The high-affinity immunoglobulin receptor FcγRI potentiates HIV-1 neutralization via antibodies against the gp41 N-heptad repeat

The HIV-1 gp41 N-heptad repeat (NHR) region of the prehairpin intermediate, which is transiently exposed during HIV-1 viral membrane fusion, is a validated clinical target in humans and is inhibited by the Food and Drug Administration (FDA)-approved drug enfuvirtide. However, vaccine candidates targeting the NHR have yielded only modest neutralization activities in animals; this inhibition has been largely restricted to tier-1 viruses, which are most sensitive to neutralization by sera from HIV-1–infected individuals. Here, we show that the neutralization activity of the well-characterized NHR-targeting antibody D5 is potentiated >5,000-fold in TZM-bl cells expressing FcγRI compared with those without, resulting in neutralization of many tier-2 viruses (which are less susceptible to neutralization by sera from HIV-1–infected individuals and are the target of current antibody-based vaccine efforts). Further, antisera from guinea pigs immunized with the NHR-based vaccine candidate (ccIZN36)₃ neutralized tier-2 viruses from multiple clades in an FcγRI-dependent manner. As FcγRI is expressed on macrophages and dendritic cells, which are present at mucosal surfaces and are implicated in the early establishment of HIV-1 infection following sexual transmission, these results may be important in the development of a prophylactic HIV-1 vaccine.

Membrane fusion between HIV-1 and host cells is mediated by the viral envelope glycoprotein (Env), a trimer consisting of the gp120 and gp41 subunits. Upon interaction with cellular receptors, Env undergoes a dramatic conformational change and forms the prehairpin intermediate (PHI) (1–3), in which the fusion peptide region at the amino terminus of gp41 inserts into the cell membrane. In the PHI, the N-heptad repeat (NHR) region of gp41 is exposed and forms a stable, three-stranded α-helical coiled coil. Subsequently, the PHI resolves when the NHR and the C-heptad repeat (CHR) regions of gp41 associate to form a trimer-of-hairpins structure that brings the viral and cell membranes into proximity, facilitating membrane fusion (Fig. 1).Open in a separate windowFig. 1.HIV-1 membrane fusion. The surface protein of the HIV-1 envelope is composed of the gp120 and gp41 subunits. After Env binds to cell-surface receptors, gp41 inserts into the host cell membrane and undergoes a conformational change to form the prehairpin intermediate. The N-heptad repeat (orange) region of gp41 is exposed in the PHI and forms a three-stranded coiled coil. To complete viral fusion, the PHI resolves to a trimer-of-hairpins structure in which the C-heptad repeat (blue) adopts a helical conformation and binds the NHR region. Fusion inhibitors such as enfuvirtide bind the NHR, preventing viral fusion by inhibiting formation of the trimer of hairpins (1–3). The membrane-proximal external region (red) is located adjacent to the transmembrane (TM) region of gp41.The NHR region of the PHI is a validated therapeutic target in humans: the Food and Drug Administration (FDA)-approved drug enfuvirtide binds the NHR and inhibits viral entry into cells (4, 5). Various versions of the three-stranded coiled coil formed by the NHR have been created and used as vaccine candidates in animals (6–10). The neutralization potencies of these antisera, as well as those of anti-NHR monoclonal antibodies (mAbs) (11–15), are modest and mostly limited to HIV-1 isolates that are highly sensitive to antibody-mediated neutralization [commonly referred to as tier-1 viruses (16)]. These results have led to skepticism about the PHI as a vaccine target.Earlier studies showed that the neutralization activities of mAbs that bound another region of gp41, the membrane-proximal external region (MPER) (Fig. 1), were enhanced as much as 5,000-fold in cells expressing FcγRI (CD64) (17, 18), an integral membrane protein that binds the Fc portion of immunoglobulin G (IgG) molecules with high (nanomolar) affinity (19, 20). This effect was not attributed to phagocytosis and occurred when the cells were preincubated with antibody and washed before adding virus (17, 18). Since the MPER is a partially cryptic epitope that is not fully exposed until after Env engages with cellular receptors (21, 22), these results suggest that by binding the Fc region, FcγRI provides a local concentration advantage for MPER mAbs at the cell surface that enhances viral neutralization (17, 18). While not expressed on T cells, FcγRI is expressed on macrophages and dendritic cells (23), which are present at mucosal surfaces and are implicated in sexual HIV-1 transmission and the early establishment of HIV-1 infection (22–34).Here we investigated whether FcγRI expression also potentiates the neutralizing activity of antibodies targeting the NHR, since that region, like the MPER, is preferentially exposed during viral fusion. We found that D5, a well-characterized anti-NHR mAb (11, 12), inhibits HIV-1 infection ∼5,000-fold more potently in TZM-bl cells expressing FcγRI (TZM-bl/FcγRI cells) than in TZM-bl cells that do not. Further, while antisera from guinea pigs immunized with (ccIZN36)₃, an NHR-based vaccine candidate (7), displayed weak neutralizing activity in TZM-bl cells, they exhibited enhanced neutralization in TZM-bl/FcγRI cells, including against some tier-2 HIV-1 isolates that are more resistant to antibody-mediated neutralization (16) and that serve as benchmarks for antibody-based vaccine efforts. These results indicate that FcγRI can play an important role in neutralization by antibodies that target the PHI. Since these receptors are expressed on cells prevalent at mucosal surfaces thought to be important for sexual HIV-1 transmission, our results motivate vaccine strategies that harness this potentiating effect.     

Mutational fitness landscapes reveal genetic and structural improvement pathways for a vaccine-elicited HIV-1 broadly neutralizing antibody

Vaccine-based elicitation of broadly neutralizing antibodies holds great promise for preventing HIV-1 transmission. However, the key biophysical markers of improved antibody recognition remain uncertain in the diverse landscape of potential antibody mutation pathways, and a more complete understanding of anti–HIV-1 fusion peptide (FP) antibody development will accelerate rational vaccine designs. Here we survey the mutational landscape of the vaccine-elicited anti-FP antibody, vFP16.02, to determine the genetic, structural, and functional features associated with antibody improvement or fitness. Using site-saturation mutagenesis and yeast display functional screening, we found that 1% of possible single mutations improved HIV-1 envelope trimer (Env) affinity, but generally comprised rare somatic hypermutations that may not arise frequently in vivo. We observed that many single mutations in the vFP16.02 Fab could enhance affinity >1,000-fold against soluble FP, although affinity improvements against the HIV-1 trimer were more measured and rare. The most potent variants enhanced affinity to both soluble FP and Env, had mutations concentrated in antibody framework regions, and achieved up to 37% neutralization breadth compared to 28% neutralization of the template antibody. Altered heavy- and light-chain interface angles and conformational dynamics, as well as reduced Fab thermal stability, were associated with improved HIV-1 neutralization breadth and potency. We also observed parallel sets of mutations that enhanced viral neutralization through similar structural mechanisms. These data provide a quantitative understanding of the mutational landscape for vaccine-elicited FP-directed broadly neutralizing antibody and demonstrate that numerous antigen-distal framework mutations can improve antibody function by enhancing affinity simultaneously toward HIV-1 Env and FP.

