The rockefeller university

Previously we introduced peptidiscs as an alternative to detergents to stabilize membrane proteins in solution (Carlson et al., 2018). Here, we present 'on-gradient' reconstitution, a new gentle approach for the reconstitution of labile membrane-protein complexes, and used it to reconstitute Rhodobacter sphaeroides reaction center complexes, demonstrating that peptidiscs can adapt to transmembrane domains of very different sizes and shapes. Using the conventional 'on-bead' approach, we reconstituted Escherichia coli proteins MsbA and MscS and find that peptidiscs stabilize them in their native conformation and allow for high-resolution structure determination by cryo-electron microscopy. The structures reveal that peptidisc peptides can arrange around transmembrane proteins differently, thus revealing the structural basis for why peptidiscs can stabilize such a large variety of membrane proteins. Together, our results establish the gentle and easy-to-use peptidiscs as a potentially universal alternative to detergents as a means to stabilize membrane proteins in solution for structural and functional studies.

Objective Long interspersed nuclear element 1 (LINE-1) encodes 2 proteins, the RNA binding protein p40 and endonuclease and reverse transcriptase (open-reading frame 2p [ORF2p]), which are both required for LINE-1 to retrotranspose. In cells expressing LINE-1, these proteins assemble with LINE-1 RNA and additional RNA binding proteins, some of which are well-known autoantigens in patients with systemic lupus erythematosus (SLE). This study was undertaken to investigate whether SLE patients also produce autoantibodies against LINE-1 p40. Methods Highly purified p40 protein was used to quantitate IgG autoantibodies in serum from 172 SLE patients and from disease controls and age-matched healthy subjects by immunoblotting and enzyme-linked immunosorbent assay (ELISA). Preparations of p40 that also contained associated proteins were analyzed by immunoblotting with patient sera. Results Antibodies reactive with p40 were detected in the majority of patients and many healthy controls. Their levels were higher in patients with SLE, but not those with systemic sclerosis, compared to healthy subjects (P = 0.01). Anti-p40 reactivity was higher in patients during a flare than in patients with disease in remission (P = 0.03); correlated with the SLE Disease Activity Index score (P = 0.0002), type I interferon score (P = 0.006), decrease in complement C3 level (P = 0.0001), the presence of anti-DNA antibodies (P < 0.0001) and anti-C1q antibodies (P = 0.004), and current or past history of nephritis (P = 0.02 and P = 0.003, respectively); and correlated inversely with age (r = -0.49, P < 0.0001). SLE patient sera also reacted with p40-associated proteins. Conclusion Autoantibodies reacting with LINE-1 p40 characterize a population of SLE patients with severe and active disease. These autoantibodies may represent an early immune response against LINE-1 p40 that does not yet by itself imply clinically significant autoimmunity, but may represent an early, and still reversible, step toward SLE pathogenesis.

T-loops are thought to hide telomeres from DNA damage signaling and DSB repair pathways. T-loop formation requires the shelterin component TRF2, which represses ATM signaling and NHEJ. Here we establish that TRF2 alone, in the absence of other shelterin proteins can form t-loops. Mouse and human cells contain two isoforms of TRF2, one of which is uncharacterized. We show that both isoforms protect telomeres and form t-loops. The isoforms are not cell cycle regulated and t-loops are present in G1, S, and G2. Using the DNA wrapping deficient TRF2 Topless mutant, we confirm its inability to form t-loops and repress ATM. However, since the mutant is also defective in repression of NHEJ and telomeric localization, the role of topological changes in telomere protection remains unclear. Finally, we show that Rad51 does not affect t-loop frequencies or telomere protection. Therefore, alternative models for how TRF2 forms t-loops should be explored.

Background: As CRH-binding protein (CRHBP) SNP rs1500 was associated with reduced cocaine abuse after 1 year in methadone maintenance treatment (MMT) for heroin addiction, we evaluated the association of additional 28 selected SNPs, in 17 stress-related genes, with MMT outcome. Methods: The distribution of genotypes of each SNP by cocaine abuse after 1 year in MMT was assessed under the dominant and recessive models using chi(2). Cumulative retention (up to 26.5 years) was studied using Kaplan-Meier analyses. Logistic regression and Cox model were used for multivariate analyses. Results: Of a nonselective sample of 404 patients, 25 patients with <50% Europeans/Middle Eastern ancestry were excluded. Of the remaining 379 patients, 330 (87.1%) stayed at least 1 year in treatment. Four SNPs were associated with cocaine abuse after 1 year in MMT. A lower proportion of cocaine abusers was found in the groups of subjects with the following genotypes: arginine vasopressin (AVP) SNP rs2282018 CC, CRHBP rs7728378 TT, galanin rs3136541 TT/TC, and neuropeptide Y receptor Y1 (NPY1R) rs4518200 AA. The following independent variables were associated with lack of cocaine in urine after 1 year (multivariate analyses): CRHBP rs7728378 TT, NPY1R rs4518200 AA, no cocaine in urine on admission, as well as opiate and benzodiazepine use after 1 year in MMT. Cumulative retention (n = 379) was longer in carriers of AVP rs2282018 CC (13.7 years, 95% CI 11.1-16.2) versus TT/TC genotypes (10.5, 95% CI 9.4-11.5) (p = 0.0230) Conclusions: The study suggests that a reduction in cocaine abuse and longer retention among MMT patients is mediated in part by variants in stress-related genes and is a step toward precision medicine.

