Antibodies are important tools for experimental research and medical applications. Most antibodies are composed of two heavy and two light chains. Both chains contribute to the antigen-binding site which is usually Xat or concave. In addition to these conventional antibodies, llamas, other camelids, and sharks also produce antibodies composed only of heavy chains. The antigen-binding site of these unusual heavy chain antibodies (hcAbs) is formed only by a single domain, designated VHH in camelid hcAbs and VNAR in shark hcAbs. VHH and VNAR are easily produced as recombinant proteins, designated single domain antibodies (sdAbs) or nanobodies. The CDR3 region of these sdAbs possesses the extraordinary capacity to form long Wngerlike extensions that can extend into cavities on antigens, e.g., the active site crevice of enzymes. Other advantageous features of nanobodies include their small size, high solubility, thermal stability, refolding capacity, and good tissue penetration in vivo. Here we review the results of several recent proofof-principle studies that open the exciting perspective of using sdAbs for modulating immune functions and for targeting toxins and microbes.

Keratins are major components of the epithelial cytoskeleton and are believed to play a vital role for mechanical integrity at the cellular and tissue level. Keratinocytes as the main cell type of the epidermis express a differentiation-specific set of type I and type II keratins forming a stable network and are major contributors of keratinocyte mechanical properties. However, owing to compensatory keratin expression, the overall contribution of keratins to cell mechanics was difficult to examine in vivo on deletion of single keratin genes. To overcome this problem, we used keratinocytes lacking all keratins. The mechanical properties of these cells were analyzed by atomic force microscopy (AFM) and magnetic tweezers experiments. We found a strong and highly significant softening of keratin-deficient keratinocytes when analyzed by AFM on the cell body and above the nucleus. Magnetic tweezers experiments fully confirmed these results showing, in addition, high viscous contributions to magnetic bead displacement in keratin-lacking cells. Keratin loss neither affected actin or microtubule networks nor their overall protein concentration. Furthermore, depolymerization of actin preserves cell softening in the absence of keratin. On reexpression of the sole basal epidermal keratin pair K5/14, the keratin filament network was reestablished, and mechanical properties were restored almost to WT levels in both experimental setups. The data presented here demonstrate the importance of keratin filaments for mechanical resilience of keratinocytes and indicate that expression of a single keratin pair is sufficient for almost complete reconstitution of their mechanical properties.

Keratins limit PKC-α phosphorylation activity and desmosome turnover to ensure the stability of epithelial intracellular adhesion.

ADP-ribosylation is a post-translational modification regulating protein function in which amino acid-specific ADP-ribosyltransferases (ARTs) transfer ADP-ribose from NAD onto specific target proteins. Attachment of the bulky ADP-ribose usually inactivates the target by sterically blocking its interaction with other proteins. P2X7, an ATP-gated ion channel with important roles in inflammation and cell death, in contrast, is activated by ADP-ribosylation. Here, we report the structural basis for this gating and present the first molecular model for the activation of a target protein by ADP-ribosylation. We demonstrate that the ecto-enzyme ART2.2 ADP-ribosylates P2X7 at arginine 125 in a prominent, cysteine-rich region at the interface of 2 receptor subunits. ADP-ribose shares an adenine-ribonucleotide moiety with ATP. Our results indicate that ADP-ribosylation of R125 positions this common chemical framework to fit into the nucleotide-binding site of P2X7 and thereby gates the channel.