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Poly(ADP-ribose) polymerase 7 (PARP-7) has emerged as a critically important member of a large enzyme family that catalyzes ADP-ribosylation in mammalian cells. PARP-7 is a critical regulator of the innate immune response. What remains unclear is the mechanism by which PARP-7 regulates this process, namely because the protein targets of PARP-7 mono-ADP-ribosylation (MARylation) are largely unknown. Here, we combine chemical genetics, proximity labeling, and proteome-wide amino acid ADP-ribosylation site profiling for identifying the direct targets and sites of PARP-7-mediated MARylation in a cellular context. We found that the inactive PARP family member, PARP-13—a critical regulator of the antiviral innate immune response—is a major target of PARP-7. PARP-13 is preferentially MARylated on cysteine residues in its RNA binding zinc finger domain. Proteome-wide ADP-ribosylation analysis reveals cysteine as a major MARylation acceptor of PARP-7. This study provides insight into PARP-7 targeting and MARylation site preference.

Membrane transporters move substrates across the membrane by alternating access of their binding sites between the opposite sides of the membrane. An emerging model of this process is the elevator mechanism, in which a substrate-binding transport domain moves a large distance across the membrane. This mechanism has been characterized by a transition between two states, but the conformational path that leads to the transition is not yet known, largely because the available structural information has been limited to the two end states. Here we present crystal structures of the inward-facing, intermediate, and outward-facing states of a concentrative nucleoside transporter from Neisseria wadsworthii . Notably, we determined the structures of multiple intermediate conformations, in which the transport domain is captured halfway through its elevator motion. Our structures present a trajectory of the conformational transition in the elevator model, revealing multiple intermediate steps and state-dependent conformational changes within the transport domain that are associated with the elevator-like motion. Multiple crystallographic structures of a concentrative nucleoside transporter show how it uses an ‘elevator’ mechanism to move its transport domain across the membrane. Multistep nucleoside transport Nucleosides that are key to DNA and RNA synthesis and nucleoside-derived drugs used in the treatment of cancer and viral infections are imported into cells by 'concentrative nucleoside transporters' (CNTs). It has been proposed that an elevator-like substrate-binding domain moves across the membrane, but the intermediate steps are still unknown. Seok-Yong Lee and colleagues now report X-ray crystallographic structures of a CNT, not only in its inward- and outward-facing states, but also in several intermediate states, thanks to a combination of ligands, ions, mutations and crystallization conditions that stabilize different conformations in the crystal lattice. The work suggests that the transport domain undergoes state-dependent conformational changes rather than a single rigid-body motion during its elevator-like movement.

BackgroundSLC29A3-related syndromes (SLC29A3-RS) are characterised by severe and multiorgan involvement that has a severe impact on the quality of life of the affected persons and therefore merit further genetic and clinical research. We investigated the clinical and genetic aspects of patients with SLC29A3-RS.MethodsSix pathogenic variants of the SLC29A3 gene were identified in eight families in the current study. RNA sequencing was used for evaluating SLC29A3 variant gene expression and protein stability by molecular dynamics (MD) simulations. This study conducted a Preferred Reporting Items for Systematic Reviews and Meta-Analyses-compliant systematic review of cases across five electronic databases.ResultsGenetic analysis revealed six pathogenic variants of the SLC29A3 gene in eight families; one variant was shared among three families, indicating a possible founder effect. The estimated most recent common ancestor for these patients lived approximately 8.5 generations ago. MD studies revealed structural instability in mutant proteins. RNA sequencing also demonstrated that the expression of SLC29A3 was downregulated while the expression of the immune markers CD68 and LYZ was upregulated. A systematic search of 197 patients of different ethnic backgrounds revealed that the following symptoms were frequent findings: hyperpigmentation, hypertrichosis, hearing loss, short stature and hepatomegaly. The age of onset of SLC29A3-RS was 5.53±5.24 years with an IQR of 1.4–8.25 years.ConclusionsThe characterisation of the founder variants and the genotype-phenotype correlations helps delineate the phenotype spectrum of SLC29A3-RS, which will facilitate the genetic counselling and screening of the high-risk population. Findings on SLC29A3 variants show the way to proceed in the process of developing the diagnostic and therapeutic methods in the management of SLC29A3-RS.

Membrane proteins play key roles in many biological processes, from acquisition of nutrients to neurotransmission, and are targets for more than 50% of current therapeutic drugs. However, their investigation is hampered by difficulties in their production and purification on a scale suitable for structural studies. In particular, the nature and location of affinity tags introduced for the purification of recombinant membrane proteins can greatly influence their expression levels by affecting their membrane insertion. The extent of such effects typically depends on the transmembrane topologies of the proteins, which for proteins of unknown structure are usually uncertain. For example, attachment of oligohistidine tags to the periplasmic termini of membrane proteins often interferes with folding and drastically impairs expression in Escherichia coli. To circumvent this problem we have employed a novel strategy to enable the rapid production of constructs bearing a range of different affinity tags compatible with either cytoplasmic or periplasmic attachment. Tags include conventional oligohistidine tags compatible with cytoplasmic attachment and, for attachment to proteins with a periplasmic terminus, either tandem Strep-tag II sequences or oligohistidine tags fused to maltose binding protein and a signal sequence. Inclusion of cleavage sites for TEV or HRV-3C protease enables tag removal prior to crystallisation trials or a second step of purification. Together with the use of bioinformatic approaches to identify members of membrane protein families with topologies favourable to cytoplasmic tagging, this has enabled us to express and purify multiple bacterial membrane transporters. To illustrate this strategy, we describe here its use to purify bacterial homologues of human membrane proteins from the Nramp and ZIP families of divalent metal cation transporters and from the concentrative nucleoside transporter family. The proteins are expressed in E. coli in a correctly folded, functional state and can be purified in amounts suitable for structural investigations.