The tremendous circulating sequence diversity of HIV-1 and its capacity to evade host immunity pose unique challenges for vaccine design (reviewed in refs. 1, 2). Broadly neutralizing antibodies (bNAbs) identified from HIV-1 patients target conserved vulnerable epitopes on the HIV-1 envelope protein (Env) to prevent HIV-1 infection, and HIV-1 bNAb elicitation has become a major goal for HIV-1 vaccine design (3, 4). Several HIV-1 vulnerable epitopes have been described (5) including the following: the V1V2 apex (6, 7), the CD4-binding site (8–10), the membrane-proximal external region (11, 12), the glycan-V3 region (also known as N332 glycan supersite) (10, 13), the highly glycosylated region at the center of the silent face on the gp120 subunit (14), and the fusion peptide (FP), which is required for viral entry (15–17). Several complementary approaches seek to develop immunogens that elicit broadly neutralizing HIV-1 antibodies, with promising results (4, 18, 19). However, vaccine-elicited HIV-1 antibodies are often either less potent or less broad than many of the bNAbs identified from human patients. There is a pressing need to better understand bNAb developmental pathways and outline the genetic and structural antibody features that can provide enhanced neutralization breadth and potency for HIV-1 vaccines.Clinical data show that broadly neutralizing serum antibodies develop naturally in around 20% of individuals with chronic HIV-1 infection (20, 21) and that a smaller number of individuals show highly potent neutralization (22, 23). bNAbs develop via the accumulation of somatic hypermutations (SHM) and affinity maturation following their initial B-cell selection and expansion. While HIV-1–infected individuals may have high titers against HIV-1 antigens, the rarity of bNAbs suggests that the mutations acquired during bNAb development are correspondingly rare and/or that the mutational pathways to effective bNAb development are explored only in the context of chronic HIV-1 antigen exposure over years of viral infection (24). Evidence that high viral load is correlated with higher HIV-1 serum breadth and potency also suggests that larger numbers of sampled antibody maturation pathways are correlated with broad HIV-1 neutralization (20). A major question for HIV-1 vaccine design is thus how to quickly and effectively induce B-cell maturation to bNAbs from a small number of controlled immunizations.HIV-1 bNAbs are often highly somatically mutated (25–28), and reverting these mutations to antibody germline sequences results in drastic reductions of neutralization breadth and potency (9, 29–31). Complementarity determining regions (CDRs) are known to be mutational hotspots and are often in direct contact with antigen to enable recognition. Antibody framework region (FR) mutations can also modulate neutralization breadth and potency by altering the structural orientation, particularly at heavy:light interface alignments, and by altering the intrinsic flexibility of the paratope (16, 32–35). Not all observed bNAb somatic mutations are required for high neutralization breadth and potency (36), and a better understanding of the critical mutations and antibody structural features (both naturally elicited and vaccine-induced) to improve neutralization breadth and potency would enhance our understanding of bNAb development and guide efficient HIV-1 vaccine strategies (16, 17, 37–41).Among various immunization approaches reported for eliciting broadly neutralizing antibodies (16–19, 38–42), one strategy targets the HIV-1 FP epitope and has elicited 59 and 31% HIV-1 neutralization breadth in rhesus macaques and mice, respectively, against a broad panel of 208 HIV-1 strains (16, 17). Priming the immune response with soluble FP followed by three Env trimer boosts elicited antibodies with FP-targeted trimer recognition and broad HIV-1 neutralization; however, the structural mechanisms that enable effective anti-FP antibody maturation and pathway selection are not fully understood. Several key unanswered questions include the following: 1) What are the structural features of FP recognition vs. trimer recognition that may appropriately guide antibody development?; 2) What critical parameters control virus neutralization of FP-targeting bNAbs?; and 3) How do beneficial mutations fit into the entire landscape of possible mutational pathways, and what fraction of possible mutations provide increased affinity, neutralization breadth, and potency?To address these questions, we characterized the genetic and functional fitness landscape of an anti-FP bNAb and used biophysical and structural techniques to follow the mechanisms of antibody improvement. We implemented these screening strategies using the anti-FP bNAb vFP16.02, a vaccine-elicited antibody that neutralizes ∼30% of HIV-1 viral isolates (16). We applied yeast display and fluorescence-activated cell sorting (FACS) coupled with next-generation sequencing (NGS) to identify single mutations that enhanced binding or fitness to multiple HIV-1 BG505 Env trimer variants (Fig. 1). We mapped possible single mutations by their functional impacts and identified a panel of mutations with enhanced binding affinity and neutralization. Structural analyses of a subset of these mutations provided insights into the mechanisms of enhanced neutralization. These data confirm that several parallel mutational pathways exist for HIV-1 bNAb improvement and underscore the importance of improved Env trimer affinity for enhancing neutralization potency and breadth in HIV-1 vaccine designs.Open in a separate windowFig. 1.Experimental workflow for comprehensive functional analysis of all possible single mutations in an HIV-1 bNAb. SSM libraries were designed for VH and VL regions of the anti–HIV-1 FP bNAb vFP16.02. SSM libraries were cloned into yeast Fab surface display libraries and screened by FACS to determine the functional impact of each possible single mutation. Single-mutation display libraries were sorted for their affinity against a BG505 DS-SOSIP HIV-1 Env trimer with two different FP sequences. Sorted yeast libraries were prepped for NGS to enable quantitative variant tracking across three screening rounds; bioinformatic analyses of NGS data were used to interpret the functional impact of each possible amino acid mutation. Selected variants were characterized for neutralization activity and affinity against soluble FP and against HIV-1 Env. Structural analyses were performed to understand the mechanistic basis of anti–HIV-1 FP bNAb improvement.     

