Patients and mice with deficiency in the SNARE protein SYNTAXIN-11 have a secondary B cell defect.

SYNTAXIN-11 (STX11) is a SNARE protein that mediates the fusion of cytotoxic granules with the plasma membrane at the immunological synapses of CD8 T or NK cells. Autosomal recessive inheritance of deleterious STX11 variants impairs cytotoxic granule exocytosis, causing familial hemophagocytic lymphohistiocytosis type 4 (FHL-4). In several FHL-4 patients, we also observed hypogammaglobulinemia, elevated frequencies of naive B cells, and increased double-negative DN2:DN1 B cell ratios, indicating a hitherto unrecognized role of STX11 in humoral immunity. Detailed analysis of Stx11-deficient mice revealed impaired CD4 T cell help for B cells, associated with disrupted germinal center formation, reduced isotype class switching, and low antibody avidity. Mechanistically, Stx11-/- CD4 T cells exhibit impaired membrane fusion leading to reduced CD107a and CD40L surface mobilization and diminished IL-2 and IL-10 secretion. Our findings highlight a critical role of STX11 in SNARE-mediated membrane trafficking and vesicle exocytosis in CD4 T cells, important for successful CD4 T cell-B cell interactions. Deficiency in STX11 impairs CD4 T cell-dependent B cell differentiation and humoral responses.

Gene editing of hematopoietic stem cells restores T-cell response in familial hemophagocytic lymphohistiocytosis.

BACKGROUND: Hemophagocytic lymphohistiocytosis (HLH) is a hyperinflammatory disorder characterized by a life-threatening cytokine storm and immunopathology. Familial HLH type 3 (FHL3) accounts for approximately 30% of all inborn HLH cases worldwide. It is caused by mutations in the UNC13D gene that result in impaired degranulation of cytotoxic vesicles and hence compromised T-cell- and natural killer-cell-mediated killing. Current treatment protocols, including allogeneic hematopoietic stem cell (HSC) transplantation, still show high mortality. OBJECTIVE: We sought to develop and evaluate a curative genome editing strategy in the preclinical FHL3 Jinx mouse model. Jinx mice harbor a cryptic splice donor site in Unc13d intron 26 and develop clinical symptoms of human FHL3 upon infection with lymphocytic choriomeningitis virus (LCMV). METHODS: We employed clustered regularly interspaced short palindromic repeats (CRISPR)-Cas technology to delete the disease-causing mutation in HSCs and transplanted Unc13d-edited stem cells into busulfan-conditioned Jinx recipient mice. Safety studies included extensive genotyping and chromosomal aberrations analysis by single targeted linker-mediated PCR sequencing (CAST-Seq)-based off-target analyses. Cure from HLH predisposition was assessed by LCMV infection. RESULTS: Hematopoietic cells isolated from transplanted mice revealed efficient gene editing (>95%), polyclonality of the T-cell receptor repertoire, and neither signs of off-target effects nor leukemogenesis. Unc13d transcription levels of edited and wild-type cells were comparable. While LCMV challenge resulted in acute HLH in Jinx mice transplanted with mock-edited HSCs, Jinx mice grafted with Unc13d-edited cells showed rapid virus clearance and protection from HLH. CONCLUSIONS: Our study demonstrates that transplantation of CRISPR-Cas edited HSCs supports the development of a functional polyclonal T-cell response in the absence of genotoxicity-associated clonal outgrowth.

Optimizing human α-galactosidase for treatment of Fabry disease.

Fabry disease is caused by a deficiency of α-galactosidase A (GLA) leading to the lysosomal accumulation of globotriaosylceramide (Gb3) and other glycosphingolipids. Fabry patients experience significant damage to the heart, kidney, and blood vessels that can be fatal. Here we apply directed evolution to generate more stable GLA variants as potential next generation treatments for Fabry disease. GLAv05 and GLAv09 were identified after screening more than 12,000 GLA variants through 8 rounds of directed evolution. Both GLAv05 and GLAv09 exhibit increased stability at both lysosomal and blood pH, stability to serum, and elevated enzyme activity in treated Fabry fibroblasts (19-fold) and GLA-/- podocytes (10-fold). GLAv05 and GLAv09 show improved pharmacokinetics in mouse and non-human primates. In a Fabry mouse model, the optimized variants showed prolonged half-lives in serum and relevant tissues, and a decrease of accumulated Gb3 in heart and kidney. To explore the possibility of diminishing the immunogenic potential of rhGLA, amino acid residues in sequences predicted to bind MHC II were targeted in late rounds of GLAv09 directed evolution. An MHC II-associated peptide proteomics assay confirmed a reduction in displayed peptides for GLAv09. Collectively, our findings highlight the promise of using directed evolution to generate enzyme variants for more effective treatment of lysosomal storage diseases.

