Parkin is a RING-between-RING E3 ligase that functions in the covalent attachment of ubiquitin to specific substrates, and mutations in Parkin are linked to Parkinson’s disease, cancer and mycobacterial infection. The RING-between-RING family of E3 ligases are suggested to function with a canonical RING domain and a catalytic cysteine residue usually restricted to HECT E3 ligases, thus termed ‘RING/HECT hybrid’ enzymes. Here we present the 1.58 Å structure of Parkin-R0RBR, revealing the fold architecture for the four RING domains, and several unpredicted interfaces. Examination of the Parkin active site suggests a catalytic network consisting of C431 and H433. In cells, mutation of C431 eliminates Parkin-catalysed degradation of mitochondria, and capture of an ubiquitin oxyester confirms C431 as Parkin’s cellular active site. Our data confirm that Parkin is a RING/HECT hybrid, and provide the first crystal structure of an RING-between-RING E3 ligase at atomic resolution, providing insight into this disease-related protein.

Monoclonal antibodies play an increasingly important role for the development of new drugs across multiple therapy areas. The term 'developability' encompasses the feasibility of molecules to successfully progress from discovery to development via evaluation of their physicochemical properties. These properties include the tendency for self-interaction and aggregation, thermal stability, colloidal stability, and optimization of their properties through sequence engineering. Selection of the best antibody molecule based on biological function, efficacy, safety, and developability allows for a streamlined and successful CMC phase. An efficient and practical high-throughput developability workflow (100 s-1,000 s of molecules) implemented during early antibody generation and screening is crucial to select the best lead candidates. This involves careful assessment of critical developability parameters, combined with binding affinity and biological properties evaluation using small amounts of purified material (<1 mg), as well as an efficient data management and database system. Herein, a panel of 152 various human or humanized monoclonal antibodies was analyzed in biophysical property assays. Correlations between assays for different sets of properties were established. We demonstrated in two case studies that physicochemical properties and key assay endpoints correlate with key downstream process parameters. The workflow allows the elimination of antibodies with suboptimal properties and a rank ordering of molecules for further evaluation early in the candidate selection process. This enables any further engineering for problematic sequence attributes without affecting program timelines.

TH17 cells enter tissues to facilitate pathogenic autoimmune responses, including multiple sclerosis (MS). However, the adhesion molecules involved in the unique migratory capacity of TH17 cells, into both inflamed and uninflamed tissues remain unclear. Herein, we characterize MCAM (CD146) as an adhesion molecule that defines human TH17 cells in the circulation; following in vitro restimulation of human memory T cells, nearly all of the capacity to secrete IL-17 is contained within the population of cells expressing MCAM. Furthermore, we identify the MCAM ligand as laminin 411, an isoform of laminin expressed within the vascular endothelial basement membranes under inflammatory as well as homeotstatic conditions. Purified MCAM-Fc binds to laminin 411 with an affinity of 27 nM, and recognizes vascular basement membranes in mouse and human tissue. MCAM-Fc binding was undetectable in tissue from mice with targeted deletion of laminin 411, indicating that laminin 411 is a major tissue ligand for MCAM. An anti-MCAM monoclonal antibody, selected for inhibition of laminin binding, as well as soluble MCAM-Fc, inhibited T cell adhesion to laminin 411 in vitro. When administered in vivo, the antibody reduced TH17 cell infiltration into the CNS and ameliorated disease in an animal model of MS. Our data suggest that MCAM and laminin 411 interact to facilitate TH17 cell entry into tissues and promote inflammation.

Immunotherapy targeting of amyloid ␤ (A␤) peptide in transgenic mouse models of Alzheimer disease (AD) has been widely demonstrated to resolve amyloid deposition as well as associated neuronal, glial, and inflammatory pathologies. These successes have provided the basis for ongoing clinical trials of immunotherapy for treatment of AD in humans. Acute as well as chronic A␤-targeted immunotherapy has also been demonstrated to reverse A␤-related behavioral deficits assessing memory in AD transgenic mouse models. We observe that three antibodies targeting the same linear epitope of A␤, A␤ 3-7 , differ in their ability to reverse contextual fear deficits in Tg2576 mice in an acute testing paradigm. Reversal of contextual fear deficit by the antibodies does not correlate with in vitro recognition of A␤ in a consistent or correlative manner. To better define differences in antigen recognition at the atomic level, we determined crystal structures of Fab fragments in complex with A␤. The conformation of the A␤ peptide recognized by all three antibodies was highly related and is also remarkably similar to that observed in independently reported A␤:antibody crystal structures. Sequence and structural differences between the antibodies, particularly in CDR3 of the heavy chain variable region, are proposed to account for differing in vivo properties of the antibodies under study. These findings provide a structural basis for immunotherapeutic strategies targeting A␤ species postulated to underlie cognitive deficits in AD. Immunotherapy targeting the amyloid ␤ (A␤)2 peptide via either active (i.e. immunization with A␤ peptide, or fragments derived from it), or passive immunization (i.e. parenteral administration of anti-A␤ antibodies) has been widely demonstrated to be efficacious for modification of AD pathology (1, 2), as well as A␤-related behavioral deficits (3-5) in transgenic mouse models of Alzheimer disease (AD) (for reviews see Refs. 6, 7). These successes in pre-clinical studies have provided the basis for clinical trials of A␤ immunotherapy for treatment of AD in humans. Results from post-mortem histological evaluation of a limited sampling of patients from clinical trials of active immunotherapy with AN1792 provided initial corroborating evidence of pre-clinical findings with respect to reversal of plaque-associated AD pathology at autopsy in brains of treated patients (8 -13). Conclusive evidence for cognitive benefits stemming from reversal of pathology in AD patients undergoing anti-A␤ immunotherapy must await results from adequately powered Phase 3 clinical trial studies. Analysis of cognitive and functional outcomes in patients from Phase 1 and Phase 2 clinical trials provide evidence supporting improvement in some (13, 14), but not all (15) clinical measures of disease.A␤-associated behavioral deficits in transgenic mouse models of AD offer a potential surrogate of the cognitive and memory decline seen in AD patients (reviewed in Ref. 16). Arguments in support of this hypothesis stem from the fact that the b...