Immature HIV-1 assembles from Gag dimers leaving partial hexamers at lattice edges as potential substrates for proteolytic maturation

The CA (capsid) domain of immature HIV-1 Gag and the adjacent spacer peptide 1 (SP1) play a key role in viral assembly by forming a lattice of CA hexamers, which adapts to viral envelope curvature by incorporating small lattice defects and a large gap at the site of budding. This lattice is stabilized by intrahexameric and interhexameric CA-CA interactions, which are important in regulating viral assembly and maturation. We applied subtomogram averaging and classification to determine the oligomerization state of CA at lattice edges and found that CA forms partial hexamers. These structures reveal the network of interactions formed by CA-SP1 at the lattice edge. We also performed atomistic molecular dynamics simulations of CA-CA interactions stabilizing the immature lattice and partial CA-SP1 helical bundles. Free energy calculations reveal increased propensity for helix-to-coil transitions in partial hexamers compared to complete six-helix bundles. Taken together, these results suggest that the CA dimer is the basic unit of lattice assembly, partial hexamers exist at lattice edges, these are in a helix-coil dynamic equilibrium, and partial helical bundles are more likely to unfold, representing potential sites for HIV-1 maturation initiation.

The polyprotein Gag is the main structural component of HIV-1, consisting of the MA (matrix), CA (capsid), NC (nucleocapsid), and p6 domains as well as the spacer peptides SP1 and SP2 (1). Gag is produced in infected host cells and trafficked to the plasma membrane, where it assembles into a hexagonal lattice via its CA domain and recruits other viral proteins and the viral RNA genome (1, 2). Assembly of the curved Gag lattice is commensurate with membrane bending at the site of assembly, after which recruitment of Endosomal Sorting Complex Required for Transport III (ESCRT-III) components by the p6 domain of Gag induces membrane scission and release of the immature virus particle (2). The hexagonal Gag lattice accommodates curvature in the growing bud by incorporating vacancy defects (3). The activity of ESCRT-III is timed such that the final immature lattice is incomplete, giving rise to an additional large gap in the lattice, resulting in a truncated spherical shape (4–6).During or after budding, the viral protease is activated and cleaves this immature Gag lattice into its component domains, which leads to structural rearrangement within the virus particle (2). The released CA domains assemble to form a closed, conical capsid around the condensed ribonucleoprotein (RNP) complex of the mature virus (1, 7). Maturation is required for the virion to become infectious (1).Within the immature virus particle, the N-terminal domain of CA (CA_NTD) forms trimeric interactions linking three Gag hexamers while the C-terminal domain of CA (CA_CTD) forms dimeric interactions mediated by helix 9 of CA, linking two Gag hexamers together (8). The CA_CTD additionally forms intrahexamer interactions around the sixfold axis of the hexamer (8, 9). Amphipathic helices formed by the C-terminal residues of CA_CTD and the N-terminal residues of SP1 junction assemble into a six-helix bundle (6HB), thereby imposing hexagonal order on the CA domains, via classical knobs-in-holes packing mediated by exposed hydrophobic side chains, as also seen in coiled coils (9, 10). In combination, these relatively weak interactions give rise to a very dynamic, reversible assembly process that prevents the assembling lattice from becoming trapped in kinetically unfavorable states (11). This robust assembly behavior is consistent with icosahedral viruses (12–15). It is not surprising, therefore, that the energetics of Gag assembly are tightly controlled and highly dependent on scaffolding effects from the viral RNA and the membrane-interacting MA domain of Gag in order to ensure productive viral assembly (11, 16). Analysis of the diffusion pattern of fluorescently labeled Gag supports the notion that Gag is trafficked to the site of assembly as low-order multimers, although it is still unclear whether these are Gag dimers, trimers, or other multimeric forms of Gag (16, 17).The primary assembly unit of the Gag lattice remains largely unknown. We can identify two hypothetical ways in which the lattice could assemble. First, the lattice could grow by addition of Gag hexamers (or sets of six component monomers), such that the CA-SP1 junction is assembled within a hexameric 6HB at all positions in the lattice. In this case, interfaces between hexamers would be unoccupied at the edge of the lattice. From a purely energetic perspective, this appears most reasonable. Second, the lattice could form via addition of Gag dimers or Gag trimers (or equivalently from sets of either two or three component monomers). This would maintain, for example, the dimeric CA-CA interhexamer interactions but leave incomplete hexamers at the lattice edges, including unoccupied hexamer-forming interfaces along the CA-SP1 bundle. It additionally remains unclear whether the unoccupied Gag-Gag interfaces at the lattice edges are simply exposed, or whether they are stabilized by alternative conformations of individual domains or proteins, or by other binding partners. Understanding the structure of the edge of the immature Gag lattice therefore has implications for understanding the mechanism of virus assembly.Viral assembly, budding, and maturation are tightly linked, and disrupting the kinetics of any of these processes can give rise to defects in maturation and formation of noninfectious viral particles (1, 18, 19). The rate-limiting proteolytic cleavage site in the maturation process resides within the CA-SP1 6HB (20). Unfolding of the helical bundle is required to allow proteolytic cleavage to proceed (21–23), but the exact mechanism for protease access to this site is not known. The spatial localization of proteolytic processing within the context of the immature Gag lattice is relevant: Does the protease act on Gag within the lattice, or does it act on the edges of the Gag lattice, causing a cascade of lattice disruption? At the lattice edge, is the substrate for the protease with a 6HB or within an incomplete hexamer? Understanding the structure of the edge of the immature Gag lattice therefore has implications for understanding the mechanism of virus maturation.High-resolution immature Gag structures have previously been determined directly from purified viruses by cryo-electron tomography (cryo-ET) and subtomogram averaging (10). These structures represent an average hexamer within the immature lattice, with a full complement of six Gag hexamer neighbors. Here, we have applied subtomogram classification and averaging approaches to an existing immature virus dataset (10) in order to determine the structures of Gag assemblies at lattice edges. We also applied atomistic molecular dynamics simulations to assess the roles of the different CA-CA interactions in immature lattice stabilization and predict the properties of the structures we observe at lattice edges. Together, our results suggest that the basic unit of immature HIV-1 assembly is a Gag dimer and partial CA-SP1 helical bundles are present at the edges of the assembled lattice and may be substrates for initiation of maturation.     