Fanconi anemia (FA) is a rare genetic disorder characterized by bone marrow failure, predisposition to cancer, and congenital abnormalities. FA is caused by pathogenic variants in any of 22 genes involved in the DNA repair pathway responsible for removing interstrand crosslinks. FANCL, an E3 ubiquitin ligase, is an integral component of the pathway, but patients affected by disease-causing FANCL variants are rare, with only nine cases reported worldwide. We report here a FANCL founder variant, anticipated to be synonymous, c.1092G>A;p.K364=, but demonstrated to induce aberrant splicing, c.1021_1092del;p.W341_K364del, that accounts for the onset of FA in 13 cases from South Asia, 12 from India and one from Pakistan. We comprehensively illustrate the pathogenic nature of the variant, provide evidence for a founder effect, and propose including this variant in genetic screening of suspected FA patients in India and Pakistan, as well as those with ancestry from these regions of South Asia.

The repair of DNA double-strand breaks occurs through nonhomologous end joining or homologous recombination in vertebrate cells-a choice that is thought to be decided by a competition between DNA-dependent protein kinase (DNA-PK) and the Mre11/Rad50/Nbs1 (MRN) complex but is not well understood. Using ensemble biochemistry and single-molecule approaches, here, we show that the MRN complex is dependent on DNA-PK and phosphorylated CtIP to perform efficient processing and resection of DNA ends in physiological conditions, thus eliminating the competition model. Endonucleolytic removal of DNA-PK-bound DNA ends is also observed at double-strand break sites in human cells. The involvement of DNA-PK in MRN-mediated end processing promotes an efficient and sequential transition from nonhomologous end joining to homologous recombination by facilitating DNA-PK removal.

The gamma-tubulin ring complex (gamma-TuRC) is an essential regulator of centrosomal and acentrosomal microtubule formation, yet its structure is not known. Here, we present a cryo-EM reconstruction of the native human gamma-TuRC at similar to 3.8 angstrom resolution, revealing an asymmetric, cone-shaped structure. Pseudoatomic models indicate that GCP4, GCP5, and GCP6 form distinct Y-shaped assemblies that structurally mimic GCP2/GCP3 subcomplexes distal to the gamma-TuRC "seam.'' We also identify an unanticipated structural bridge that includes an actin-like protein and spans the gamma-TuRC lumen. Despite its asymmetric architecture, the gamma-TuRC arranges gamma-tubulins into a helical geometry poised to nucleate microtubules. Diversity in the gamma-TuRC subunits introduces large (>100,000 angstrom(2)) surfaces in the complex that allow for interactions with different regulatory factors. The observed compositional complexity of the gamma-TuRC could self-regulate its assembly into a cone-shaped structure to control microtubule formation across diverse contexts, e.g., within biological condensates or alongside existing filaments.

Identifying the molecular mechanisms that give rise to genetic variation is essential for the understanding of evolutionary processes. Previously, we have used adaptive laboratory evolution to enable biomass synthesis from CO2 in Escherichia coli. Genetic analysis of adapted clones from two independently evolving populations revealed distinct enrichment for insertion and deletion mutational events. Here, we follow these observations to show that mutations in the gene encoding for DNA topoisomerase I (topA) give rise to mutator phenotypes with characteristic mutational spectra. Using genetic assays and mutation accumulation lines, we find that point mutations in topA increase the rate of sequence deletion and duplication events. Interestingly, we observe that a single residue substitution (R168C) results in a high rate of head-to-tail (tandem) short sequence duplications, which are independent of existing sequence repeats. Finally, we show that the unique mutation spectrum of topA mutants enhances the emergence of antibiotic resistance in comparison to mismatch-repair (mutS) mutators, and leads to new resistance genotypes. Our findings highlight a potential link between the catalytic activity of topoisomerases and the fundamental question regarding the emergence of de novo tandem repeats, which are known modulators of bacterial evolution.

The NlpC/p60-family of peptidoglycan hydrolases are key enzymes that facilitate bacterial cell division and also modulate microbe-host interactions. These endopeptidases utilize conserved Cys-His residues in their active site and are expressed in most bacterial species as well as some eukaryotes. Here we describe methods for biochemical analysis of Enterococcus faecium SagA-NlpC/p60 peptidoglycan hydrolase activity (Kim et al., 2019; Rangan et al., 2016), which includes recombinant protein preparation and biochemical analysis using both gel-based and LC-MS profiling of peptidoglycan fragments. These protocols should also facilitate the biochemical analysis of other NlpC/p60 peptidoglycan hydrolases.