Publications by authors named "Joseph A Marsh"

69 Publications

PRIM1 deficiency causes a distinctive primordial dwarfism syndrome.

Genes Dev 2020 Oct 15. Epub 2020 Oct 15.

MRC Human Genetics Unit, MRC Institute of Genetics and Molecular Medicine, the University of Edinburgh, Edinburgh EH4 2XU, United Kingdom.

DNA replication is fundamental for cell proliferation in all organisms. Nonetheless, components of the replisome have been implicated in human disease, and here we report encoding the catalytic subunit of DNA primase as a novel disease gene. Using a variant classification agnostic approach, biallelic mutations in PRIM1 were identified in five individuals. PRIM1 protein levels were markedly reduced in patient cells, accompanied by replication fork asymmetry, increased interorigin distances, replication stress, and prolonged S-phase duration. Consequently, cell proliferation was markedly impaired, explaining the patients' extreme growth failure. Notably, phenotypic features distinct from those previously reported with DNA polymerase genes were evident, highlighting differing developmental requirements for this core replisome component that warrant future investigation.
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http://dx.doi.org/10.1101/gad.340190.120DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7608753PMC
October 2020

Heterozygous lamin B1 and lamin B2 variants cause primary microcephaly and define a novel laminopathy.

Genet Med 2021 Feb 9;23(2):408-414. Epub 2020 Oct 9.

MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK.

Purpose: Lamins are the major component of nuclear lamina, maintaining structural integrity of the nucleus. Lamin A/C variants are well established to cause a spectrum of disorders ranging from myopathies to progeria, termed laminopathies. Phenotypes resulting from variants in LMNB1 and LMNB2 have been much less clearly defined.

Methods: We investigated exome and genome sequencing from the Deciphering Developmental Disorders Study and the 100,000 Genomes Project to identify novel microcephaly genes.

Results: Starting from a cohort of patients with extreme microcephaly, 13 individuals with heterozygous variants in the two human B-type lamins were identified. Recurrent variants were established to be de novo in nine cases and shown to affect highly conserved residues within the lamin ɑ-helical rod domain, likely disrupting interactions required for higher-order assembly of lamin filaments.

Conclusion: We identify dominant pathogenic variants in LMNB1 and LMNB2 as a genetic cause of primary microcephaly, implicating a major structural component of the nuclear envelope in its etiology and defining a new form of laminopathy. The distinct nature of this lamin B-associated phenotype highlights the strikingly different developmental requirements for lamin paralogs and suggests a novel mechanism for primary microcephaly warranting future investigation.
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http://dx.doi.org/10.1038/s41436-020-00980-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7862057PMC
February 2021

Identification of pathogenic missense mutations using protein stability predictors.

Sci Rep 2020 09 21;10(1):15387. Epub 2020 Sep 21.

MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, EH4 2XU, UK.

Attempts at using protein structures to identify disease-causing mutations have been dominated by the idea that most pathogenic mutations are disruptive at a structural level. Therefore, computational stability predictors, which assess whether a mutation is likely to be stabilising or destabilising to protein structure, have been commonly used when evaluating new candidate disease variants, despite not having been developed specifically for this purpose. We therefore tested 13 different stability predictors for their ability to discriminate between pathogenic and putatively benign missense variants. We find that one method, FoldX, significantly outperforms all other predictors in the identification of disease variants. Moreover, we demonstrate that employing predicted absolute energy change scores improves performance of nearly all predictors in distinguishing pathogenic from benign variants. Importantly, however, we observe that the utility of computational stability predictors is highly heterogeneous across different proteins, and that they are all inferior to the best performing variant effect predictors for identifying pathogenic mutations. We suggest that this is largely due to alternate molecular mechanisms other than protein destabilisation underlying many pathogenic mutations. Thus, better ways of incorporating protein structural information and molecular mechanisms into computational variant effect predictors will be required for improved disease variant prioritisation.
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http://dx.doi.org/10.1038/s41598-020-72404-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7506547PMC
September 2020

Mutations in COPA lead to abnormal trafficking of STING to the Golgi and interferon signaling.