SLFN11 promotes CDT1 degradation by CUL4 in response to replicative DNA damage,while its absence leads to synthetic lethality with ATR/CHK1 inhibitors

Schlafen-11 (SLFN11) inactivation in ∼50% of cancer cells confers broad chemoresistance. To identify therapeutic targets and underlying molecular mechanisms for overcoming chemoresistance, we performed an unbiased genome-wide RNAi screen in SLFN11-WT and -knockout (KO) cells. We found that inactivation of Ataxia Telangiectasia- and Rad3-related (ATR), CHK1, BRCA2, and RPA1 overcome chemoresistance to camptothecin (CPT) in SLFN11-KO cells. Accordingly, we validate that clinical inhibitors of ATR (M4344 and M6620) and CHK1 (SRA737) resensitize SLFN11-KO cells to topotecan, indotecan, etoposide, cisplatin, and talazoparib. We uncover that ATR inhibition significantly increases mitotic defects along with increased CDT1 phosphorylation, which destabilizes kinetochore-microtubule attachments in SLFN11-KO cells. We also reveal a chemoresistance mechanism by which CDT1 degradation is retarded, eventually inducing replication reactivation under DNA damage in SLFN11-KO cells. In contrast, in SLFN11-expressing cells, SLFN11 promotes the degradation of CDT1 in response to CPT by binding to DDB1 of CUL4^CDT2 E3 ubiquitin ligase associated with replication forks. We show that the C terminus and ATPase domain of SLFN11 are required for DDB1 binding and CDT1 degradation. Furthermore, we identify a therapy-relevant ATPase mutant (E669K) of the SLFN11 gene in human TCGA and show that the mutant contributes to chemoresistance and retarded CDT1 degradation. Taken together, our study reveals new chemotherapeutic insights on how targeting the ATR pathway overcomes chemoresistance of SLFN11-deficient cancers. It also demonstrates that SLFN11 irreversibly arrests replication by degrading CDT1 through the DDB1–CUL4^CDT2 ubiquitin ligase.

Schlafen-11 (SLFN11) is an emergent restriction factor against genomic instability acting by eliminating cells with replicative damage (1–6) and potentially acting as a tumor suppressor (6, 7). SLFN11-expressing cancer cells are consistently hypersensitive to a broad range of chemotherapeutic drugs targeting DNA replication, including topoisomerase inhibitors, alkylating agents, DNA synthesis, and poly(ADP-ribose) polymerase (PARP) inhibitors compared to SLFN11-deficient cancer cells, which are chemoresistant (1, 2, 4, 8–17). Profiling SLFN11 expression is being explored for patients to predict survival and guide therapeutic choice (8, 13, 18–24).The Cancer Genome Atlas (TCGA) and cancer cell databases demonstrate that SLFN11 mRNA expression is suppressed in a broad fraction of common cancer tissues and in ∼50% of all established cancer cell lines across multiple histologies (1, 2, 5, 8, 13, 25, 26). Silencing of the SLFN11 gene, like known tumor suppressor genes, is under epigenetic mechanisms through hypermethylation of its promoter region and activation of histone deacetylases (HDACs) (21, 23, 25, 26). A recent study in small-cell lung cancer patient-derived xenograft models also showed that SLFN11 gene silencing is caused by local chromatin condensation related to deposition of H3K27me3 in the gene body of SLFN11 by EZH2, a histone methyltransferase (11). Targeting epigenetic regulators is therefore an attractive combination strategy to overcome chemoresistance of SLFN11-deficient cancers (10, 25, 26). An alternative approach is to attack SLFN11-negative cancer cells by targeting the essential pathways that cells use to overcome replicative damage and replication stress. Along these lines, a prior study showed that inhibition of ATR (Ataxia Telangiectasia- and Rad3-related) kinase reverses the resistance of SLFN11-deficient cancer cells to PARP inhibitors (4). However, targeting the ATR pathway in SLFN11-deficient cells has not yet been fully explored.SLFN11 consists of two functional domains: A conserved nuclease motif in its N terminus and an ATPase motif (putative helicase) in its C terminus (2, 6). The N terminus nuclease has been implicated in the selective degradation of type II tRNAs (including those coding for ATR) and its nuclease structure can be derived from crystallographic analysis of SLFN13 whose N terminus domain is conserved with SLFN11 (27, 28). The C terminus is only present in the group III Schlafen family (24, 29). Its potential ATPase activity and relationship to chemosensitivity to DNA-damaging agents (3–5) imply that the ATPase/helicase of SLFN11 is involved specifically in DNA damage response (DDR) to replication stress. Indeed, inactivation of the Walker B motif of SLFN11 by the mutation E669Q suppresses SLFN11-mediated replication block (5, 30). In addition, SLFN11 contains a binding site for the single-stranded DNA binding protein RPA1 (replication protein A1) at its C terminus (3, 31) and is recruited to replication damage sites by RPA (3, 5). The putative ATPase activity of SLFN11 is not required for this recruitment (5) but is required for blocking the replication helicase complex (CMG-CDC45) and inducing chromatin accessibility at replication origins and promoter sites (5, 30). Based on these studies, our current model is that SLFN11 is recruited to “stressed” replication forks by RPA filaments formed on single-stranded DNA (ssDNA), and that the ATPase/helicase activity of SLFN11 is required for blocking replication progression and remodeling chromatin (5, 30). However, underlying mechanisms of how SLFN11 irreversibly blocks replication in DNA damage are still unclear.Increased RPA-coated ssDNA caused by DNA damage and replication fork stalling also triggers ATR kinase activation, promoting subsequent phosphorylation of CHK1, which transiently halts cell cycle progression and enables DNA repair (32). ATR inhibitors are currently in clinical development in combination with DNA replication damaging drugs (33, 34), such as topoisomerase I (TOP1) inhibitors, which are highly synergistic with ATR inhibitors in preclinical models (35). ATR inhibitors not only inhibit DNA repair, but also lead to unscheduled replication origin firing (36), which kills cancer cells (37, 38) by inducing genomic alterations due to faulty replication and mitotic catastrophe (33).The replication licensing factor CDT1 orchestrates the initiation of replication by assembling prereplication complexes (pre-RC) in G1-phase before cells enter S-phase (39). Once replication is started by loading and activation of the MCM helicase, CDT1 is degraded by the ubiquitin proteasomal pathway to prevent additional replication initiation and ensure precise genome duplication and the firing of each origin only once per cell cycle (39, 40). At the end of G2 and during mitosis, CDT1 levels rise again to control kinetochore-microtubule attachment for accurate chromosome segregation (41). Deregulated overexpression of CDT1 results in rereplication, genome instability, and tumorigenesis (42). The cellular CDT1 levels are tightly regulated by the damage-specific DNA binding protein 1 (DDB1)–CUL4^CDT2 E3 ubiquitin ligase complex in G1-phase (43) and in response to DNA damage (44, 45). How CDT1 is recognized by CUL4^CDT2 in response to DNA damage remains incompletely known.In the present study, starting with a human genome-wide RNAi screen, bioinformatics analyses, and mechanistic validations, we explored synthetic lethal interactions that overcome the chemoresistance of SLFN11-deficient cells to the TOP1 inhibitor camptothecin (CPT). The strongest synergistic interaction was between depletion of the ATR/CHK1-mediated DNA damage response pathways and DNA-damaging agents in SLFN11-deficient cells. We validated and expanded our molecular understanding of combinatorial strategies in SLFN11-deficient cells with the ATR (M4344 and M6620) and CHK1 (SRA737) inhibitors in clinical development (33, 46, 47) and found that ATR inhibition leads to CDT1 stabilization and hyperphosphorylation with mitotic catastrophe. Our study also establishes that SLFN11 promotes the degradation of CDT1 by binding to DDB1, an adaptor molecule of the CUL4^CDT2 E3 ubiquitin ligase complex, leading to an irreversible replication block in response to replicative DNA damage.     

