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Since their discovery, the matrix metalloproteinase (MMP) family proteases have been considered as therapeutic targets in numerous diseases and disorders. Unfortunately, clinical trials with MMP inhibitors have failed to yield any clinical benefits of these inhibitors. These failures were largely due to a lack of MMP-selective agents; accordingly, it has become important to identify a platform with which high selectivity can be achieved. To this end, we propose using MMP-targeting antibodies that can achieve high specificity in interactions with their targets. Using a scaffold of single-domain antibodies, here we raised a panel of MMP10-selective antibodies through immunization of llamas, a member of the camelid family, whose members generate conventional heavy/light-chain antibodies and also smaller antibodies lacking light-chain and CH1 domains. We report the generation of a highly selective and tightly binding MMP10 inhibitor (K(i) < 2 nm). Using bio-layer interferometry–based binding assays, we found that this antibody interacts with the MMP10 active site. Activity assays demonstrated that the antibody selectively inhibits MMP10 over its closest relative, MMP3. The ability of a single-domain antibody to discriminate between the most conserved MMP pair via an active site–directed mechanism of inhibition reported here supports the potential of this antibody as a broadly applicable scaffold for the development of selective, tightly binding MMP inhibitors.

X-linked inhibitor of apoptosis (XIAP) is a potent antagonist of caspase apoptotic activity. XIAP also functions as an E3 ubiquitin ligase, targeting caspases for degradation. However, molecular pathways controlling XIAP activities remain unclear. Here we report that nitric oxide (NO) reacts with XIAP by S-nitrosylating its RING domain (forming SNO-XIAP), thereby inhibiting E3 ligase and antiapoptotic activity. NO-mediated neurotoxicity and caspase activation have been linked to several neurodegenerative disorders, including Alzheimer’s, Parkinson’s, and Huntington’s diseases. We find significant SNO-XIAP formation in brains of patients with these diseases, implicating this reaction in the etiology of neuronal damage. Conversely, S-nitrosylation of caspases is known to inhibit apoptotic activity. Unexpectedly, we find that SNO-caspase transnitrosylates (transfers its NO group) to XIAP, forming SNO-XIAP, and thus promotes cell injury and death. These findings provide unique insights into the regulation of caspase activation in neurodegenerative disorders mediated, at least in part, by nitrosative stress.

We describe the peptide-binding specificity of the baculoviral IAP repeat (BIR) domains of the human inhibitor of apoptosis (IAP) proteins, X-linked IAP, cellular IAP1 and neuronal apoptosis inhibitory protein (NAIP). Synthetic peptide libraries were used to profile each domain, and we distinguish two types of binding specificity, which we refer to as type II and type III BIR domains. Both types have a dominant selectivity for Ala in the first position of the four N-terminal residues of the peptide ligands, which constitute a core recognition motif. Our analysis allows us to define the signature of type III BIRs that demonstrate a preference for Pro in the third residue of the ligand, resembling the classic IAP-binding motif (IBM). The signature of the type II BIRs was similar to type III, but with a striking absence of specificity for Pro in the third position, suggesting that the definition of an IBM must be modified depending on the type of BIR in question. These findings explain how subtle changes in the peptide-binding groove of IAP BIR domains can significantly alter the target protein selectivity. Our analysis allows for prediction of BIR domain protein-binding preferences, provides a context for understanding the mechanism of peptide selection and heightens our knowledge of the specificity of IAP antagonists that are being developed as cancer therapeutics.

Several of the inhibitor of apoptosis protein (IAP) family members regulate apoptosis in response to various cellular assaults. Some members are also involved in cell signalling, mitosis and targeting proteins to the ubiquitin-proteasome degradation machinery. The most intensively studied family member, X-linked IAP (XIAP), is a potent inhibitor of caspase activity; hence, it is generally assumed that direct caspase inhibition is an important conserved function of most members of the family. Biochemical and structural studies have precisely mapped the elements of XIAP required for caspase inhibition. Intriguingly, these elements are not conserved among IAPs. Here, we review current knowledge of the caspase-inhibitory potential of the human IAPs and show that XIAP is probably the only bona fide caspase inhibitor, suggesting that the other family members never gained the ability to directly inhibit caspase activity.

cIAPs (cellular inhibitor of apoptosis proteins) 1 and 2 are able to regulate apoptosis when ectopically expressed in recipient cells and probably also in vivo. Previous work suggested that this is at least partially due to direct caspase inhibition, mediated by two of the three baculovirus IAP repeat (BIR) domains that are contained in these proteins. In support of this we show that the BIR domains 2 and 3 of the two cIAPs are able to bind caspases-7 and -9. However, we demonstrate that neither of these BIR domains is able to inhibit caspases because of critical substitutions in the regions that target caspase inhibition in the X-linked IAP, a tight binding caspase inhibitor. The cIAP BIR domains can be converted to tight binding caspase inhibitors by substituting these critical residues with XIAP residues. Thus, cIAPs maintain protein scaffolds suitable for direct caspase inhibition but have lost or never acquired specific caspase inhibitory interaction sites. Consequently, although the binding function of the cIAP BIRs may be important for their physiologic function, caspase inhibition is not.