J Exp Med 2020 11;217(11)

Laboratory of Neurogenetics and Neuroinflammation, Imagine Institute, Paris, France.

Heterozygous missense mutations in coatomer protein subunit α, COPA, cause a syndrome overlapping clinically with type I IFN-mediated disease due to gain-of-function in STING, a key adaptor of IFN signaling. Recently, increased levels of IFN-stimulated genes (ISGs) were described in COPA syndrome. However, the link between COPA mutations and IFN signaling is unknown. We observed elevated levels of ISGs and IFN-α in blood of symptomatic COPA patients. In vitro, both overexpression of mutant COPA and silencing of COPA induced STING-dependent IFN signaling. We detected an interaction between COPA and STING, and mutant COPA was associated with an accumulation of ER-resident STING at the Golgi. Given the known role of the coatomer protein complex I, we speculate that loss of COPA function leads to enhanced type I IFN signaling due to a failure of Golgi-to-ER STING retrieval. These data highlight the importance of the ER-Golgi axis in the control of autoinflammation and inform therapeutic strategies in COPA syndrome.
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http://dx.doi.org/10.1084/jem.20200600DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7596811PMC
November 2020

Using deep mutational scanning to benchmark variant effect predictors and identify disease mutations.

Mol Syst Biol 2020 07;16(7):e9380

MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK.

To deal with the huge number of novel protein-coding variants identified by genome and exome sequencing studies, many computational variant effect predictors (VEPs) have been developed. Such predictors are often trained and evaluated using different variant data sets, making a direct comparison between VEPs difficult. In this study, we use 31 previously published deep mutational scanning (DMS) experiments, which provide quantitative, independent phenotypic measurements for large numbers of single amino acid substitutions, in order to benchmark and compare 46 different VEPs. We also evaluate the ability of DMS measurements and VEPs to discriminate between pathogenic and benign missense variants. We find that DMS experiments tend to be superior to the top-ranking predictors, demonstrating the tremendous potential of DMS for identifying novel human disease mutations. Among the VEPs, DeepSequence clearly stood out, showing both the strongest correlations with DMS data and having the best ability to predict pathogenic mutations, which is especially remarkable given that it is an unsupervised method. We further recommend SNAP2, DEOGEN2, SNPs&GO, SuSPect and REVEL based upon their performance in these analyses.
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http://dx.doi.org/10.15252/msb.20199380DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7336272PMC
July 2020

Genetic and functional insights into CDA-I prevalence and pathogenesis.

J Med Genet 2021 Mar 9;58(3):185-195. Epub 2020 Jun 9.

MRC Molecular Haematology Unit, MRC Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK

Background: Congenital dyserythropoietic anaemia type I (CDA-I) is a hereditary anaemia caused by biallelic mutations in the widely expressed genes and . Little is understood about either protein and it is unclear in which cellular pathways they participate.

Methods: Genetic analysis of a cohort of patients with CDA-I identifies novel pathogenic variants in both known causative genes. We analyse the mutation distribution and the predicted structural positioning of amino acids affected in Codanin-1, the protein encoded by . Using western blotting, immunoprecipitation and immunofluorescence, we determine the effect of particular mutations on both proteins and interrogate protein interaction, stability and subcellular localisation.

Results: We identify six novel mutations and one novel mutation in and uncover evidence of further genetic heterogeneity in CDA-I. Additionally, population genetics suggests that CDA-I is more common than currently predicted. Mutations are enriched in six clusters in Codanin-1 and tend to affect buried residues. Many missense and in-frame mutations do not destabilise the entire protein. Rather C15orf41 relies on Codanin-1 for stability and both proteins, which are enriched in the nucleolus, interact to form an obligate complex in cells.

