Rare BANF1 Alleles and Relatively Frequent EMD Alleles Including `Healthy Lipid' Emerin p.D149H in the ExAC Cohort

Emerin (EMD) and barrier to autointegration factor 1 (BANF1) each bind A-type lamins (LMNA) as fundamental components of nuclear lamina structure. Mutations in LMNA, EMD and BANF1 are genetically linked to many tissue-specific disorders including Emery-Dreifuss muscular dystrophy and cardiomyopathy (LMNA, EMD), lipodystrophy, insulin resistance and type 2 diabetes (LMNA) and progeria (LMNA, BANF1). To explore human genetic variation in these genes, we analyzed EMD and BANF1 alleles in the Exome Aggregation Consortium (ExAC) cohort of 60,706 unrelated individuals. We identified 13 rare heterozygous BANF1 missense variants (p.T2S, p.H7Y, p.D9N, p.S22R, p.G25E, p.D55N, p.D57Y, p.L63P, p.N70T, p.K72R, p.R75W, p.R75Q, p.G79R), and one homozygous variant (p.D9H). Several variants are known (p.G25E) or predicted (e.g., p.D9H, p.D9N, p.L63P) to perturb BANF1 and warrant further study. Analysis of EMD revealed two previously identified variants associated with adult-onset cardiomyopathy (p.K37del, p.E35K) and one deemed `benign' in an Emery-Dreifuss patient (p.D149H). Interestingly p.D149H was the most frequent emerin variant in ExAC, identified in 58 individuals (overall allele frequency 0.06645%), of whom 55 were East Asian (allele frequency 0.8297%). Furthermore, p.D149H associated with four `healthy' traits: reduced triglycerides (-0.336; p = 0.0368), reduced waist circumference (-0.321; p = 0.0486), reduced cholesterol (-0.572; p = 0.000346) and reduced LDL cholesterol (-0.599; p = 0.000272). These traits are distinct from LMNA-associated metabolic disorders and provide the first insight that emerin influences metabolism. We also identified one novel in-frame deletion (p.F39del) and 62 novel emerin missense variants, many of which were relatively frequent and potentially disruptive including p.N91S and p.S143F (0.041% and -0.034% of non-Finnish Europeans, respectively), p.G156S (-0.39% of Africans), p.R204G (-0.18% of Latinx), p.R207P (-0.08% of South Asians) and p.R221L (-0.15% of Latinx). Many novel BANF1 variants are predicted to disrupt dimerization or binding to DNA, histones, emerin or A-type lamins. Many novel emerin variants are predicted to disrupt emerin filament dynamics or binding to BANF1, HDAC3, A-type lamins or other partners. These new human variants provide a foundational resource for future studies to test the molecular mechanisms of BANF1 and emerin function, and to understand the link between emerin variant p.D149H and a `healthy' lipid profile

LMNA Sequences of 60,706 Unrelated Individuals Reveal 132 Novel Missense Variants in A-Type Lamins and Suggest a Link between Variant p.G602S and Type 2 Diabetes

Mutations in LMNA, encoding nuclear intermediate filament proteins lamins A and C, cause multiple diseases (‘laminopathies’) including muscular dystrophy, dilated cardiomyopathy, familial partial lipodystrophy (FPLD2), insulin resistance syndrome and progeria. To assess the prevalence of LMNA missense mutations (‘variants’) in a broad, ethnically diverse population, we compared missense alleles found among 60,706 unrelated individuals in the ExAC cohort to those identified in 1,404 individuals in the laminopathy database (UMD-LMNA). We identified 169 variants in the ExAC cohort, of which 37 (∼22%) are disease-associated including p.I299V (allele frequency 0.0402%), p.G602S (allele frequency 0.0262%) and p.R644C (allele frequency 0.124%), suggesting certain LMNA mutations are more common than previously recognized. Independent analysis of LMNA variants via the type 2 diabetes (T2D) Knowledge Portal showed that variant p.G602S associated significantly with type 2 diabetes (p = 0.02; odds ratio = 4.58), and was more frequent in African Americans (allele frequency 0.297%). The FPLD2-associated variant I299V was most prevalent in Latinos (allele frequency 0.347%). The ExAC cohort also revealed 132 novel LMNA missense variants including p.K108E (limited to individuals with psychiatric disease; predicted to perturb coil-1B), p.R397C and p.R427C (predicted to perturb filament biogenesis), p.G638R and p.N660D (predicted to perturb prelamin A processing), and numerous Ig-fold variants predicted to perturb phenotypically characteristic protein–protein interactions. Overall, this two-pronged strategy— mining a large database for missense variants in a single gene (LMNA), coupled to knowledge about the structure, biogenesis and functions of A-type lamins— revealed an unexpected number of LMNA variants, including novel variants predicted to perturb lamin assembly or function. Interestingly, this study also correlated novel variant p.K108E with psychiatric disease, identified known variant p.I299V as a potential risk factor for metabolic disease in Latinos, linked variant p.G602 with type 2 diabetes, and identified p.G602S as a predictor of diabetes risk in African Americans

