Delta-aminolevulinic acid (ALA) is the precursor of porphyrin synthesis and has been recently used in vitro and in clinical studies as an endogenous photosensitizer for photodynamic therapy in the treatment of various tumors. For this purpose, ALA is given topically, systemically, or orally. When administered by the oral route, it shows excellent intestinal absorption. ALA is also efficiently reabsorbed in the renal proximal tubule after glomerular filtration. However, the pathways and mechanisms for its transmembrane transport into epithelial cells of intestine and kidney are unknown. Here we demonstrate that ALA uses the intestinal and renal apical peptide transporters for entering into epithelial cells. Kinetics and characteristics of ALA transport were determined in Xenopus laevis ooyctes and Pichia pastoris yeast cells expressing either the cloned intestinal peptide transporter PEPT1 or the renal form PEPT2. By using radiolabeled ALA and electrophysiological techniques in these heterologous expression systems, we established that: (a) PEPT1 and PEPT2 translocate 3H-ALA by saturable and pH-dependent transport mechanisms, (b) that ALA and di-/tripeptides, but not GABA or related amino acids, compete at the same substrate-binding site of the carriers, and (c) that ALA transport is electrogenic in nature as a consequence of H+/ALA cotransport. Reverse transcriptase-PCR analysis performed with specific primers for PEPT1 and PEPT2 in rabbit tissues demonstrates that, in particular, the PEPT2 mRNA is expressed in a variety of other tissues including lung, brain, and mammary gland, which have been shown to accumulate ALA. This suggests that these tissues could take up the porphyrin precusor via expressed peptide transporters, providing the endogenous photosensitizers for efficient photodynamic therapy.

The epithelial anion transporter SLC26A9 contributes to airway surface hydration and gastric acid production. Colocalizing with CFTR, SLC26A9 has been proposed as a target for the treatment of cystic fibrosis. To provide molecular details of its transport mechanism, we present cryo-EM structures and a functional characterization of murine Slc26a9. These structures define the general architecture of eukaryotic SLC26 family members and reveal an unusual mode of oligomerization which relies predominantly on the cytosolic STAS domain. Our data illustrates conformational transitions of Slc26a9, supporting a rapid alternate-access mechanism which mediates uncoupled chloride transport with negligible bicarbonate or sulfate permeability. The characterization of structure-guided mutants illuminates the properties of the ion transport path, including a selective anion binding site located in the center of a mobile module within the transmembrane domain. This study thus provides a structural foundation for the understanding of the entire SLC26 family and potentially facilitates their therapeutic exploitation.

The COVID-19 pandemic, caused by the novel coronavirus SARS-CoV-2, has resulted in a global health and economic crisis of unprecedented scale. The high transmissibility of SARS-CoV-2, combined with a lack of population immunity and prevalence of severe clinical outcomes, urges the rapid development of effective therapeutic countermeasures. Here, we report the generation of synthetic nanobodies, known as sybodies, against the receptor-binding domain (RBD) of SARS-CoV-2. In an expeditious process taking only twelve working days, sybodies were selected entirely in vitro from three large combinatorial libraries, using ribosome and phage display. We obtained six strongly enriched sybody pools against the isolated RBD and identified 63 unique anti-RBD sybodies which also interact in the context of the full-length SARS-CoV-2 spike protein. It is anticipated that compact binders such as these sybodies could feasibly be developed into an inhalable drug that can be used as a convenient prophylaxis against COVID-19. Moreover, generation of polyvalent antivirals, via fusion of anti-RBD sybodies to additional small binders recognizing secondary epitopes, could enhance the therapeutic potential and guard against escape mutants. We present full sequence information and detailed protocols for the identified sybodies, as a freely accessible resource. This report will be updated as we further characterize the identified sybodies, in terms of affinities, scaled-up purification yields, and their potential to neutralize SARS-CoV-2 infections.

Key Points Antibodies against the factor VIII C2 domain inhibit procoagulant function. Crystal structure analysis of a C2 domain/antibody ternary complex describes epitopes for classical and nonclassical inhibitory antibodies.

Thiostrepton, a macrocyclic thiopeptide antibiotic, inhibits prokaryotic translation by interfering with the function of elongation factor G (EF-G). Here, we have used 70S ribosome binding and GTP hydrolysis assays to study the effects of thiostrepton on EF-G and a newly described translation factor, elongation factor 4 (EF4). In the presence of thiostrepton, ribosome-dependent GTP hydrolysis is inhibited for both EF-G and EF4, with IC(50) values equivalent to the 70S ribosome concentration (0.15 µM). Further studies indicate the mode of thiostrepton inhibition is to abrogate the stable binding of EF-G and EF4 to the 70S ribosome. In support of this model, an EF-G truncation variant that does not possess domains IV and V was shown to possess ribosome-dependent GTP hydrolysis activity that was not affected by the presence of thiostrepton (>100 µM). Lastly, chemical footprinting was employed to examine the nature of ribosome interaction and tRNA movements associated with EF4. In the presence of non-hydrolyzable GTP, EF4 showed chemical protections similar to EF-G and stabilized a ratcheted state of the 70S ribosome. These data support the model that thiostrepton inhibits stable GTPase binding to 70S ribosomal complexes, and a model for the first step of EF4-catalyzed reverse-translocation is presented.