Conclusion: Stability and interaction data suggest that C15orf41 may be the key determinant of CDA-I and offer insight into the mechanism underlying this disease. Both proteins share a common pathway likely to be present in a wide variety of cell types; however, nucleolar enrichment may provide a clue as to the erythroid specific nature of CDA-I. The surprisingly high predicted incidence of CDA-I suggests that better ascertainment would lead to improved patient care.
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http://dx.doi.org/10.1136/jmedgenet-2020-106880DOI Listing
March 2021

Recurrent heterozygous PAX6 missense variants cause severe bilateral microphthalmia via predictable effects on DNA-protein interaction.

Genet Med 2020 03 8;22(3):598-609. Epub 2019 Nov 8.

MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK.

Purpose: Most classical aniridia is caused by PAX6 haploinsufficiency. PAX6 missense variants can be hypomorphic or mimic haploinsufficiency. We hypothesized that missense variants also cause previously undescribed disease by altering the affinity and/or specificity of PAX6 genomic interactions.

Methods: We screened PAX6 in 372 individuals with bilateral microphthalmia, anophthalmia, or coloboma (MAC) from the Medical Research Council Human Genetics Unit eye malformation cohort (HGU) and reviewed data from the Deciphering Developmental Disorders study. We performed cluster analysis on PAX6-associated ocular phenotypes by variant type and molecular modeling of the structural impact of 86 different PAX6 causative missense variants.

Results: Eight different PAX6 missense variants were identified in 17 individuals (15 families) with MAC, accounting for 4% (15/372) of our cohort. Seven altered the paired domain (p.[Arg26Gln]x1, p.[Gly36Val]x1, p.[Arg38Trp]x2, p.[Arg38Gln]x1, p.[Gly51Arg]x2, p.[Ser54Arg]x2, p.[Asn124Lys]x5) and one the homeodomain (p.[Asn260Tyr]x1). p.Ser54Arg and p.Asn124Lys were exclusively associated with severe bilateral microphthalmia. MAC-associated variants were predicted to alter but not ablate DNA interaction, consistent with the electrophoretic mobility shifts observed using mutant paired domains with well-characterized PAX6-binding sites. We found no strong evidence for novel PAX6-associated extraocular disease.

Conclusion: Altering the affinity and specificity of PAX6-binding genome-wide provides a plausible mechanism for the worse-than-null effects of MAC-associated missense variants.
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http://dx.doi.org/10.1038/s41436-019-0685-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7056646PMC
March 2020

Finding Diagnostically Useful Patterns in Quantitative Phenotypic Data.

Am J Hum Genet 2019 11 10;105(5):933-946. Epub 2019 Oct 10.

Wellcome Sanger Institute, Hinxton, Cambridgeshire CB10 1SA, UK.

Trio-based whole-exome sequence (WES) data have established confident genetic diagnoses in ∼40% of previously undiagnosed individuals recruited to the Deciphering Developmental Disorders (DDD) study. Here we aim to use the breadth of phenotypic information recorded in DDD to augment diagnosis and disease variant discovery in probands. Median Euclidean distances (mEuD) were employed as a simple measure of similarity of quantitative phenotypic data within sets of ≥10 individuals with plausibly causative de novo mutations (DNM) in 28 different developmental disorder genes. 13/28 (46.4%) showed significant similarity for growth or developmental milestone metrics, 10/28 (35.7%) showed similarity in HPO term usage, and 12/28 (43%) showed no phenotypic similarity. Pairwise comparisons of individuals with high-impact inherited variants to the 32 individuals with causative DNM in ANKRD11 using only growth z-scores highlighted 5 likely causative inherited variants and two unrecognized DNM resulting in an 18% diagnostic uplift for this gene. Using an independent approach, naive Bayes classification of growth and developmental data produced reasonably discriminative models for the 24 DNM genes with sufficiently complete data. An unsupervised naive Bayes classification of 6,993 probands with WES data and sufficient phenotypic information defined 23 in silico syndromes (ISSs) and was used to test a "phenotype first" approach to the discovery of causative genotypes using WES variants strictly filtered on allele frequency, mutation consequence, and evidence of constraint in humans. This highlighted heterozygous de novo nonsynonymous variants in SPTBN2 as causative in three DDD probands.
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http://dx.doi.org/10.1016/j.ajhg.2019.09.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6848993PMC
November 2019

Ligand Binding Site Structure Shapes Folding, Assembly and Degradation of Homomeric Protein Complexes.