IFNγ binding to extracellular matrix prevents fatal systemic toxicity.

Interferon-γ (IFNγ) is an important mediator of cellular immune responses, but high systemic levels of this cytokine are associated with immunopathology. IFNγ binds to its receptor (IFNγR) and to extracellular matrix (ECM) via four positively charged C-terminal amino acids (KRKR), the ECM-binding domain (EBD). Across evolution, IFNγ is not well conserved, but the EBD is highly conserved, suggesting a critical function. Here, we show that IFNγ lacking the EBD (IFNγΔKRKR) does not bind to ECM but still binds to the IFNγR and retains bioactivity. Overexpression of IFNγΔKRKR in tumors reduced local ECM binding, increased systemic levels and induced sickness behavior, weight loss and toxicity. To analyze the function of the EBD during infection, we generated IFNγΔKRKR mice lacking the EBD by using CRISPR-Cas9. Infection with lymphocytic choriomeningitis virus resulted in higher systemic IFNγΔKRKR levels, enhanced sickness behavior, weight loss and fatal toxicity. We conclude that local retention of IFNγ is a pivotal mechanism to protect the organism from systemic toxicity during prolonged immune stimulation.

Repurposing the aldose reductase inhibitor and diabetic neuropathy drug epalrestat for the congenital disorder of glycosylation PMM2-CDG.

Phosphomannomutase 2 deficiency, or PMM2-CDG, is the most common congenital disorder of glycosylation and affects over 1000 patients globally. There are no approved drugs that treat the symptoms or root cause of PMM2-CDG. To identify clinically actionable compounds that boost human PMM2 enzyme function, we performed a multispecies drug repurposing screen using a novel worm model of PMM2-CDG, followed by PMM2 enzyme functional studies in PMM2-CDG patient fibroblasts. Drug repurposing candidates from this study, and drug repurposing candidates from a previously published study using yeast models of PMM2-CDG, were tested for their effect on human PMM2 enzyme activity in PMM2-CDG fibroblasts. Of the 20 repurposing candidates discovered in the worm-based phenotypic screen, 12 were plant-based polyphenols. Insights from structure-activity relationships revealed epalrestat, the only antidiabetic aldose reductase inhibitor approved for use in humans, as a first-in-class PMM2 enzyme activator. Epalrestat increased PMM2 enzymatic activity in four PMM2-CDG patient fibroblast lines with genotypes R141H/F119L, R141H/E139K, R141H/N216I and R141H/F183S. PMM2 enzyme activity gains ranged from 30% to 400% over baseline, depending on genotype. Pharmacological inhibition of aldose reductase by epalrestat may shunt glucose from the polyol pathway to glucose-1,6-bisphosphate, which is an endogenous stabilizer and coactivator of PMM2 homodimerization. Epalrestat is a safe, oral and brain penetrant drug that was approved 27 years ago in Japan to treat diabetic neuropathy in geriatric populations. We demonstrate that epalrestat is the first small molecule activator of PMM2 enzyme activity with the potential to treat peripheral neuropathy and correct the underlying enzyme deficiency in a majority of pediatric and adult PMM2-CDG patients.

Yeast Models of Phosphomannomutase 2 Deficiency, a Congenital Disorder of Glycosylation.

Phosphomannomutase 2 Deficiency (PMM2-CDG) is the most common monogenic congenital disorder of glycosylation (CDG) affecting at least 800 patients globally. PMM2 orthologs are present in model organisms, including the budding yeast Saccharomyces cerevisiae gene SEC53 Here we describe conserved genotype-phenotype relationships across yeast and human patients between five PMM2 loss-of-function missense mutations and their orthologous SEC53 mutations. These alleles range in severity from folding defective (hypomorph) to dimerization defective (severe hypomorph) to catalytic dead (null). We included the first and second most common missense mutations - R141H, F119L respectively- and the most common compound heterozygote genotype - PMM2R141H/F119L - observed in PMM2-CDG patients. Each mutation described is expressed in haploid as well as homozygous and heterozygous diploid yeast cells at varying protein expression levels as either SEC53 protein variants or PMM2 protein variants. We developed a 384-well-plate, growth-based assay for use in a screen of the 2,560-compound Microsource Spectrum library of approved drugs, experimental drugs, tool compounds and natural products. We identified three compounds that suppress growth defects of SEC53 variants, F126L and V238M, based on the biochemical defect of the allele, protein abundance or ploidy. The rare PMM2 E139K protein variant is fully functional in yeast cells, suggesting that its pathogenicity in humans is due to the underlying DNA mutation that results in skipping of exon 5 and a nonfunctional truncated protein. Together, these results demonstrate that yeast models can be used to characterize known and novel PMM2 patient alleles in quantitative growth and enzymatic activity assays, and used as patient avatars for PMM2-CDG drug screens yielding compounds that could be rapidly cross-validated in zebrafish, rodent and human organoid models.