The human SLC29 family of proteins contains four members, designated equilibrative nucleoside transporters (ENTs) because of the properties of the first-characterised family member, hENT1. They belong to the widely-distributed eukaryotic ENT family of equilibrative and concentrative nucleoside/nucleobase transporters and are distantly related to a lysosomal membrane protein, CLN3, mutations in which cause neuronal ceroid lipofuscinosis. A predicted topology of 11 transmembrane helices with a cytoplasmic N-terminus and an extracellular C-terminus has been experimentally confirmed for hENT1. The best-characterised members of the family, hENT1 and hENT2, possess similar broad substrate specificities for purine and pyrimidine nucleosides, but hENT2 in addition efficiently transports nucleobases. The ENT3 and ENT4 isoforms have more recently also been shown to be genuine nucleoside transporters. All four isoforms are widely distributed in mammalian tissues, although their relative abundance varies: ENT2 is particularly abundant in skeletal muscle. In polarised cells ENT1 and ENT2 are found in the basolateral membrane and, in tandem with concentrative transporters of the SLC28 family, may play a role in transepithelial nucleoside transport. The transporters play key roles in nucleoside and nucleobase uptake for salvage pathways of nucleotide synthesis, and are also responsible for the cellular uptake of nucleoside analogues used in the treatment of cancers and viral diseases. In addition, by regulating the concentration of adenosine available to cell surface receptors, they influence many physiological processes ranging from cardiovascular activity to neurotransmission.

Concentrative nucleoside transporters (CNTs) are responsible for cellular entry of nucleosides, which serve as precursors to nucleic acids and act as signaling molecules. CNTs also play a crucial role in the uptake of nucleoside-derived drugs, including anticancer and antiviral agents. Understanding how CNTs recognize and import their substrates could not only lead to a better understanding of nucleoside-related biological processes but also the design of nucleoside-derived drugs that can better reach their targets. Here, we present a combination of X-ray crystallographic and equilibrium-binding studies probing the molecular origins of nucleoside and nucleoside drug selectivity of a CNT from Vibrio cholerae. We then used this information in chemically modifying an anticancer drug so that it is better transported by and selective for a single human CNT subtype. This work provides proof of principle for utilizing transporter structural and functional information for the design of compounds that enter cells more efficiently and selectively. DNA molecules are made from four bases—often named ‘G’, ‘A’, ‘C’, and ‘T’—that are arranged along a backbone made of sugars and phosphate groups. Chemicals called nucleosides are essentially the same as these four building blocks of DNA (and other similar molecules) but without the phosphate groups. Proteins called nucleoside transporters are found in the membranes that surround cells and can pump nucleosides into the cell. These transporters also allow drugs that are made from modified nucleosides to enter cells; however, it was previously unclear how different transporters recognized and imported specific nucleosides. Like other proteins, nucleoside transporters are basically strings of amino acids that have folded into a specific three-dimensional shape. A protein's shape is often important for defining what that protein can do, as often other molecules must bind to proteins—much like a key fitting into a lock. Johnson et al. have now revealed the three-dimensional structure of one nucleoside transporter protein bound to different nucleosides and nucleoside-derived chemicals, including three anti-cancer drugs and one anti-viral drug. Some of these chemicals were shown to bind more strongly to the transporter protein than others, and examining the three-dimensional structures revealed that the different chemicals interacted with slightly different amino acids in the transporter protein. Johnson et al. then used this information to chemically modify an anticancer drug so that it is transported more easily into cells and is imported by only one of the subtypes of nucleoside transporters that are found in humans. This provides proof of principle that information about the structure and function of a transporter protein can help to redesign chemicals such that they can enter cells more efficiently, and to tailor them for transport by specific transporters. A similar approach may in the future allow researchers to design new nucleoside-derived drugs that are better at getting inside specific cells and, as such, provide effective treatments against cancers and viral infections.