J Mol Biol 2019 09 12;431(19):3871-3888. Epub 2019 Jul 12.

MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine, University of Edinburgh, Crewe Road, Edinburgh, EH4 2XU, UK.

Ligand binding site structure has profound consequences for the evolution of function of protein complexes, particularly in homomers-complexes comprising multiple copies of the same protein. Previously, we have shown that homomers with multichain binding sites (MBSs) are characterized by more conserved binding sites and quaternary structure, and qualitatively different allosteric pathways than homomers with single-chain binding sites (SBSs) or monomers. Here, using computational methods, we show that the folds of single-domain MBS and SBS homomers are different, and SBS homomers are likely to be folded cotranslationally, while MBS homomers are more likely to form post-translationally and rely on more advanced folding-assistance and quality control mechanisms, which include chaperonins. In addition, our findings demonstrate that MBS homomers are qualitatively different from monomers, while SBS homomers are much less distinct, supporting the hypothesis that the evolution of quaternary structure in SBS homomers is significantly influenced by stochastic processes.
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http://dx.doi.org/10.1016/j.jmb.2019.07.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6739599PMC
September 2019

The role of protein complexes in human genetic disease.

Protein Sci 2019 08 1;28(8):1400-1411. Epub 2019 Jul 1.

MRC Human Genetics Unit, Institute of Genetics & Molecular Medicine, University of Edinburgh, Edinburgh, EH4 2XU, United Kingdom.

Many human genetic disorders are caused by mutations in protein-coding regions of DNA. Taking protein structure into account has therefore provided key insight into the molecular mechanisms underlying human genetic disease. Although most studies have focused on the intramolecular effects of mutations, the critical role of the assembly of proteins into complexes is being increasingly recognized. Here, we review multiple ways in which consideration of protein complexes can help us to understand and explain the effects of pathogenic mutations. First, we discuss disorders caused by mutations that perturb intersubunit interactions in homomeric and heteromeric complexes. Second, we address how protein complex assembly can facilitate a dominant-negative mechanism, whereby mutated subunits can disrupt the activity of wild-type protein. Third, we show how mutations that change protein expression levels can lead to damaging stoichiometric imbalances. Finally, we review how mutations affecting different subunits of the same heteromeric complex often cause similar diseases, whereas mutations in different interfaces of the same subunit can cause distinct phenotypes.
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http://dx.doi.org/10.1002/pro.3667DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6635777PMC
August 2019

Evolution of protein interfaces in multimers and fibrils.

J Chem Phys 2019 Jun;150(22):225102

Department of Physics and Astronomy, McMaster University, Hamilton, Ontario L8S 4M1, Canada.

A majority of cellular proteins function as part of multimeric complexes of two or more subunits. Multimer formation requires interactions between protein surfaces that lead to closed structures, such as dimers and tetramers. If proteins interact in an open-ended way, uncontrolled growth of fibrils can occur, which is likely to be detrimental in most cases. We present a statistical physics model that allows aggregation of proteins as either closed dimers or open fibrils of all lengths. We use pairwise amino-acid contact energies to calculate the energies of interacting protein surfaces. The probabilities of all possible aggregate configurations can be calculated for any given sequence of surface amino acids. We link the statistical physics model to a population genetics model that describes the evolution of the surface residues. When proteins evolve neutrally, without selection for or against multimer formation, we find that a majority of proteins remain as monomers at moderate concentrations, but strong dimer-forming or fibril-forming sequences are also possible. If selection is applied in favor of dimers or in favor of fibrils, then it is easy to select either dimer-forming or fibril-forming sequences. It is also possible to select for oriented fibrils with protein subunits all aligned in the same direction. We measure the propensities of amino acids to occur at interfaces relative to noninteracting surfaces and show that the propensities in our model are strongly correlated with those that have been measured in real protein structures. We also show that there are significant differences between amino acid frequencies at isologous and heterologous interfaces in our model, and we observe that similar effects occur in real protein structures.
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http://dx.doi.org/10.1063/1.5086042DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6561775PMC
June 2019

Protein aggregation mediates stoichiometry of protein complexes in aneuploid cells.

