Structure, sequon recognition and mechanism of tryptophan C-mannosyltransferase.

C-linked glycosylation is essential for the trafficking, folding and function of secretory and transmembrane proteins involved in cellular communication processes. The tryptophan C-mannosyltransferase (CMT) enzymes that install the modification attach a mannose to the first tryptophan of WxxW/C sequons in nascent polypeptide chains by an unknown mechanism. Here, we report cryogenic-electron microscopy structures of Caenorhabditis elegans CMT in four key states: apo, acceptor peptide-bound, donor-substrate analog-bound and as a trapped ternary complex with both peptide and a donor-substrate mimic bound.

Molecular basis for glycan recognition and reaction priming of eukaryotic oligosaccharyltransferase.

Oligosaccharyltransferase (OST) is the central enzyme of N-linked protein glycosylation. It catalyzes the transfer of a pre-assembled glycan, GlcNAcManGlc, from a dolichyl-pyrophosphate donor to acceptor sites in secretory proteins in the lumen of the endoplasmic reticulum. Precise recognition of the fully assembled glycan by OST is essential for the subsequent quality control steps of glycoprotein biosynthesis.

Generation of nanobodies targeting the human, transcobalamin-mediated vitamin B uptake route.

Cellular uptake of vitamin B in humans is mediated by the endocytosis of the B carrier protein transcobalamin (TC) via its cognate cell surface receptor TCblR, encoded by the CD320 gene. Because CD320 expression is associated with the cell cycle and upregulated in highly proliferating cells including cancer cells, this uptake route is a potential target for cancer therapy. We developed and characterized four camelid nanobodies that bind holo-TC (TC in complex with B ) or the interface of the human holo-TC:TCblR complex with nanomolar affinities.

Development of a universal nanobody-binding Fab module for fiducial-assisted cryo-EM studies of membrane proteins.

With conformation-specific nanobodies being used for a wide range of structural, biochemical, and cell biological applications, there is a demand for antigen-binding fragments (Fabs) that specifically and tightly bind these nanobodies without disturbing the nanobody-target protein interaction. Here, we describe the development of a synthetic Fab (termed NabFab) that binds the scaffold of an alpaca-derived nanobody with picomolar affinity. We demonstrate that upon complementary-determining region grafting onto this parent nanobody scaffold, nanobodies recognizing diverse target proteins and derived from llama or camel can cross-react with NabFab without loss of affinity.

Structure and mechanism of the ER-based glucosyltransferase ALG6.

In eukaryotic protein N-glycosylation, a series of glycosyltransferases catalyse the biosynthesis of a dolichylpyrophosphate-linked oligosaccharide before its transfer onto acceptor proteins. The final seven steps occur in the lumen of the endoplasmic reticulum (ER) and require dolichylphosphate-activated mannose and glucose as donor substrates. The responsible enzymes-ALG3, ALG9, ALG12, ALG6, ALG8 and ALG10-are glycosyltransferases of the C-superfamily (GT-Cs), which are loosely defined as containing membrane-spanning helices and processing an isoprenoid-linked carbohydrate donor substrate.

Structure of the human transcobalamin beta domain in four distinct states.

Vitamin B12 (cyanocobalamin, CNCbl) is an essential cofactor-precursor for two biochemical reactions in humans. When ingested, cobalamins (Cbl) are transported via a multistep transport system into the bloodstream, where the soluble protein transcobalamin (TC) binds Cbl and the complex is taken up into the cells via receptor mediated endocytosis. Crystal structures of TC in complex with CNCbl have been solved previously.

Structural basis of transcobalamin recognition by human CD320 receptor.

Cellular uptake of vitamin B12 (cobalamin) requires capture of transcobalamin (TC) from the plasma by CD320, a ubiquitous cell surface receptor of the LDLR family. Here we present the crystal structure of human holo-TC in complex with the extracellular domain of CD320, visualizing the structural basis of the TC-CD320 interaction. The observed interaction chemistry can rationalize the high affinity of CD320 for TC and lack of haptocorrin binding.

Ribosomal Antibiotics: Contemporary Challenges.

Most ribosomal antibiotics obstruct distinct ribosomal functions. In selected cases, in addition to paralyzing vital ribosomal tasks, some ribosomal antibiotics are involved in cellular regulation. Owing to the global rapid increase in the appearance of multi-drug resistance in pathogenic bacterial strains, and to the extremely slow progress in developing new antibiotics worldwide, it seems that, in addition to the traditional attempts at improving current antibiotics and the intensive screening for additional natural compounds, this field should undergo substantial conceptual revision.

Intralymphatic CCL21 Promotes Tissue Egress of Dendritic Cells through Afferent Lymphatic Vessels.

To induce adaptive immunity, dendritic cells (DCs) migrate through afferent lymphatic vessels (LVs) to draining lymph nodes (dLNs). This process occurs in several consecutive steps. Upon entry into lymphatic capillaries, DCs first actively crawl into downstream collecting vessels.

Intravenous aflibercept administered in combination with irinotecan, 5-fluorouracil and leucovorin in patients with advanced solid tumours: results from the expansion cohort of a phase I study.

Background: Following the dose-escalation stage, this double-blind expansion stage of the phase I study evaluated the safety, pharmacodynamics, pharmacokinetics, anti-vascular effects and antitumour activity of aflibercept 4 mg/kg with irinotecan, 5-fluorouracil and leucovorin (LV5FU2).

Patients And Methods: Patients with advanced solid tumours were randomised at cycle-1 to placebo or aflibercept (4 mg/kg) on day 1 then irinotecan-LV5FU2 on days 1 and 2. Subsequently, all patients received aflibercept with irinotecan-LV5FU2 every 2 weeks.

Treatment for metastatic malignant melanoma: old drugs and new strategies.

The number of melanoma cases worldwide is increasing faster than any other cancer and remains one of the most treatment-refractory malignancies. Despite decades of clinical trials testing chemotherapy and immunotherapy, a standard first-line treatment for metastatic melanoma has not yet been established; tough single agent dacarbazine represents the most common option. This review will focus on metastatic malignant melanoma treatment from single agent until new therapies.