Testing preexisting antibodies prior to AAV gene transfer therapy: rationale, lessons and future considerations.

Given the increasing number of gene transfer therapy studies either completed or underway, there is growing attention to the importance of preexisting adaptive immunity to the viral vectors used. The recombinant viral vectors developed for gene transfer therapy share structural features with naturally occurring wild-type virus. Antibodies generated against viral vectors obtained through a previous exposure to wild-type virus can potentially compromise transgene expression by blocking transduction, thereby limiting the therapeutic efficacy of the gene transfer therapy; they may also pose potential safety concerns. Therefore, systemic gene transfer delivery requires testing patients for preexisting antibodies. Two different assays have been used: (1) binding assays that focus on total antibodies (both neutralizing and non-neutralizing) and (2) neutralizing assays that detect neutralizing antibodies. In this review we focus on adeno-associated virus-based gene therapies, describing the immune response that occurs to naturally occurring adeno-associated viruses, the implications for patients with this exposure, the assays used to detect preexisting immune responses, and strategies to circumvent preexisting adaptive immunity to expand the patient base that could benefit from such therapies.

Combining Electron Microscopy (EM) and Cross-Linking Mass Spectrometry (XL-MS) for Structural Characterization of Protein Complexes.

Structural biology has recently witnessed the benefits of the combined use of two complementary techniques: electron microscopy (EM) and cross-linking mass spectrometry (XL-MS). EM (especially its cryogenic variant cryo-EM) has proven to be a very powerful tool for the structural determination of proteins and protein complexes, even at an atomic level. In a complementary way, XL-MS allows the precise characterization of particular interactions when residues are located in close proximity. When working from low-resolution, negative-staining images and less-defined regions of flexible domains (whose mapping is made possible by cryo-EM), XL-MS can provide critical information on specific amino acids, thus identifying interacting regions and helping to deduce the overall protein structure. The protocol described here is particularly well suited for the study of protein complexes whose intrinsically flexible or transient nature prevents their high-resolution characterization by any structural technique itself.

The cochaperone CHIP marks Hsp70- and Hsp90-bound substrates for degradation through a very flexible mechanism.

Some molecular chaperones are involved not only in assisting the folding of proteins but also, given appropriate conditions, in their degradation. This is the case for Hsp70 and Hsp90 which, in concert with the cochaperone CHIP, direct their bound substrate to degradation through ubiquitination. We generated complexes between the chaperones (Hsp70 or Hsp90), the cochaperone CHIP and, as substrate, a p53 variant containing the GST protein (p53-TMGST). Both ternary complexes (Hsp70:p53-TMGST:CHIP and Hsp90:p53-TMGST:CHIP) ubiquitinated the substrate at a higher efficiency than in the absence of the chaperones. The 3D structures of the two complexes, obtained using a combination of cryoelectron microscopy and crosslinking mass spectrometry, showed the substrate located between the chaperone and the cochaperone, suggesting a ubiquitination mechanism in which the chaperone-bound substrate is presented to CHIP. These complexes are inherently flexible, which is important for the ubiquitination process.

Macrophage-specific MHCII expression is regulated by a remote Ciita enhancer controlled by NFAT5.

MHCII in antigen-presenting cells (APCs) is a key regulator of adaptive immune responses. Expression of MHCII genes is controlled by the transcription coactivator CIITA, itself regulated through cell type-specific promoters. Here we show that the transcription factor NFAT5 is needed for expression of Ciita and MHCII in macrophages, but not in dendritic cells and other APCs. NFAT5-deficient macrophages showed defective activation of MHCII-dependent responses in CD4+ T lymphocytes and attenuated capacity to elicit graft rejection in vivo. Ultrasequencing analysis of NFAT5-immunoprecipitated chromatin uncovered an NFAT5-regulated region distally upstream of Ciita This region was required for CIITA and hence MHCII expression, exhibited NFAT5-dependent characteristics of active enhancers such as H3K27 acetylation marks, and required NFAT5 to interact with Ciita myeloid promoter I. Our results uncover an NFAT5-regulated mechanism that maintains CIITA and MHCII expression in macrophages and thus modulates their T lymphocyte priming capacity.

Hsp70 - a master regulator in protein degradation.

Proteostasis, the controlled balance of protein synthesis, folding, assembly, trafficking and degradation, is a paramount necessity for cell homeostasis. Impaired proteostasis is a hallmark of ageing and of many human diseases. Molecular chaperones are essential for proteostasis in eukaryotic cells, and their function has traditionally been linked to protein folding, assembly and disaggregation. More recent findings suggest that chaperones also contribute to key steps in protein degradation. In particular, Hsp70 has an essential role in substrate degradation through the ubiquitin-proteasome system, as well as through different autophagy pathways. Accumulated knowledge suggests that the fate of an Hsp70 substrate is dictated by the combination of partners (cochaperones and other chaperones) that interact with Hsp70 in a given cell context.

Modulation of the chaperone DnaK allosterism by the nucleotide exchange factor GrpE.

Hsp70 chaperones comprise two domains, the nucleotide-binding domain (Hsp70NBD), responsible for structural and functional changes in the chaperone, and the substrate-binding domain (Hsp70SBD), involved in substrate interaction. Substrate binding and release in Hsp70 is controlled by the nucleotide state of DnaKNBD, with ATP inducing the open, substrate-receptive DnaKSBD conformation, whereas ADP forces its closure. DnaK cycles between the two conformations through interaction with two cofactors, the Hsp40 co-chaperones (DnaJ in Escherichia coli) induce the ADP state, and the nucleotide exchange factors (GrpE in E. coli) induce the ATP state. X-ray crystallography showed that the GrpE dimer is a nucleotide exchange factor that works by interaction of one of its monomers with DnaKNBD. DnaKSBD location in this complex is debated; there is evidence that it interacts with the GrpE N-terminal disordered region, far from DnaKNBD. Although we confirmed this interaction using biochemical and biophysical techniques, our EM-based three-dimensional reconstruction of the DnaK-GrpE complex located DnaKSBD near DnaKNBD. This apparent discrepancy between the functional and structural results is explained by our finding that the tail region of the GrpE dimer in the DnaK-GrpE complex bends and its tip contacts DnaKSBD, whereas the DnaKNBD-DnaKSBD linker contacts the GrpE helical region. We suggest that these interactions define a more complex role for GrpE in the control of DnaK function.