Neurotransmission is based on the exocytic fusion of synaptic vesicles (SVs) followed by endocytic membrane retrieval and the reformation of SVs. Recent data suggest that at physiological temperature SVs are internalized via clathrin-independent ultrafast endocytosis (UFE) within hundreds of milliseconds, while other studies have postulated a key role for clathrin-mediated endocytosis (CME) of SV proteins on a timescale of seconds to tens of seconds. Here we demonstrate using cultured hippocampal neurons as a model that at physiological temperature SV endocytosis occurs on several timescales from less than a second to several seconds, yet, is largely independent of clathrin. Clathrin-independent endocytosis (CIE) of SV membranes is mediated by actin-nucleating formins such as mDia1, which are required for the formation of presynaptic endosome-like vacuoles from which SVs reform. Our results resolve previous discrepancies in the field and suggest that SV membranes are predominantly retrieved via CIE mediated by formin-dependent actin assembly.

Autophagy induction by starvation and stress involves the enzymatic activation of the class III phosphatidylinositol (PI) 3-kinase complex I (PI3KC3-C1). The inactive basal state of PI3KC3-C1 is maintained by inhibitory contacts between the VPS15 protein kinase and VPS34 lipid kinase domains that restrict the conformation of the VPS34 activation loop. Here, the proautophagic MIT domain-containing protein NRBF2 was used to map the structural changes leading to activation. Cryoelectron microscopy was used to visualize a 2-step PI3KC3-C1 activation pathway driven by NRFB2 MIT domain binding. Binding of a single NRBF2 MIT domain bends the helical solenoid of the VPS15 scaffold, displaces the protein kinase domain of VPS15, and releases the VPS34 kinase domain from the inhibited conformation. Binding of a second MIT stabilizes the VPS34 lipid kinase domain in an active conformation that has an unrestricted activation loop and is poised for access to membranes.

Malaria, a mosquito-borne disease caused by Plasmodium species, claims more than 400,000 lives globally each year. The increasing drug resistance of the parasite renders the development of new anti-malaria drugs necessary. Alternatively, better delivery systems for already marketed drugs could help to solve the resistance problem. Herein, we report glucose-based ultra-small gold nanoparticles (Glc-NCs) that bind to cysteine-rich domains of Plasmodium falciparum surface proteins . Microscopy shows that Glc-NCs bind specifically to extracellular and all intra-erythrocytic stages of P. falciparum . Glc-NCs may be used as drug delivery agents as illustrated for ciprofloxacin, a poorly soluble antibiotic with low antimalarial activity. Ciprofloxacin conjugated to Glc-NCs is more water-soluble than the free drug and is more potent. Glyco-gold nanoparticles that target cysteine-rich domains on parasites may be helpful for the prevention and treatment of malaria.

Hetero- and carbocyclic anellated cyclopropanes were converted in one step by a visible light induced photooxidation to their corresponding polycyclic endoperoxides, which show promising antimalarial activity.

No abstract

Plasmodium parasites cause malaria disease, one of the leading global health burdens for humanity, infecting hundreds of millions of people each year. Different glycans on the parasite and the host cell surface play significant roles in both malaria pathogenesis and host defense mechanisms. So far, only small, truncated N- and O-glycans have been identified in Plasmodium species. In contrast, complex glycosylphosphatidylinositol (GPI) glycolipids are highly abundant on the parasite’s cell membrane and are essential for its survival. Moreover, the parasites express lectins that bind and exploit the host cell surface glycans for different aspects of the parasite life cycle, such as adherence, invasion, and evasion of the host immune system. In parallel, the host cell glycocalyx and lectin expression serve as the first line of defense against Plasmodium parasites and directly dictate susceptibility to Plasmodium infection. This review provides an overview of the glycobiology involved in Plasmodium-host interactions and its contribution to malaria pathogenesis. Recent findings are presented and evaluated in the context of potential therapeutic exploitation.

Autophagy induction by starvation and stress involves the enzymatic activation of the class III phosphatidylinositol 3-kinase complex I (PI3KC3-C1). The inactive basal state of PI3KC3-C1 is maintained by inhibitory contacts between the VPS15 protein kinase and VPS34 lipid kinase domains that restrict the conformation of the VPS34 activation loop. Here, the pro-autophagic MIT domain-containing protein NRBF2 was used to map the structural changes leading to activation. Cryo-EM was used to visualize stepwise PI3KC3-C1 activating effects of binding the NRFB2 MIT domains. Binding of a single NRBF2 MIT domain to bends the helical solenoid of the VPS15 scaffold, displaces the protein kinase domain of VPS15, and releases the VPS34 kinase domain from the inhibited conformation. Binding of a second MIT stabilizes the VPS34 lipid kinase domain in an active conformation that has an unrestricted activation loop and is poised for access to membranes.Autophagy is a core cellular process, conserved throughout eukaryotes, which is the central recycling system for the removal of misfolded proteins, damaged organelles, and the recycling of nutrients in starvation. Autophagic dysfunction is implicated in many disease states, including neurodegeneration, immune disorders, cancer, and aging, among others (1). The class III phosphatidylinositol-3 kinase complexes (PI3KC3) I and II (PI3KC3-C1 and -C2), respectively, are essential for the initiation and expansion of autophagosomes (2-5). PI3KC3 generates the lipid phosphatidylinositol-3-phosphate, PI(3)P, which is recognized by the WIPI proteins. WIPIs in turn recruit the machinery that conjugates the autophagosomal marker LC3 to the expanding autophagosomal membrane (6). PI3KC3-C1 has been proposed to be a promising therapeutic target for autophagy activators (7) because the generation of PI(3)P is absolutely required for the recruitment of downstream autophagy proteins. There is considerable medical interest in selectively activating this pathway to promote human health and treat disease, yet there are no FDA approved pharmaceuticals that uniquely activate autophagy.PI3KC3-C1 consists of the lipid kinase VPS34, the putative serine/threonine protein kinase VPS15, the regulatory subunit BECN1, and the early autophagy-specific targeting subunit ATG14 (8,9). In PI3KC3-C2, ATG14 is replaced with UVRAG (10), while the other three subunits are preserved. The overall architecture of both PI3KC3-C1 and -C2 has the shape of the letter V (11, 12) (Fig. 1A). The long coiled-coils of BECN1 and ATG14 scaffold the left arm in the standard view, with the membrane binding BARA domain of BECN1 located at the outermost tip of the arm. PI3KC3-C2 is inhibited by Rubicon and the HIV-1 protein Nef (13-15), which regulate membrane docking by the tip of the left arm. The catalytic domains of the kinases VPS34 and VPS15 are at the tip of the right arm (11,12). PI3KC3 complexes are phosphoregulated by the Unc-51 like autophagy-activating kinase 1 (ULK1) complex (16),

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.