Replisome bypass of a protein-based R-loop block by Pif1

Efficient and faithful replication of the genome is essential to maintain genome stability. Replication is carried out by a multiprotein complex called the replisome, which encounters numerous obstacles to its progression. Failure to bypass these obstacles results in genome instability and may facilitate errors leading to disease. Cells use accessory helicases that help the replisome bypass difficult barriers. All eukaryotes contain the accessory helicase Pif1, which tracks in a 5′–3′ direction on single-stranded DNA and plays a role in genome maintenance processes. Here, we reveal a previously unknown role for Pif1 in replication barrier bypass. We use an in vitro reconstituted Saccharomyces cerevisiae replisome to demonstrate that Pif1 enables the replisome to bypass an inactive (i.e., dead) Cas9 (dCas9) R-loop barrier. Interestingly, dCas9 R-loops targeted to either strand are bypassed with similar efficiency. Furthermore, we employed a single-molecule fluorescence visualization technique to show that Pif1 facilitates this bypass by enabling the simultaneous removal of the dCas9 protein and the R-loop. We propose that Pif1 is a general displacement helicase for replication bypass of both R-loops and protein blocks.

Efficient and faithful replication of the genome is essential to maintain genome stability and is carried out by a multiprotein complex called the replisome (1–4). There are numerous obstacles to progression of the replisome during the process of chromosome duplication. These obstacles include RNA-DNA hybrids (R-loops), DNA secondary structures, transcribing RNA polymerases, and other tightly bound proteins (5–9). Failure to bypass these barriers may result in genome instability, which can lead to cellular abnormalities and genetic disease. Cells contain various accessory helicases that help the replisome bypass these difficult barriers (10–20). A subset of these helicases act on the opposite strand of the replicative helicase (1, 2, 14, 19).All eukaryotes contain an accessory helicase, Pif1, which tracks in a 5′–3′ direction on single-stranded DNA (ssDNA) (11–16). Pif1 is important in pathways such as Okazaki-fragment processing and break-induced repair that require the removal of DNA-binding proteins as well as potential displacement of R-loops (11–13, 21, 15–18, 22–25). Genetic studies and immunoprecipitation pull-down assays indicate that Pif1 interacts with PCNA (the DNA sliding clamp), Pol ε (the leading-strand polymerase), the MCMs (the motor subunits of the replicative helicase CMG), and RPA (the single-stranded DNA-binding protein) (15, 26, 27). Pif1 activity in break-induced repair strongly depends on its interaction with PCNA (26). These interactions with replisomal components suggest that Pif1 could interact with the replisome during replication. In Escherichia coli, the replicative helicase is the DnaB homohexamer that encircles the lagging strand and moves in a 5′–3′ direction (20). E. coli accessory helicases include the monomeric UvrD (helicase II) and Rep, which move in the 3′–5′ direction and operate on the opposite strand from the DnaB hexamer. It is known that these monomeric helicases promote the bypass of barriers during replication such as stalled RNA polymerases (5). The eukaryotic replicative helicase is the 11-subunit CMG (Cdc45, Mcm2–7, GINS) and tracks in the 3′–5′ direction, opposite to the direction of Pif1 (25, 28). Once activated by Mcm10, the MCM motor domains of CMG encircle the leading strand (29–32). We hypothesized that, similar to UvrD and Rep in E. coli, Pif1 interacts with the replisome tracking in the opposite direction to enable bypass of replication obstacles.In this report, we use an in vitro reconstituted Saccharomyces cerevisiae replisome to study the role of Pif1 in bypass of a “dead” Cas9 (dCas9), which is a Cas9 protein that is deactivated in DNA cleavage but otherwise fully functional in DNA binding. As with Cas9, dCas9 is a single-turnover enzyme that can be programmed with a guide RNA (gRNA) to target either strand. The dCas9–gRNA complex forms a roadblock consisting of an R-loop and a tightly bound protein (dCas9), a construct that is similar to a stalled RNA polymerase. This roadblock (hereafter dCas9 R-loop) arrests replisomes independent of whether the dCas9 R-loop is targeted to the leading or lagging strand (30). Besides its utility due to its programmable nature (33), the use of the dCas9 R-loop allows us to answer several mechanistic questions. For example, the ability to program the dCas9 R-loop block to any specific sequence enables us to observe whether block removal is different depending on whether the block is on the leading or lagging strand. Furthermore, the inner diameter of CMG can accommodate double-stranded DNA (dsDNA) and possibly an R-loop, but not a dCas9 protein. Using the dCas9 R-loop block allows us to determine the fate of each of its components.Here, we report that Pif1 enables the bypass of the dCas9 R-loop by the replisome. Interestingly, dCas9 R-loops targeted to either the leading or lagging strand are bypassed with similar efficiency. In addition, the PCNA clamp is not required for bypass of the block, indicating that Pif1 does not need to interact with PCNA during bypass of the block. We used a single-molecule fluorescence imaging to show that both the dCas9 and the R-loop are displaced as an intact nucleoprotein complex. We propose that Pif1 is a general displacement helicase for replication bypass of both R-loops and protein blocks.     