Bacteriophage and Bacterial Susceptibility, Resistance, and Tolerance to Antibiotics

Bacteriophages, viruses that infect and replicate within bacteria, impact bacterial responses to antibiotics in complex ways. Recent studies using lytic bacteriophages to treat bacterial infections (phage therapy) demonstrate that phages can promote susceptibility to chemical antibiotics and that phage/antibiotic synergy is possible. However, both lytic and lysogenic bacteriophages can contribute to antimicrobial resistance. In particular, some phages mediate the horizontal transfer of antibiotic resistance genes between bacteria via transduction and other mechanisms. In addition, chronic infection filamentous phages can promote antimicrobial tolerance, the ability of bacteria to persist in the face of antibiotics. In particular, filamentous phages serve as structural elements in bacterial biofilms and prevent the penetration of antibiotics. Over time, these contributions to antibiotic tolerance favor the selection of resistance clones. Here, we review recent insights into bacteriophage contributions to antibiotic susceptibility, resistance, and tolerance. We discuss the mechanisms involved in these effects and address their impact on bacterial fitness

Native lamin A/C proteomes and novel partners from heart and skeletal muscle in a mouse chronic inflammation model of human frailty

Tandem Mass Tag (TMT) proteomicsProtein extracts (40 ug total at 1 ug/ul in 1% SDS) were reduced with 15 uL of 15 mM DTT for 1 hour at 56°C, alkylated by adding 15 uL 100 mM iodoacetamide and incubating in the dark for 30 min, and then TCA/Acetone precipitated. The protein pellet from each sample was digested overnight at 37°C by adding 100 uL Trypsin/LysC mixture (40 ug proteases in 1.2 mL of 100 mM triethylammonium bicarbonate (TEAB; Promega #V5071) or approximately 3.33 ug protease per sample. Individual samples (40 ug) were labeled with a unique isobaric mass tag reagent (TMT 10-plex, Thermo Scientific) according to manufacturer instructions. Both pairing and labeling order of TMT reagent and peptide sample were randomized. Briefly, the TMT reagents (0.8 ug vials) were allowed to come to room temperature before adding 41 uL anhydrous acetonitrile, then vortexed and centrifuged. The entire TMT reagent vial was added to the 100 ug peptide sample and reacted at room temperature for 1 hour. The reaction was quenched by adding hydroxylamine (8 uL) to a final concentration of 5%. All TMT-labeled samples were combined and vacuum centrifuged to dryness removing the entire liquid.Basic Reverse Phase (bRP) fractionation— Labeled peptide samples were fractionated by basic reverse phase (bRP) chromatography on Oasis HLB uElution plates (Waters). TMT labeled peptides (5%, approximately 20 ug) were bound to HLB resin in 10 mM triethylammonium bicarbonate (TEAB) buffer and step eluted with 0%, 5%, 10%, 25%, and 75% acetonitrile in 10 mM TEAB (0 and 5% fractions were combined). Fractions were dried by vacuum centrifugation.Mass Spectrometry Analysis— The peptide fractions were resuspended in 20 uL 2% acetonitrile in 0.1% formic acid; approximately 0.5 ug (2 uL) was loaded onto a C18 trap (S-10 uM, 120Å, 75 um x 2 cm; YMC Co., LTD, Kyoto, Japan) and then separated on an in-house packed PicoFrit column (75 um x 200 mm, 15 um, +/-1 um tip, New Objective) with C18 phase (ReproSil-Pur C18-AQ, 3 um, 120Å, www.dr-maisch.com) using 2-90% acetonitrile gradient at 300 nL/min over 120 min on a EasyLC nanoLC 1000 (Thermo Scientific). Eluting peptides were sprayed at 2.0 kV directly into an Orbitrap Fusion Lumos (Thermo Scientific) mass spectrometer. Survey scans (full ms) were acquired from 360-1700 m/z with a cycle time of 3 sec. Precursor ions isolated in a 0.7 Da window and fragmented using HCD activation collision energy 39 and 15s dynamic exclusion, with a scan range of 116m/z-2000m/z. Precursor and fragment ions were analyzed at resolutions 120,000 and 30,000, respectively, with automatic gain control (AGC) target values at 4 x 105 with 50 ms maximum injection time (IT) and 1 x 105 with 118ms maximum IT, respectively.Data analysis— Isotopically resolved masses in precursor (MS) and fragmentation (MS/MS) spectra were extracted from raw MS data using spectrum selector with recalibration in Proteome Discoverer (PD) software (version 2.4.0.305, Thermo Scientific) and searched using Mascot (2.6.2; www.matrixscience.com) against a Mus musculus protein database (RefSeq2017_83, created 5/23/2019, containing 76,508 sequences. The following criteria were set for all database searches: (a) all species in database; (b) trypsin as the enzyme, (c) two missed cleavages allowed; (d) N-terminal TMT6plex and cysteine carbamidomethylation as fixed modifications; (e) lysine TMT6plex, methionine oxidation, serine, threonine and tyrosine phosphorylation, asparagine and glutamine deamidation, HexNAc on serine or threonine, as variable modifications; and (f) precursor and fragment ion tolerances were set to 5ppm and 0.03Da, respectively. Peptide identifications from Mascot searches were filtered at 5% False Discovery Rate (FDR) confidence threshold, based on a concatenated decoy database search, using the Proteome Discoverer. Proteome Discoverer uses only the peptide identifications with the highest Mascot score for the same peptide matched spectrum from the different extraction methods. The protein intensities were reported as S/N of each peptide and relative protein comparisons were calculated using the peptide grouping in Proteome Discoverer. Quan value correction factors were used (Lot TK271715) with a co-isolation threshold of 30. Peptide abundances were normalized against a custom sequence .FASTA file containing only prelamin A (XP_006501136.1 PREDICTED: prelamin-A/C isoform X1 [Mus musculus]) to ensure there was no experimental bias in protein quantification that depended on the total amount of lamin A/C immunoprecipitated from each sample.</p