The Yeast DNA Damage Checkpoint Kinase Rad53 Targets the Exoribonuclease, Xrn1.

The highly conserved DNA damage response (DDR) pathway monitors the genomic integrity of the cell and protects against genotoxic stresses. The apical kinases, Mec1 and Tel1 (ATR and ATM in human, respectively), initiate the DNA damage signaling cascade through the effector kinases, Rad53 and Chk1, to regulate a variety of cellular processes including cell cycle progression, DNA damage repair, chromatin remodeling, and transcription. The DDR also regulates other cellular pathways, but direct substrates and mechanisms are still lacking. Using a mass spectrometry-based phosphoproteomic screen in Saccharomyces cerevisiae, we identified novel targets of Rad53, many of which are proteins that are involved in RNA metabolism. Of the 33 novel substrates identified, we verified that 12 are directly phosphorylated by Rad53 in vitro: Xrn1, Gcd11, Rps7b, Ded1, Cho2, Pus1, Hst1, Srv2, Set3, Snu23, Alb1, and Scp160. We further characterized Xrn1, a highly conserved 5' exoribonuclease that functions in RNA degradation and the most enriched in our phosphoproteomics screen. Phosphorylation of Xrn1 by Rad53 does not appear to affect Xrn1's intrinsic nuclease activity in vitro, but may affect its activity or specificity in vivo.

Meiotic crossover control by concerted action of Rad51-Dmc1 in homolog template bias and robust homeostatic regulation.

During meiosis, repair of programmed DNA double-strand breaks (DSBs) by recombination promotes pairing of homologous chromosomes and their connection by crossovers. Two DNA strand-exchange proteins, Rad51 and Dmc1, are required for meiotic recombination in many organisms. Studies in budding yeast imply that Rad51 acts to regulate Dmc1's strand exchange activity, while its own exchange activity is inhibited. However, in a dmc1 mutant, elimination of inhibitory factor, Hed1, activates Rad51's strand exchange activity and results in high levels of recombination without participation of Dmc1. Here we show that Rad51-mediated meiotic recombination is not subject to regulatory processes associated with high-fidelity chromosome segregation. These include homolog bias, a process that directs strand exchange between homologs rather than sister chromatids. Furthermore, activation of Rad51 does not effectively substitute for Dmc1's chromosome pairing activity, nor does it ensure formation of the obligate crossovers required for accurate homolog segregation. We further show that Dmc1's dominance in promoting strand exchange between homologs involves repression of Rad51's strand-exchange activity. This function of Dmc1 is independent of Hed1, but requires the meiotic kinase, Mek1. Hed1 makes a relatively minor contribution to homolog bias, but nonetheless this is important for normal morphogenesis of synaptonemal complexes and efficient crossing-over especially when DSB numbers are decreased. Super-resolution microscopy shows that Dmc1 also acts to organize discrete complexes of a Mek1 partner protein, Red1, into clusters along lateral elements of synaptonemal complexes; this activity may also contribute to homolog bias. Finally, we show that when interhomolog bias is defective, recombination is buffered by two feedback processes, one that increases the fraction of events that yields crossovers, and a second that we propose involves additional DSB formation in response to defective homolog interactions. Thus, robust crossover homeostasis is conferred by integrated regulation at initiation, strand-exchange and maturation steps of meiotic recombination.

Native/Denaturing two-dimensional DNA electrophoresis and its application to the analysis of recombination intermediates.

Two-dimensional (2D) gel electrophoresis employs distinct electrophoretic conditions to better resolve complex mixtures of molecules. In combination with Southern analysis, 2D agarose gel electrophoresis is routinely employed to detect and analyze DNA intermediates that arise during the replication and repair of chromosomes. By separating intermediates into their component single-strands, native/denaturing 2D gels can reveal structure that is not apparent under native conditions alone. Here, we describe a general method for native/denaturing two-dimensional gel electrophoresis and its application to understanding the DNA strand-composition of recombination intermediates formed during meiosis.

A new protein complex promoting the assembly of Rad51 filaments.