CAP1 binds and activates adenylyl cyclase in mammalian cells

CAP1 (Cyclase-Associated Protein 1) is highly conserved in evolution. Originally identified in yeast as a bifunctional protein involved in Ras-adenylyl cyclase and F-actin dynamics regulation, the adenylyl cyclase component seems to be lost in mammalian cells. Prompted by our recent identification of the Ras-like small GTPase Rap1 as a GTP-independent but geranylgeranyl-specific partner for CAP1, we hypothesized that CAP1-Rap1, similar to CAP-Ras-cyclase in yeast, might play a critical role in cAMP dynamics in mammalian cells. In this study, we report that CAP1 binds and activates mammalian adenylyl cyclase in vitro, modulates cAMP in live cells in a Rap1-dependent manner, and affects cAMP-dependent proliferation. Utilizing deletion and mutagenesis approaches, we mapped the interaction of CAP1-cyclase with CAP’s N-terminal domain involving critical leucine residues in the conserved RLE motifs and adenylyl cyclase’s conserved catalytic loops (e.g., C1a and/or C2a). When combined with a FRET-based cAMP sensor, CAP1 overexpression–knockdown strategies, and the use of constitutively active and negative regulators of Rap1, our studies highlight a critical role for CAP1-Rap1 in adenylyl cyclase regulation in live cells. Similarly, we show that CAP1 modulation significantly affected cAMP-mediated proliferation in an RLE motif–dependent manner. The combined study indicates that CAP1-cyclase-Rap1 represents a regulatory unit in cAMP dynamics and biology. Since Rap1 is an established downstream effector of cAMP, we advance the hypothesis that CAP1-cyclase-Rap1 represents a positive feedback loop that might be involved in cAMP microdomain establishment and localized signaling.

CAP/srv2 was originally identified in yeast biochemically as an adenylyl cyclase–associated protein (1) and genetically as a suppressor of the hyperactive Ras2-V19 allele (2). CAP/srv2-deficient yeast cells are unresponsive to active Ras2, and adenylyl cyclase activity is no longer regulated by Ras2 in these cells (1, 2), indicating the involvement of CAP/srv2 in the Ras/cyclase pathway. However, some mutant CAP/srv2 alleles presented phenotypes not observed in strains with impaired Ras/cyclase pathway (1–3), indicating the existence of Ras/cyclase-independent functions downstream of CAP/srv2. These two phenotype groups, that is, Ras/cyclase-linked and Ras/cyclase-independent, could be suppressed by expression of an N-terminal half and a C-terminal half of CAP/srv2, respectively (4). Subsequent studies showed that the C-terminal half of CAP/srv2 was able to bind monomeric G-actin (5–8) and other actin regulators establishing a role in F-actin dynamics (9–16). Thus, CAP/srv2 is a bifunctional protein with an N-terminal domain involved in Ras/cyclase regulation and a C-terminal domain involved with F-actin dynamics regulation (16–18).CAP1 is structurally conserved in all eukaryotes (18–22); however, their functions are not. Expression of the closely related Schizosaccharomyces pombe cap or mammalian CAP1 in yeast can only suppress the phenotypes associated with deletion of CAP/srv2’s C-terminal but not its N-terminal domain (19, 20, 22), suggesting that only the F-actin dynamics function was conserved while the Ras/cyclase regulation diverged early on in evolution (16–18). CAP/srv2’s N-terminal 1 to 36 domain was sufficient for cyclase binding in yeast involving a conserved RLE motif with predicted coiled-coil folding (23). Interestingly, this domain is also involved in CAP1 oligomerization both in yeast and mammalian cells (24–26), where it purifies as a high-molecular complex of ∼600 kDa consistent with a 1:1 stoichiometric CAP1-actin hexameric organization (12, 25, 27, 28). Importantly, removal of this domain disrupted CAP1 oligomerization, reduced F-actin turnover in vitro and caused defects in cell growth, cell morphology, and F-actin organization in vivo (24, 29). However, whether the conserved RLE motif in mammalian CAP1 interacts with other coiled-coil–containing proteins is for the moment unknown.Ras2-mediated cyclase regulation in yeast requires its farnesylation (30–32). However, the lipid target involved was not identified in the original studies. We have recently shown that mammalian CAP1 interacts with the small GTPase Rap1. The interaction involves Rap1’s C-terminal hypervariable region (HVR) and its lipid moiety in a geranylgeranyl-specific manner; that is, neither the closely related Ras1 nor engineered farnesylated Rap1 interacted with CAP1 (33). Thus, we raised the question whether CAP1-Rap1, similar to CAP/srv2-Ras2 in yeast, plays a role in cAMP dynamics in mammalian cells.In this study, we report that CAP1 binds to and activates mammalian adenylyl cyclase in vitro. The interaction involves CAP1’s conserved RLE motifs and cyclase’s conserved catalytic subdomains (e.g., C1a and/or C2a). Most importantly, we show that both CAP1 and Rap1 modulate cAMP dynamics in live cells and are critical players in cAMP-dependent proliferation.     

Neuromodulator release in neurons requires two functionally redundant calcium sensors

Neuropeptides and neurotrophic factors secreted from dense core vesicles (DCVs) control many brain functions, but the calcium sensors that trigger their secretion remain unknown. Here, we show that in mouse hippocampal neurons, DCV fusion is strongly and equally reduced in synaptotagmin-1 (Syt1)- or Syt7-deficient neurons, but combined Syt1/Syt7 deficiency did not reduce fusion further. Cross-rescue, expression of Syt1 in Syt7-deficient neurons, or vice versa, completely restored fusion. Hence, both sensors are rate limiting, operating in a single pathway. Overexpression of either sensor in wild-type neurons confirmed this and increased fusion. Syt1 traveled with DCVs and was present on fusing DCVs, but Syt7 supported fusion largely from other locations. Finally, the duration of single DCV fusion events was reduced in Syt1-deficient but not Syt7-deficient neurons. In conclusion, two functionally redundant calcium sensors drive neuromodulator secretion in an expression-dependent manner. In addition, Syt1 has a unique role in regulating fusion pore duration.

