exhibits two distinct conformations that correlates with the different binding states of the di-iron center in cADO structures. Therefore, our results provide a structural explanation for the highly labile feature of cADO di-iron center, which we proposed to be related to its low enzymatic activity. On the basis of our structural and biochemical data, a possible catalytic process of cADO was proposed, which could aid the design of cADO with improved activity.

Structural insights into energy regulation of light-harvesting complex CP29 from spinach.

CP29, one of the minor light-harvesting complexes of higher-plant photosystem II, absorbs and transfers solar energy for photosynthesis and also has important roles in photoprotection. We have solved the crystal structure of spinach CP29 at 2.80-Å resolution. Each CP29 monomer contains 13 chlorophyll and 3 carotenoid molecules, which differs considerably from the major light-harvesting complex LHCII and the previously proposed CP29 model. The 13 chlorophyll-binding sites are assigned as eight chlorophyll a sites, four chlorophyll b and one putative mixed site occupied by both chlorophylls a and b. Based on the present X-ray structure, an integrated pigment network in CP29 is constructed. Two special clusters of pigment molecules, namely a615-a611-a612-Lut and Vio(Zea)-a603-a609, have been identified and might function as potential energy-quenching centers and as the exit or entrance in energy-transfer pathways.