OGT (O-GlcNAC transferase) selectively modifies multiple residues unique to Lamin A

The LMNA gene encodes lamins A and C with key roles in nuclear structure, signaling, gene regulation, and genome integrity. Mutations in LMNA cause over 12 diseases (‘laminopathies’). Lamins A and C are identical for their first 566 residues. However, they form separate filaments in vivo, with apparently distinct roles. We report that lamin A is β-O-linked N-acetylglucosamine-(O-GlcNAc)-modified in human hepatoma (Huh7) cells and in mouse liver. In vitro assays with purified O-GlcNAc transferase (OGT) enzyme showed robust O-GlcNAcylation of recombinant mature lamin A tails (residues 385–646), with no detectable modification of lamin B1, lamin C, or ‘progerin’ (Δ50) tails. Using mass spectrometry, we identified 11 O-GlcNAc sites in a ‘sweet spot’ unique to lamin A, with up to seven sugars per peptide. Most sites were unpredicted by current algorithms. Double-mutant (S612A/T643A) lamin A tails were still robustly O-GlcNAc-modified at seven sites. By contrast, O-GlcNAcylation was undetectable on tails bearing deletion Δ50, which causes Hutchinson–Gilford progeria syndrome, and greatly reduced by deletion Δ35. We conclude that residues deleted in progeria are required for substrate recognition and/or modification by OGT in vitro. Interestingly, deletion Δ35, which does not remove the majority of identified O-GlcNAc sites, does remove potential OGT-association motifs (lamin A residues 622–625 and 639–645) homologous to that in mouse Tet1. These biochemical results are significant because they identify a novel molecular pathway that may profoundly influence lamin A function. The hypothesis that lamin A is selectively regulated by OGT warrants future testing in vivo, along with two predictions: genetic variants may contribute to disease by perturbing OGT-dependent regulation, and nutrient or other stresses might cause OGT to misregulate wildtype lamin A

Native lamin A/C proteomes and novel partners from heart and skeletal muscle in a mouse chronic inflammation model of human frailty.

