DOMAIN OF UNKNOWN FUNCTION581-9 negatively regulates SnRK1 kinase activity.

In plants, sucrose nonfermenting 1 (SNF1)-related protein kinase 1 (SnRK1) is a key energy sensor that orchestrates large-scale transcriptional reprograming to maintain cellular homeostasis under energy deficit. SnRK1 activity is under tight negative control, although the exact mechanisms leading to its activation are not well understood. We show that the Arabidopsis (Arabidopsis thaliana) DOMAIN OF UNKNOWN FUNCTION (DUF581) protein DUF581-9/FCS-like zinc finger 3 binds to the catalytic SnRK1.1 α subunit (KIN10) to inhibit its activation by geminivirus rep-interacting kinase (GRIK)-dependent T-loop phosphorylation. Overexpression of DUF581-9 in Arabidopsis dampens SnRK1 signaling and interferes with adaptation to dark-induced starvation. The presence of DUF581-9 significantly reduced SnRK1 activity in protoplasts and in vitro. This was accompanied by a reduction in T175 T-loop phosphorylation and also diminished KIN10 auto-phosphorylation. Furthermore, DUF581-9 reduced binding of the upstream activating kinase GRIK2 to KIN10, explaining the reduced KIN10 T-loop phosphorylation. Ectopically expressed DUF581-9 protein was rapidly turned over by the proteasome when Arabidopsis plants were subjected to starvation treatment, likely releasing its inhibitory activity on the SnRK1 complex. Taken together, our results support a model in which DUF581-9 negatively regulates SnRK1 activity under energy sufficient conditions. Turnover of the protein provides a rapid way for SnRK1 activation under energy deficit without the need of de novo protein synthesis.

Thiol-Mediated Uptake of a Cysteine-Containing Nanobody for Anticancer Drug Delivery.

The identification of tumor-specific biomarkers is one of the bottlenecks in the development of cancer therapies. Previous work revealed altered surface levels of reduced/oxidized cysteines in many cancers due to overexpression of redox-controlling proteins such as protein disulfide isomerases on the cell surface. Alterations in surface thiols can promote cell adhesion and metastasis, making thiols attractive targets for treatment. Few tools are available to study surface thiols on cancer cells and exploit them for theranostics. Here, we describe a nanobody (CB2) that specifically recognizes B cell lymphoma and breast cancer in a thiol-dependent manner. CB2 binding strictly requires the presence of a nonconserved cysteine in the antigen-binding region and correlates with elevated surface levels of free thiols on B cell lymphoma compared to healthy lymphocytes. Nanobody CB2 can induce complement-dependent cytotoxicity against lymphoma cells when functionalized with synthetic rhamnose trimers. Lymphoma cells internalize CB2 via thiol-mediated endocytosis which can be exploited to deliver cytotoxic agents. CB2 internalization combined with functionalization forms the basis for a wide range of diagnostic and therapeutic applications, rendering thiol-reactive nanobodies promising tools for targeting cancer.

Smaller size packs a stronger punch - Recent advances in small antibody fragments targeting tumour-associated carbohydrate antigens.

Attached to proteins, lipids, or forming long, complex chains, glycans represent the most versatile post-translational modification in nature and surround all human cells. Unique glycan structures are monitored by the immune system and differentiate self from non-self and healthy from malignant cells. Aberrant glycosylations, termed tumour-associated carbohydrate antigens (TACAs), are a hallmark of cancer and are correlated with all aspects of cancer biology. Therefore, TACAs represent attractive targets for monoclonal antibodies for cancer diagnosis and therapy. However, due to the thick and dense glycocalyx as well as the tumour micro-environment, conventional antibodies often suffer from restricted access and limited effectiveness in vivo. To overcome this issue, many small antibody fragments have come forth, showing similar affinity with better efficiency than their full-length counterparts. Here we review small antibody fragments against specific glycans on tumour cells and highlight their advantages over conventional antibodies.

Generation of glycan-specific nanobodies.

The development of antibodies that target specific glycan structures on cancer cells or human pathogens poses a significant challenge due to the immense complexity of naturally occurring glycans. Automated glycan assembly enables the production of structurally homogeneous glycans in amounts that are difficult to derive from natural sources. Nanobodies (Nbs) are the smallest antigen-binding domains of heavy-chain-only antibodies (hcAbs) found in camelids. To date, the development of glycan-specific Nbs using synthetic glycans has not been reported. Here, we use defined synthetic glycans for alpaca immunization to elicit glycan-specific hcAbs, and describe the identification, isolation, and production of a Nb specific for the tumor-associated carbohydrate antigen Globo-H. The Nb binds the terminal fucose of Globo-H and recognizes synthetic Globo-H in solution and native Globo-H on breast cancer cells with high specificity. These results demonstrate the potential of our approach for generating glycan-targeting Nbs to be used in biomedical and biotechnological applications.