Genes Dev 2019 08 13;33(15-16):1031-1047. Epub 2019 Jun 13.

David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.

Aneuploidy, a condition characterized by chromosome gains and losses, causes reduced fitness and numerous cellular stresses, including increased protein aggregation. Here, we identify protein complex stoichiometry imbalances as a major cause of protein aggregation in aneuploid cells. Subunits of protein complexes encoded on excess chromosomes aggregate in aneuploid cells, which is suppressed when expression of other subunits is coordinately altered. We further show that excess subunits are either degraded or aggregate and that protein aggregation is nearly as effective as protein degradation at lowering levels of excess proteins. Our study explains why proteotoxic stress is a universal feature of the aneuploid state and reveals protein aggregation as a form of dosage compensation to cope with disproportionate expression of protein complex subunits.
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http://dx.doi.org/10.1101/gad.327494.119DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6672052PMC
August 2019

Ligands and Receptors with Broad Binding Capabilities Have Common Structural Characteristics: An Antibiotic Design Perspective.

J Med Chem 2019 11 25;62(21):9357-9374. Epub 2019 Jun 25.

MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine , University of Edinburgh , Crewe Road , Edinburgh EH4 2XU , U.K.

The spread of antibiotic resistance is one of the most serious global public-health problems. Here we show that a particular class of homomers with binding sites spanning multiple protein chains is particularly suitable for targeting by broad-spectrum antibacterial agents because due to the slow evolutionary change of such binding pockets, ligands of such homomers are much more likely to bind their homologs than ligands of monomers, or homomers with a single-chain binding site. Additionally, using de novo ligand design and deep learning, we show that the chemical compounds that can bind several different receptors have common structural characteristics and that halogens and fragments similar to the building blocks existing antimicrobials are overrepresented in them. Finally, we show that binding multiple receptors selects for flexible compounds, which are less likely to accumulate in Gram-negative bacteria; thus there is trade-off between reducing the emergence of resistance by multitargeting and broad-spectrum antibacterial activity.
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http://dx.doi.org/10.1021/acs.jmedchem.9b00220DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6858282PMC
November 2019

Biallelic variants in DNA2 cause microcephalic primordial dwarfism.

Hum Mutat 2019 08 23;40(8):1063-1070. Epub 2019 Jun 23.

MRC Human Genetics Unit, MRC Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, United Kingdom.

Microcephalic primordial dwarfism (MPD) is a group of rare single-gene disorders characterized by the extreme reduction in brain and body size from early development onwards. Proteins encoded by MPD-associated genes play important roles in fundamental cellular processes, notably genome replication and repair. Here we report the identification of four MPD individuals with biallelic variants in DNA2, which encodes an adenosine triphosphate (ATP)-dependent helicase/nuclease involved in DNA replication and repair. We demonstrate that the two intronic variants (c.1764-38_1764-37ins(53) and c.74+4A>C) found in these individuals substantially impair DNA2 transcript splicing. Additionally, we identify a missense variant (c.1963A>G), affecting a residue of the ATP-dependent helicase domain that is highly conserved between humans and yeast, with the resulting substitution (p.Thr655Ala) predicted to directly impact ATP/ADP (adenosine diphosphate) binding by DNA2. Our findings support the pathogenicity of these variants as biallelic hypomorphic mutations, establishing DNA2 as an MPD disease gene.
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http://dx.doi.org/10.1002/humu.23776DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6773220PMC
August 2019

Ligand-Binding-Site Structure Shapes Allosteric Signal Transduction and the Evolution of Allostery in Protein Complexes.

Mol Biol Evol 2019 08;36(8):1711-1727

MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, United Kingdom.