To date, over 100 genes encoding neuropeptides and neurotrophic factors, together referred to as neuromodulators, are identified, and most neurons express neuromodulators and neuromodulator receptors (1). Neuromodulators travel through neurons in dense core vesicles (DCVs) and, upon secretion, regulate neuronal excitability, synaptic plasticity, and neurite outgrowth (2–4). Dysregulation of DCV secretion is linked to many brain disorders (5–7). However, the molecular mechanisms that regulate neuromodulator secretion remain largely elusive.Neuromodulator secretion, like neurotransmitter secretion from synaptic vesicles (SVs), is tightly controlled by Ca²⁺. The Ca²⁺ sensors that regulate secretion have been described for other secretory pathways but not for DCV exocytosis in neurons. Synaptotagmin (Syt) and Doc2a/b are good candidate sensors due to their interaction with SNARE complexes, phospholipids, and Ca²⁺ (8–11). The Syt family consists of 17 paralogs (12, 13). Eight show Ca²⁺-dependent lipid binding: Syt1 to 3, Syt5 to 7, and Syt9 and 10 (14, 15). Syt1 mediates synchronous SV fusion (8), consistent with its low Ca²⁺-dependent lipid affinity (15, 16) and fast Ca²⁺/membrane dissociation kinetics (16, 17). Syt1 is also required for the fast fusion in chromaffin cells (18) and fast striatal dopamine release (19). Synaptotagmin-7 (Syt7), in contrast, drives asynchronous SV fusion (20), in line with its a higher Ca²⁺ affinity (15) and slower dissociation kinetics (16). Syt7 is also a major calcium sensor for neuroendocrine secretion (21) and secretion in pancreatic cells (22–24). Other sensors include Syt4, which negatively regulates brain-derived neurothropic factor (25) and oxytocin release (26), in line with its Ca²⁺ independency. Syt9 regulates hormone secretion in the anterior pituitary (27) and, together with Syt1, secretion from PC12 cells (28, 29). Syt10 controls growth factor secretion (30). However, Syt9 and Syt10 expression is highly restricted in the brain (31–33). Hence, the calcium sensors for neuronal DCV fusion remain largely elusive. Because DCVs are generally not located close to Ca²⁺ channels (34), we hypothesized that DCV fusion is triggered by high-affinity Ca²⁺ sensors. Because of their important roles in vesicle secretion, their Ca²⁺ binding ability, and their high expression levels in the brain (20, 31, 35–38), we addressed the roles of Doc2a/b, Syt1, and Syt7 in neuronal DCV fusion.In this study, we used primary Doc2a/b-, Syt1-, and Syt7-null (knockout, KO) neurons expressing DCV fusion reporters (34, 39–41) with single-vesicle resolution. We show that both Syt1 and Syt7, but not Doc2a/b, are required for ∼60 to 90% of DCV fusion events. Deficiency of both Syt1 and Syt7 did not produce an additive effect, suggesting they function in the same pathway. Syt1 overexpression (Syt1-OE) rescued DCV fusion in Syt7-null neurons, and vice versa, indicating that the two proteins compensate for each other in DCV secretion. Moreover, overexpression of Syt1 or Syt7 in wild-type (WT) neurons increased DCV fusion, suggesting they are both rate limiting for DCV secretion. We conclude that DCV fusion requires two calcium sensors, Syt1 and Syt7, that act in a single/serial pathway and that both sensors regulate fusion in a rate-limiting and dose-dependent manner.     

Structure and activity of SLAC1 channels for stomatal signaling in leaves

Stomata in leaves regulate gas exchange between the plant and its atmosphere. Various environmental stimuli elicit abscisic acid (ABA); ABA leads to phosphoactivation of slow anion channel 1 (SLAC1); SLAC1 activity reduces turgor pressure in aperture-defining guard cells; and stomatal closure ensues. We used electrophysiology for functional characterizations of Arabidopsis thaliana SLAC1 (AtSLAC1) and cryoelectron microscopy (cryo-EM) for structural analysis of Brachypodium distachyon SLAC1 (BdSLAC1), at 2.97-Å resolution. We identified 14 phosphorylation sites in AtSLAC1 and showed nearly 330-fold channel-activity enhancement with 4 to 6 of these phosphorylated. Seven SLAC1-conserved arginines are poised in BdSLAC1 for regulatory interaction with the N-terminal extension. This BdSLAC1 structure has its pores closed, in a basal state, spring loaded by phenylalanyl residues in high-energy conformations. SLAC1 phosphorylation fine-tunes an equilibrium between basal and activated SLAC1 trimers, thereby controlling the degree of stomatal opening.