The X-ray crystal structure of a bacterial concentrative nucleoside transporter reveals the overall architecture of this class of transporter and provides a framework for understanding how nucleosides and nucleoside-derived drugs traverse cell membranes. Cholera pathogen's nucleoside transporter Concentrative nucleoside transporters are integral membrane proteins that are responsible for the selective uptake of nucleosides and nucleoside-derived anticancer and antiviral drugs into cells. This paper presents the first X-ray crystal structure of a member of a concentrative nucleoside transporter family, from the cholera pathogen Vibrio cholerae , in complex with uridine. The structure reveals the overall architecture of this class of transporter and the molecular determinants for nucleoside- and sodium-binding. Nucleosides are required for DNA and RNA synthesis, and the nucleoside adenosine has a function in a variety of signalling processes 1 , 2 . Transport of nucleosides across cell membranes provides the major source of nucleosides in many cell types and is also responsible for the termination of adenosine signalling. As a result of their hydrophilic nature, nucleosides require a specialized class of integral membrane proteins, known as nucleoside transporters (NTs), for specific transport across cell membranes. In addition to nucleosides, NTs are important determinants for the transport of nucleoside-derived drugs across cell membranes 3 , 4 , 5 . A wide range of nucleoside-derived drugs, including anticancer drugs (such as Ara-C and gemcitabine) and antiviral drugs (such as zidovudine and ribavirin), have been shown to depend, at least in part, on NTs for transport across cell membranes 4 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 . Concentrative nucleoside transporters, members of the solute carrier transporter superfamily SLC28, use an ion gradient in the active transport of both nucleosides and nucleoside-derived drugs against their chemical gradients. The structural basis for selective ion-coupled nucleoside transport by concentrative nucleoside transporters is unknown. Here we present the crystal structure of a concentrative nucleoside transporter from Vibrio cholerae in complex with uridine at 2.4 Å. Our functional data show that, like its human orthologues, the transporter uses a sodium-ion gradient for nucleoside transport. The structure reveals the overall architecture of this class of transporter, unravels the molecular determinants for nucleoside and sodium binding, and provides a framework for understanding the mechanism of nucleoside and nucleoside drug transport across cell membranes.

The SLC28 family consists of three subtypes of sodium-dependent, concentrative nucleoside transporters, CNT1, CNT2, and CNT3 (SLC28A1, SLC28A2, and SLC28A3, respectively), that transport both naturally occurring nucleosides and synthetic nucleoside analogs used in the treatment of various diseases. These subtypes differ in their substrate specificities: CNT1 is pyrimidine-nucleoside preferring, CNT2 is purine-nucleoside preferring, and CNT3 transports both pyrimidine and purine nucleosides. Recent studies have identified key amino acid residues that are determinants of pyrimidine and purine specificity of CNT1 and CNT2. The tissue distributions of the CNTs vary: CNT1 is localized primarily in epithelia, whereas CNT2 and CNT3 have more generalized distributions. Nucleoside transporters in the SLC28 and SLC29 families play critical roles in nucleoside salvage pathways where they mediate the first step of nucleotide biosynthesis. In addition, these transporters work in concert to terminate adenosine signaling. SLC28 family members are crucial determinants of response to a variety of anticancer and antiviral nucleoside analogs, as they modulate the entry of these analogs into target tissues. Further, this family is involved in the absorption and disposition of many nucleoside analogs. Several CNT single nucleoside polymorphisms (SNPs) have been identified, but have yet to be characterized.

Adenosine has been reported to be transported by equilibrative nucleoside transporter 4 (ENT4), encoded by the SLC29A4 gene, in an acidic pH-dependent manner. This makes hENT4 of interest as a therapeutic target in acidic pathologies where adenosine is protective (e.g. vascular ischaemia). We examined the pH-sensitivity of nucleoside influx and efflux by hENT4 using a recombinant transfection model that lacks the confounding influences of other nucleoside transporters (PK15-NTD). We established that [ 3 H]2-chloroadenosine, which is resistant to metabolism by adenosine deaminase, is a substrate for hENT4. Transport of [ 3 H]2-chloroadenosine at a pH of 6.0 in PK15-NTD cells stably transfected with SLC29A4 was biphasic, with a low capacity (V max ~ 30 pmol/mg/min) high-affinity component (K m ~ 50 µM) apparent at low substrate concentrations, which shifted to a high capacity (V max ~ 500 pmol/mg/min) low affinity system (K m  > 600 µM) displaying positive cooperativity at concentrations above 200 µM. Only the low affinity component was observed at a neutral pH of 7.5 (K m ~ 2 mM). Efflux of [ 3 H]2-chloroadenosine from these cells was also enhanced by more than 4-fold at an acidic pH. Enhanced influx and efflux of nucleosides by hENT4 under acidic conditions supports its potential as a therapeutic target in pathologies such as ischaemia-reperfusion injury.

Purine salvage is an essential function for all obligate parasitic protozoa studied to date and most are also capable of efficient uptake of preformed pyrimidines. Much progress has been made in the identification and characterisation of protozoan purine and pyrimidine transporters. While the genes encoding protozoan or metazoan pyrimidine transporters have yet to be identified, numerous purine transporters have now been cloned. All protozoan purine transporter-encoding genes characterised to date have been of the Equilibrative Nucleoside Transporter family conserved in a great variety of eukaryote organisms. However, these protozoan transporters have been shown to be sufficiently different from mammalian transporters to mediate selective uptake of therapeutic agents. Recent studies are increasingly addressing the structure and substrate recognition mechanisms of these vital transport proteins.