Tandem Mass Tag (TMT) proteomicsProtein extracts (40 ug total at 1 ug/ul in 1% SDS) were reduced with 15 uL of 15 mM DTT for 1 hour at 56°C, alkylated by adding 15 uL 100 mM iodoacetamide and incubating in the dark for 30 min, and then TCA/Acetone precipitated. The protein pellet from each sample was digested overnight at 37°C by adding 100 uL Trypsin/LysC mixture (40 ug proteases in 1.2 mL of 100 mM triethylammonium bicarbonate (TEAB; Promega #V5071) or approximately 3.33 ug protease per sample. Individual samples (40 ug) were labeled with a unique isobaric mass tag reagent (TMT 10-plex, Thermo Scientific) according to manufacturer instructions. Both pairing and labeling order of TMT reagent and peptide sample were randomized. Briefly, the TMT reagents (0.8 ug vials) were allowed to come to room temperature before adding 41 uL anhydrous acetonitrile, then vortexed and centrifuged. The entire TMT reagent vial was added to the 100 ug peptide sample and reacted at room temperature for 1 hour. The reaction was quenched by adding hydroxylamine (8 uL) to a final concentration of 5%. All TMT-labeled samples were combined and vacuum centrifuged to dryness removing the entire liquid.Basic Reverse Phase (bRP) fractionation— Labeled peptide samples were fractionated by basic reverse phase (bRP) chromatography on Oasis HLB uElution plates (Waters). TMT labeled peptides (5%, approximately 20 ug) were bound to HLB resin in 10 mM triethylammonium bicarbonate (TEAB) buffer and step eluted with 0%, 5%, 10%, 25%, and 75% acetonitrile in 10 mM TEAB (0 and 5% fractions were combined). Fractions were dried by vacuum centrifugation.Mass Spectrometry Analysis— The peptide fractions were resuspended in 20 uL 2% acetonitrile in 0.1% formic acid; approximately 0.5 ug (2 uL) was loaded onto a C18 trap (S-10 uM, 120Å, 75 um x 2 cm; YMC Co., LTD, Kyoto, Japan) and then separated on an in-house packed PicoFrit column (75 um x 200 mm, 15 um, +/-1 um tip, New Objective) with C18 phase (ReproSil-Pur C18-AQ, 3 um, 120Å, www.dr-maisch.com) using 2-90% acetonitrile gradient at 300 nL/min over 120 min on a EasyLC nanoLC 1000 (Thermo Scientific). Eluting peptides were sprayed at 2.0 kV directly into an Orbitrap Fusion Lumos (Thermo Scientific) mass spectrometer. Survey scans (full ms) were acquired from 360-1700 m/z with a cycle time of 3 sec. Precursor ions isolated in a 0.7 Da window and fragmented using HCD activation collision energy 39 and 15s dynamic exclusion, with a scan range of 116m/z-2000m/z. Precursor and fragment ions were analyzed at resolutions 120,000 and 30,000, respectively, with automatic gain control (AGC) target values at 4 x 105 with 50 ms maximum injection time (IT) and 1 x 105 with 118ms maximum IT, respectively.Data analysis— Isotopically resolved masses in precursor (MS) and fragmentation (MS/MS) spectra were extracted from raw MS data using spectrum selector with recalibration in Proteome Discoverer (PD) software (version 2.4.0.305, Thermo Scientific) and searched using Mascot (2.6.2; www.matrixscience.com) against a Mus musculus protein database (RefSeq2017_83, created 5/23/2019, containing 76,508 sequences. The following criteria were set for all database searches: (a) all species in database; (b) trypsin as the enzyme, (c) two missed cleavages allowed; (d) N-terminal TMT6plex and cysteine carbamidomethylation as fixed modifications; (e) lysine TMT6plex, methionine oxidation, serine, threonine and tyrosine phosphorylation, asparagine and glutamine deamidation, HexNAc on serine or threonine, as variable modifications; and (f) precursor and fragment ion tolerances were set to 5ppm and 0.03Da, respectively. Peptide identifications from Mascot searches were filtered at 5% False Discovery Rate (FDR) confidence threshold, based on a concatenated decoy database search, using the Proteome Discoverer. Proteome Discoverer uses only the peptide identifications with the highest Mascot score for the same peptide matched spectrum from the different extraction methods. The protein intensities were reported as S/N of each peptide and relative protein comparisons were calculated using the peptide grouping in Proteome Discoverer. Quan value correction factors were used (Lot TK271715) with a co-isolation threshold of 30. Peptide abundances were normalized against a custom sequence .FASTA file containing only prelamin A (XP_006501136.1 PREDICTED: prelamin-A/C isoform X1 [Mus musculus]) to ensure there was no experimental bias in protein quantification that depended on the total amount of lamin A/C immunoprecipitated from each sample.</p

Native lamin A/C brain proteome and novel partners in a mouse chronic inflammation model of human frailty