The stomatal pore, formed by a pair of specialized guard cells in plant leaves, serves as the gateway for water transpiration and atmospheric CO₂ influx for photosynthesis (1, 2). These pores need to be tightly controlled, as inadequate CO₂ intake is harmful and excessive water loss is devastating for plants (3). The guard cells respond to a wide range of environmental stimuli, including high CO₂ levels, O₃, low air humidity, and drought, and transduce these signals into appropriate turgor pressure changes that can adjust stomatal pore aperture (4, 5). It is well established that turgor pressure of guard cells is regulated by ion transport across the membrane, notably anions and potassium ions (6–8).The phytohormone abscisic acid (ABA) plays a central role in controlling stomatal closure via activation of a complex signaling pathway that is mediated by receptors, kinase/phosphatases, and ion channels (5, 9). It has been known that the activation of a slow anion current in guard cells is a key event leading to stomatal closure (10–13). The corresponding gene, slow anion channel 1 (SLAC1), was discovered in genetic screens for O₃ or CO₂ sensitivity in Arabidopsis; when this particular channel was mutated out, the mutants showed impaired response to stimuli (14–16).Arabidopsis thaliana SLAC1 (AtSLAC1) contains 556 amino acid (aa) residues, with a conserved transmembrane domain (TMD) between hydrophilic tails at both the N and C termini (15). Within the ABA signaling pathway, SLAC1 channel activity is controlled by a protein kinase-phosphatase pair (OST1/ABI1) (17–19). In the absence of ABA, OST1 kinase is bound and inhibited by ABI1 phosphatase (20–22). Under drought condition, ABA is accumulated into guard cells and perceived by the PYR1 receptor (23, 24). This resulting hormone–receptor complex then binds to phosphatase ABI1 to form a ternary hormone–receptor–phosphatase complex (25–28), which activates OST1 kinase, and leads to phosphorylation of SLAC1 (17, 18). Subsequently, SLAC1 releases anions (Cl⁻ and NO₃⁻) from guard cells, leading to membrane depolarization. Depolarization in turn activates potassium channels to release K⁺ via K⁺ channels and drives water out of the cell, decreasing guard cell turgor to close down the stomatal pore (8).It has been shown that SLAC1 activation is due to phosphorylation at its N terminus (17, 18, 29–31). The phosphorylation of S120 by OST1 is essential but not sufficient for SLAC1 activation monitored in Xenopus oocytes (18). S59 has been shown to be the target for both OST1 and other kinases (29, 31). In another phosphoproteomic analysis, using the fragment SLAC1¹⁻¹⁸⁶ as substrate, S86 and S113 were also shown to be phosphorylated by OST1 (32). OST1 also phosphorylates SLAC1 at the C terminus (17), with T513 identified as a potential phosphorylation site for SLAC1 activation (31). Although multiple phosphorylation sites have been proposed, the underlying mechanism for SLAC1 activation remains elusive.Our previous study on a SLAC1 bacterial homolog from Haemophilus influenzae (HiTehA) revealed a superfamily of trimeric anion channels (33) and found each protomer gated by a highly conserved phenylalanine. We observed an unusual high-energy rotameric conformation for this gating phenylalanine (F262 in HiTehA), and further mutational study on the corresponding site in AtSLAC1 (F450) suggested a unique mechanism for channel activation (33). Nevertheless, since HiTehA shares a mere 19% in sequence identity with AtSLAC1 and lacks the substantial N and C termini that are essential for channel activation, questions remain about SLAC1 structure and its regulation by kinase phosphorylation (7).Here we describe the cryoelectron microscopy (cryo-EM) structure of SLAC1 from Brachypodium distachyon (BdSLAC1) at 2.97-Å resolution, and we further characterize the channel activity on Arabidopsis SLAC1. BdSLAC1 shares 63% overall sequence identity with AtSLAC1, with 73% for the pore-forming TMD and 48% for the regulatory N and C termini. As for the bacterial homolog, each BdSLAC1 TMD comprises five tandemly repeated helical hairpins that surround a transmembrane pore in each protomer of the trimeric assembly. The SLAC1 pores are electropositive, consistent with its function as an anion channel. The regulatory N-terminal domain (190 aa) and C-terminal domain (65 aa) are flexibly associated and not discernible in the structure.We employed a SLAC1::OST1 fusion design for examining SLAC1 phosphorylation, whereby we systematically characterized critical sites responsible for channel activation. Inspired by the structure, we also conducted mutagenesis on the highly conserved phenylalanine residues related to channel gating. Altogether, our structural and functional analyses provide insights into SLAC1 gating and activity modulation. These findings allow us to propose a mechanism for finely tuned SLAC1 control of the stomatal pore in response to environmental stimuli.     

A limited role of NKCC1 in telencephalic glutamatergic neurons for developing hippocampal network dynamics and behavior

NKCC1 is the primary transporter mediating chloride uptake in immature principal neurons, but its role in the development of in vivo network dynamics and cognitive abilities remains unknown. Here, we address the function of NKCC1 in developing mice using electrophysiological, optical, and behavioral approaches. We report that NKCC1 deletion from telencephalic glutamatergic neurons decreases in vitro excitatory actions of γ-aminobutyric acid (GABA) and impairs neuronal synchrony in neonatal hippocampal brain slices. In vivo, it has a minor impact on correlated spontaneous activity in the hippocampus and does not affect network activity in the intact visual cortex. Moreover, long-term effects of the developmental NKCC1 deletion on synaptic maturation, network dynamics, and behavioral performance are subtle. Our data reveal a neural network function of NKCC1 in hippocampal glutamatergic neurons in vivo, but challenge the hypothesis that NKCC1 is essential for major aspects of hippocampal development.

Intracellular chloride concentration ([Cl⁻]_i) is a major determinant of neuronal excitability, as synaptic inhibition is primarily mediated by chloride-permeable receptors (1). In the mature brain, [Cl⁻]_i is maintained at low levels by chloride extrusion, which renders γ-aminobutyric acid (GABA) hyperpolarizing (2) and counteracts activity-dependent chloride loads (3). GABAergic inhibition in the adult is crucial not only for preventing runaway excitation of glutamatergic cells (4) but also for entraining neuronal assemblies into oscillations underlying cognitive processing (5). However, the capacity of chloride extrusion is low during early brain development (6, 7). Additionally, immature neurons are equipped with chloride uptake mechanisms, particularly with the Na⁺/K⁺/2Cl⁻ cotransporter NKCC1 (8–12). NKCC1 contributes to the maintenance of high [Cl⁻]_i in the developing brain (13), favoring depolarization through GABA_A receptor (GABA_AR) activation in vivo (14, 15).When GABA acts as a depolarizing neurotransmitter, neural circuits generate burst-like spontaneous activity (16–20), which is crucial for their developmental refinement (21–24). In vitro evidence indicates that GABAergic interneurons promote neuronal synchrony in an NKCC1-dependent manner (10, 12, 25–28). However, the in vivo developmental functions of NKCC1 are far from understood (29, 30). One fundamental question is to what extent NKCC1 and GABAergic depolarization supports correlated spontaneous activity in the neonatal brain. In the neocortex, GABA imposes spatiotemporal inhibition on network activity already in the neonatal period (14, 25, 31, 32). Whether a similar situation applies to other brain regions is unknown, as two recent chemo- and optogenetic studies in the hippocampus yielded opposing results (25, 33). Manipulations of the chloride driving force are potentially suited to resolve these divergent findings, but pharmacological (34–36) or conventional knockout (10, 11, 37) strategies suffer from unspecific effects that complicate interpretations.Here, we overcome this limitation by selectively deleting Slc12a2 (encoding NKCC1) from telencephalic glutamatergic neurons. We show that chloride uptake via NKCC1 promotes synchronized activity in acute hippocampal slices, but has weak and event type-dependent effects in CA1 in vivo. Long-term loss of NKCC1 leads to subtle changes of network dynamics in the adult, leaving synaptic development unperturbed and behavioral performance intact. Our data suggest that NKCC1-dependent chloride uptake is largely dispensable for several key aspects of hippocampal development in vivo.