B-cell lymphoma is one of the most frequently observed non-cutaneous neoplasms in dogs. For both human and canine BCL, the standard of care treatment typically involves a combination chemotherapy, e.g. "CHOP" therapy. Treatment for human lymphoma greatly benefited from the addition of anti-CD20 targeted biological therapeutics to these chemotherapy protocols; this type of therapeutic has not been available to the veterinary oncologist. Here, we describe the generation and characterization of a rituximab-like anti-CD20 antibody intended as a candidate treatment for canine B-cell lymphoma. A panel of anti-canine CD20 monoclonal antibodies was generated using a mouse hybridoma approach. Mouse monoclonal antibody 1E4 was selected for construction of a canine chimeric molecule based on its rank ordering in a flow cytometry-based affinity assay. 1E4 binds to approximately the same location in the extracellular domain of CD20 as rituximab, and 1E4-based chimeric antibodies co-stain canine B cells in flow cytometric analysis of canine leukocytes using an anti-canine CD21 antibody. We show that two of the four reported canine IgG subclasses (cIgGB and cIgGC) can bind to canine CD16a, a receptor involved in antibody-dependent cellular cytotoxicity (ADCC). Chimeric monoclonal antibodies were assembled using canine heavy chain constant regions that incorporated the appropriate effector function along with the mouse monoclonal 1E4 anti-canine CD20 variable regions, and expressed in CHO cells. We observed that 1E4-cIgGB and 1E4-cIgGC significantly deplete B-cell levels in healthy beagle dogs. The in vivo half-life of 1E4-cIgGB in a healthy dog was ∼14 days. The antibody 1E4-cIgGB has been selected for further testing and development as an agent for the treatment of canine B-cell lymphoma.

ML-IAP (melanoma inhibitor of apoptosis) is a potent anti-apoptotic protein that is strongly up-regulated in melanoma and confers protection against a variety of pro-apoptotic stimuli. The mechanism by which ML-IAP regulates apoptosis is unclear, although weak inhibition of caspases 3 and 9 has been reported. Here, the binding to and inhibition of caspase 9 by the single BIR (baculovirus IAP repeat) domain of ML-IAP has been investigated and found to be significantly less potent than the ubiquitously expressed XIAP (X-linked IAP). Engineering of the ML-IAP-BIR domain, based on comparisons with the third BIR domain of XIAP, resulted in a chimeric BIR domain that binds to and inhibits caspase 9 significantly better than either ML-IAP-BIR or XIAP-BIR3. Mutational analysis of the ML-IAP-BIR domain demonstrated that similar enhancements in caspase 9 affinity can be achieved with only three amino acid substitutions. However, none of these modifications affected binding of the ML-IAP-BIR domain to the IAP antagonist Smac (second mitochondrial activator of caspases). ML-IAP-BIR was found to bind mature Smac with low nanomolar affinity, similar to that of XIAP-BIR2-BIR3. Correspondingly, increased expression of ML-IAP results in formation of a ML-IAP–Smac complex and disruption of the endogenous interaction between XIAP and mature Smac. These results suggest that ML-IAP might regulate apoptosis by sequestering Smac and preventing it from antagonizing XIAP-mediated inhibition of caspases, rather than by direct inhibition of caspases.

Dogma has it that suppression of the programmed cell death pathway by the IAP (inhibitor of apoptosis) proteins is achieved by their direct enzymic inhibition of the chief executioners of the apoptotic process, the caspases. In turn, the IAPs themselves can be neutralized by Smac/DIABLO (second mitochondrial activator of caspases/direct IAP binding protein with low pI), a protein which in healthy cells is thought to be sequestered in the mitochondria, but which, in response to apoptotic stimuli, is released from the mitochondria into the cytosol where it can bind to IAPs, displacing caspases and thus perpetuating the apoptotic signal. While this is an elegant and attractive model, recent studies have suggested that IAPs can also suppress apoptotic cell death independently of their ability to inhibit caspases, and two reports in this issue of the Biochemical Journal reach the interesting conclusion that the cytoprotective IAPs, ML-IAP (melanoma IAP) and ILP-2 (IAP-like protein 2), exert their effects not through direct caspase inhibition, but through the neutralization of Smac/DIABLO. The predicted outcome of these studies is a delicately controlled equilibrium between the activities of IAPs and Smac/DIABLO, leading to a dynamic regulation of the apoptotic threshold.

Sodium (Na+) is toxic to most plants, but the molecular mechanisms of plant Na+ uptake and distribution remain largely unknown. Here we analyze Arabidopsis lines disrupted in the Na+ transporter AtHKT1. AtHKT1 is expressed in the root stele and leaf vasculature. athkt1 null plants exhibit lower root Na+ levels and are more salt resistant than wild-type in short-term root growth assays. In shoot tissues, however, athkt1 disruption produces higher Na+ levels, and athkt1 and athkt1/sos3 shoots are Na+-hypersensitive in long-term growth assays. Thus wild-type AtHKT1 controls root/shoot Na+ distribution and counteracts salt stress in leaves by reducing leaf Na+ accumulation.