Tandem Mass Tag (TMT) proteomicsProtein extracts (40 ug total at 1 ug/ul in 1% SDS) were reduced with 15 uL of 15 mM DTT for 1 hour at 56°C, alkylated by adding 15 uL 100 mM iodoacetamide and incubating in the dark for 30 min, and then TCA/Acetone precipitated. The protein pellet from each sample was digested overnight at 37°C by adding 100 uL Trypsin/LysC mixture (40 ug proteases in 1.2 mL of 100 mM triethylammonium bicarbonate (TEAB; Promega #V5071) or approximately 3.33 ug protease per sample. Individual samples (40 ug) were labeled with a unique isobaric mass tag reagent (TMT 10-plex, Thermo Scientific) according to manufacturer instructions. Both pairing and labeling order of TMT reagent and peptide sample were randomized. Briefly, the TMT reagents (0.8 ug vials) were allowed to come to room temperature before adding 41 uL anhydrous acetonitrile, then vortexed and centrifuged. The entire TMT reagent vial was added to the 100 ug peptide sample and reacted at room temperature for 1 hour. The reaction was quenched by adding hydroxylamine (8 uL) to a final concentration of 5%. All TMT-labeled samples were combined and vacuum centrifuged to dryness removing the entire liquid.Basic Reverse Phase (bRP) fractionation— Labeled peptide samples were fractionated by basic reverse phase (bRP) chromatography on Oasis HLB uElution plates (Waters). TMT labeled peptides (5%, approximately 20 ug) were bound to HLB resin in 10 mM triethylammonium bicarbonate (TEAB) buffer and step eluted with 0%, 5%, 10%, 25%, and 75% acetonitrile in 10 mM TEAB (0 and 5% fractions were combined). Fractions were dried by vacuum centrifugation.Mass Spectrometry Analysis— The peptide fractions were resuspended in 20 uL 2% acetonitrile in 0.1% formic acid; approximately 0.5 ug (2 uL) was loaded onto a C18 trap (S-10 uM, 120Å, 75 um x 2 cm; YMC Co., LTD, Kyoto, Japan) and then separated on an in-house packed PicoFrit column (75 um x 200 mm, 15 um, +/-1 um tip, New Objective) with C18 phase (ReproSil-Pur C18-AQ, 3 um, 120Å, www.dr-maisch.com) using 2-90% acetonitrile gradient at 300 nL/min over 120 min on a EasyLC nanoLC 1000 (Thermo Scientific). Eluting peptides were sprayed at 2.0 kV directly into an Orbitrap Fusion Lumos (Thermo Scientific) mass spectrometer. Survey scans (full ms) were acquired from 360-1700 m/z with a cycle time of 3 sec. Precursor ions isolated in a 0.7 Da window and fragmented using HCD activation collision energy 39 and 15s dynamic exclusion, with a scan range of 116m/z-2000m/z. Precursor and fragment ions were analyzed at resolutions 120,000 and 30,000, respectively, with automatic gain control (AGC) target values at 4 x 105 with 50 ms maximum injection time (IT) and 1 x 105 with 118ms maximum IT, respectively.Data analysis— Isotopically resolved masses in precursor (MS) and fragmentation (MS/MS) spectra were extracted from raw MS data using spectrum selector with recalibration in Proteome Discoverer (PD) software (version 2.4.0.305, Thermo Scientific) and searched using Mascot (2.6.2; www.matrixscience.com) against a Mus musculus protein database (RefSeq2017_83, created 5/23/2019, containing 76,508 sequences. The following criteria were set for all database searches: (a) all species in database; (b) trypsin as the enzyme, (c) two missed cleavages allowed; (d) N-terminal TMT6plex and cysteine carbamidomethylation as fixed modifications; (e) lysine TMT6plex, methionine oxidation, serine, threonine and tyrosine phosphorylation, asparagine and glutamine deamidation, HexNAc on serine or threonine, as variable modifications; and (f) precursor and fragment ion tolerances were set to 5ppm and 0.03Da, respectively. Peptide identifications from Mascot searches were filtered at 5% False Discovery Rate (FDR) confidence threshold, based on a concatenated decoy database search, using the Proteome Discoverer. Proteome Discoverer uses only the peptide identifications with the highest Mascot score for the same peptide matched spectrum from the different extraction methods. The protein intensities were reported as S/N of each peptide and relative protein comparisons were calculated using the peptide grouping in Proteome Discoverer. Quan value correction factors were used (Lot TK271715) with a co-isolation threshold of 30. Peptide abundances were normalized against a custom sequence .FASTA file containing only prelamin A (XP_006501136.1 PREDICTED: prelamin-A/C isoform X1 [Mus musculus]) to ensure there was no experimental bias in protein quantification that depended on the total amount of lamin A/C immunoprecipitated from each sample.</p