The structure of ligand-binding sites has been shown to profoundly influence the evolution of function in homomeric protein complexes. Complexes with multichain binding sites (MBSs) have more conserved quaternary structure, more similar binding sites and ligands between homologs, and evolve new functions slower than homomers with single-chain binding sites (SBSs). Here, using in silico analyses of protein dynamics, we investigate whether ligand-binding-site structure shapes allosteric signal transduction pathways, and whether the structural similarity of binding sites influences the evolution of allostery. Our analyses show that: 1) allostery is more frequent among MBS complexes than in SBS complexes, particularly in homomers; 2) in MBS homomers, semirigid communities and critical residues frequently connect interfaces and thus they are characterized by signal transduction pathways that cross protein-protein interfaces, whereas SBS homomers usually not; 3) ligand binding alters community structure differently in MBS and SBS homomers; and 4) except MBS homomers, allosteric proteins are more likely to have homologs with similar binding site than nonallosteric proteins, suggesting that binding site similarity is an important factor driving the evolution of allostery.
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http://dx.doi.org/10.1093/molbev/msz093DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6657754PMC
August 2019

Intrinsic lipid binding activity of ATG16L1 supports efficient membrane anchoring and autophagy.

EMBO J 2019 05 1;38(9). Epub 2019 Apr 1.

Cancer Research UK Edinburgh Centre, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK

Membrane targeting of autophagy-related complexes is an important step that regulates their activities and prevents their aberrant engagement on non-autophagic membranes. ATG16L1 is a core autophagy protein implicated at distinct phases of autophagosome biogenesis. In this study, we dissected the recruitment of ATG16L1 to the pre-autophagosomal structure (PAS) and showed that it requires sequences within its coiled-coil domain (CCD) dispensable for homodimerisation. Structural and mutational analyses identified conserved residues within the CCD of ATG16L1 that mediate direct binding to phosphoinositides, including phosphatidylinositol 3-phosphate (PI3P). Mutating putative lipid binding residues abrogated the localisation of ATG16L1 to the PAS and inhibited LC3 lipidation. On the other hand, enhancing lipid binding of ATG16L1 by mutating negatively charged residues adjacent to the lipid binding motif also resulted in autophagy inhibition, suggesting that regulated recruitment of ATG16L1 to the PAS is required for its autophagic activity. Overall, our findings indicate that ATG16L1 harbours an intrinsic ability to bind lipids that plays an essential role during LC3 lipidation and autophagosome maturation.
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http://dx.doi.org/10.15252/embj.2018100554DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6484409PMC
May 2019

Exploring wearable sensors as an alternative to marker-based motion capture in the pitching delivery.

PeerJ 2019 24;7:e6365. Epub 2019 Jan 24.

Research and Development, Driveline Baseball, Inc, Kent, WA, USA.

Background: Improvements in data processing, increased understanding of the biomechanical background behind kinetics and kinematics, and technological advancements in inertial measurement unit (IMU) sensors have enabled high precision in the measurement of joint angles and acceleration on human subjects. This has resulted in new devices that reportedly measure joint angles, arm speed, and stresses to the pitching arms of baseball players. This study seeks to validate one such sensor, the MotusBASEBALL unit, with a marker-based motion capture laboratory.

Hypothesis: We hypothesize that the joint angle measurements ("arm slot" and "shoulder rotation") of the MotusBASEBALL device will hold a statistically significant level of reliability and accuracy, but that the "arm speed" and "stress" metrics will not be accurate due to limitations in IMU technology.

Methods: A total of 10 healthy subjects threw five to seven fastballs followed by five to seven breaking pitches (slider or curveball) in the motion capture lab. Subjects wore retroreflective markers and the MotusBASEBALL sensor simultaneously.