During homologous recombination, eukaryotic RecA homologue Rad51 assembles into a nucleoprotein filament on single-stranded DNA to catalyse homologous pairing and DNA-strand exchange with a homologous template. Rad51 nucleoprotein filaments are highly dynamic and regulated via the coordinated actions of various accessory proteins including Rad51 mediators. Here, we identify a new Rad51 mediator complex. The PCSS complex, comprising budding yeast Psy3, Csm2, Shu1 and Shu2 proteins, binds to recombination sites and is required for Rad51 assembly and function during meiosis. Within the hetero-tetramer, Psy3-Csm2 constitutes a core sub-complex with DNA-binding activity. In vitro, purified Psy3-Csm2 stabilizes the Rad51-single-stranded DNA complex independently of nucleotide cofactor. The mechanism of Rad51 stabilization is inferred by our high-resolution crystal structure, which reveals Psy3-Csm2 to be a structural mimic of the Rad51-dimer, a fundamental unit of the Rad51-filament. Together, these results reveal a novel molecular mechanism for this class of Rad51-mediators, which includes the human Rad51 paralogues.

Stabilization and electrophoretic analysis of meiotic recombination intermediates in Saccharomyces cerevisiae.

Joint Molecule (JM) recombination intermediates result from DNA strand-exchange between homologous chromosomes. Physical monitoring of JM formation in budding yeast has provided a wealth of information about the timing and mechanism of meiotic recombination. These assays are especially informative when applied to the analysis of mutants for which genetic analysis of recombination is impossible, i.e. mutants that die during meiosis. This chapter describes three distinct methods to stabilize JMs against thermally driven dissolution as well as electrophoretic approaches to resolve and detect JMs at two well-characterized recombination hotspots.

RecQ helicase, Sgs1, and XPF family endonuclease, Mus81-Mms4, resolve aberrant joint molecules during meiotic recombination.

Saccharomyces cerevisiae RecQ helicase, Sgs1, and XPF family endonuclease, Mus81-Mms4, are implicated in processing joint molecule (JM) recombination intermediates. We show that cells lacking either enzyme frequently experience chromosome segregation problems during meiosis and that when both enzymes are absent attempted segregation fails catastrophically. In all cases, segregation appears to be impeded by unresolved JMs. Analysis of the DNA events of recombination indicates that Sgs1 limits aberrant JM structures that result from secondary strand-invasion events and often require Mus81-Mms4 for their normal resolution. Aberrant JMs contain high levels of single Holliday junctions and include intersister JMs, multichromatid JMs comprising three and four chromatids, and newly identified recombinant JMs containing two chromatids, one of which has undergone crossing over. Despite persistent JMs in sgs1 mms4 double mutants, crossover and noncrossover products still form at high levels. We conclude that Sgs1 and Mus81-Mms4 collaborate to eliminate aberrant JMs, whereas as-yet-unidentified enzymes process normal JMs.

Rad52 promotes postinvasion steps of meiotic double-strand-break repair.

During DNA double-strand-break (DSB) repair by recombination, the broken chromosome uses a homologous chromosome as a repair template. Early steps of recombination are well characterized: DSB ends assemble filaments of RecA-family proteins that catalyze homologous pairing and strand-invasion reactions. By contrast, the postinvasion steps of recombination are poorly characterized. Rad52 plays an essential role during early steps of recombination by mediating assembly of a RecA homolog, Rad51, into nucleoprotein filaments. The meiosis-specific RecA-homolog Dmc1 does not show this dependence, however. By exploiting the Rad52 independence of Dmc1, we reveal that Rad52 promotes postinvasion steps of both crossover and noncrossover pathways of meiotic recombination in Saccharomyces cerevisiae. This activity resides in the N-terminal region of Rad52, which can anneal complementary DNA strands, and is independent of its Rad51-assembly function. Our findings show that Rad52 functions in temporally and biochemically distinct reactions and suggest a general annealing mechanism for reuniting DSB ends during recombination.

BLM ortholog, Sgs1, prevents aberrant crossing-over by suppressing formation of multichromatid joint molecules.

Bloom's helicase (BLM) is thought to prevent crossing-over during DNA double-strand-break repair (DSBR) by disassembling double-Holliday junctions (dHJs) or by preventing their formation. We show that the Saccharomyces cerevisiae BLM ortholog, Sgs1, prevents aberrant crossing-over during meiosis by suppressing formation of joint molecules (JMs) comprising three and four interconnected duplexes. Sgs1 and procrossover factors, Msh5 and Mlh3, are antagonistic since Sgs1 prevents dHJ formation in msh5 cells and sgs1 mutation alleviates crossover defects of both msh5 and mlh3 mutants. We propose that differential activity of Sgs1 and procrossover factors at the two DSB ends effects productive formation of dHJs and crossovers and prevents multichromatid JMs and counterproductive crossing-over. Strand invasion of different templates by both DSB ends may be a common feature of DSBR that increases repair efficiency but also the likelihood of associated crossing-over. Thus, by disrupting aberrant JMs, BLM-related helicases maximize repair efficiency while minimizing the risk of deleterious crossing-over.