Background: Nanobodies represent a specific means to modulate enzyme function. Results: HypE-specific nanobodies modulate AMPylation activity and identify HypE as localized to the nuclear envelope. Conclusion: HypE function can be interfered with and probed for with new tools. Significance: HypE nanobodies are the first known HypE activators and inhibitors.

In vivo protein ligation is of emerging interest as a means of endowing proteins with new properties in a controlled fashion. Tools to site-specifically and covalently modify proteins with small molecules, peptides, or other proteins in living cells are few and far between. Here, we describe the development of a Staphylococcus aureus sortase (SrtA)-based protein ligation approach for site-specific conjugation of fluorescent dyes and ubiquitin (Ub) to modify proteins in Caenorhabditis elegans. Hepta-mutant SrtA (SrtA7m) expressed in C. elegans is functional and supports in vitro sortase reactions in a low-Ca2+ environment. Feeding SrtA7m-expressing C. elegans with small peptide-based probes such as (Gly)3- biotin or (Gly)3-fluorophores enables in vivo target protein modification. SrtA7m also catalyzes the circularization of suitably modified linear target proteins in vivo and allows the installation of F-box domains on targets to induce their degradation in a ubiquitin-dependent manner. This is a noninvasive method to achieve in vivo protein labeling, protein circularization, and targeted degradation in C. elegans. This technique should improve our ability to monitor and alter the function of intracellular proteins in vivo.

Background：Chikungunya virus (CHIKV) is a re-emerged mosquito-borne alphavirus that can cause musculoskeletal disease and impose a substantial threat to public health globally. It would be desirable to develop a high-affinity antibodies for the diagnosis and therapy of CHIKV infection. As potential diagnostic and therapeutic agents, multivalent nanobodies hold a significant promise towards nanomedicine. Here, we developed the highly potent multivalent nanobodies from an alpaca naïve phage display library targeting the E2 glycoprotein of CHIKV virus. Multivalent nanobodies play an important role in the promotion of high-affinity binding to E2 protein.Results: In the present study, we generated 20 nanobodies using a naïve phage display library for binders to the CHIKV E2 glycoprotein. Of which, multivalent nanobodies of Nb-2E8 and Nb-3C5 had specific high-affinity binding to E2 protein with nanomolar range, showing the equilibrium dissociation constant (KD) of 2.59-20.7 nM, which is 100-fold stronger than monovalent nanobodies’ affinity. Moreover, epitope mapping showed that the Nb-2E8 and Nb-3C5 recognized different linear epitopes located on the E2 glycoprotein domain C and A, respectively. A facile protocol of sandwich ELISA was established using the BiNb-2E8 as a capture antibody and HRP-conjugated BiNb-3C5 as a detection antibody. A good linear correlation was achieved between the OD450 value and the E2 protein concentration in the 5-1000 ng/mL range (r=0.9864, P<0.0001), indicating that it can be used for the quantitative detection of E2 protein.Conclusions: Multivalent nanobodies Nb-2E8 and Nb-3C5 exhibit functional features and high affinity distinct from monovalent nanobodies, showing new candidate diagnostic applications to detect sera binding protein and/or virions.