Results: It was found that the arm slot ( = 0.975, < 0.001), shoulder rotation ( = 0.749, < 0.001), and stress ( = 0.667, = 0.001 when compared to elbow torque; = 0.653, = 0.002 when compared to shoulder torque) measurements were all significantly correlated with the results from the motion capture lab. Arm speed showed significant correlations to shoulder internal rotation speed ( = 0.668, = 0.001) and shoulder velocity magnitude ( = 0.659, = 0.002). For the entire sample, arm slot and shoulder rotation measurements were on a similar scale, or within 5-15% in absolute value, of magnitude to measurements from the motion capture test, averaging eight degrees less (12.9% relative differences) and nine degrees (5.4%) less, respectively. Arm speed had a much larger difference, averaging 3,745 deg/s (80.2%) lower than shoulder internal rotation velocity, and 3,891 deg/s (80.8%) less than the shoulder velocity magnitude. The stress metric was found to be 41 Newton meter (Nm; 38.7%) less when compared to elbow torque, and 42 Nm (39.3%) less when compared to shoulder torque. Despite the differences in magnitude, the correlations were extremely strong, indicating that the MotusBASEBALL sensor had high reliability for casual use.

Conclusion: This study attempts to validate the use of the MotusBASEBALL for future studies that look at the arm slot, shoulder rotation, arm speed, and stress measurements from the MotusBASEBALL sensor. Excepting elbow extension velocity, all metrics from the MotusBASEBALL unit showed significant correlations to their corresponding metrics from motion capture and while some magnitudes differ substantially and therefore fall short in validity, the link between the metrics is strong enough to indicate reliable casual use. Further research should be done to further investigate the validity and reliability of the arm speed metric.
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http://dx.doi.org/10.7717/peerj.6365DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6348088PMC
January 2019

Effects of a six-week weighted-implement throwing program on baseball pitching velocity, kinematics, arm stress, and arm range of motion.

PeerJ 2018 23;6:e6003. Epub 2018 Nov 23.

Research and Development, Driveline Baseball, Inc, Kent, WA, United States of America.

Background: Weighted-baseball training programs are used at the high school, collegiate, and professional levels of baseball. The purpose of this study was to evaluate the effects of a six-week training period consisting of weighted implements, manual therapy, weightlifting, and other modalities on shoulder external rotation, elbow valgus stress, pitching velocity, and kinematics.

Hypothesis: A six-week training program that includes weighted implements will increase pitching velocity along with concomitant increases in arm angular velocities, joint kinetics, and shoulder external rotation.

Methods: Seventeen collegiate and professional baseball pitchers (age range 18-23, average: 19.9 ± 1.3) training at Driveline Baseball were evaluated via a combination of an eight-camera motion-capture system, range-of-motion measurements and radar- and pitch-tracking equipment, both before and after a six-week training period. Each participant received individualized training programs, with significant overlap in training methods for all athletes. Twenty-eight biomechanical parameters were computed for each bullpen trial, four arm range-of-motion measurements were taken, and pitching velocities were recorded before and after the training period. Pre- and post-training period data were compared via post-hoc paired tests.

Results: There was no change in pitching velocity across the seventeen subjects. Four biomechanical parameters for the holistic group were significantly changed after the training period: internal rotational velocity was higher (from 4,527 ± 470 to 4,759 ± 542 degrees/second), shoulder abduction was lower at ball release (96 ± 7.6 to 93 ± 5.4°), the shoulder was less externally rotated at ball release (95 ± 15 to 86 ± 18°) and shoulder adduction torque was higher (from 103 ± 39 to 138 ± 53 N-m). Among the arm range of motion measurements, four were significantly different after the training period: the shoulder internal rotation range of motion and total range of motion for both the dominant and non-dominant arm. When the group was divided into those who gained pitching velocity and those who did not, neither group showed a significant increase in shoulder external rotation, or elbow valgus stress.

Conclusions: Following a six-week weighted implement program, pitchers did not show a significant change in velocity, joint kinetics, or shoulder external rotation range of motion. When comparing pitchers who gained velocity versus pitchers who did not, no statistically significant changes were seen in joint kinetics and shoulder range of motion.
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http://dx.doi.org/10.7717/peerj.6003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6254244PMC
November 2018

Gain-of-function DNMT3A mutations cause microcephalic dwarfism and hypermethylation of Polycomb-regulated regions.

Nat Genet 2019 01 26;51(1):96-105. Epub 2018 Nov 26.

MRC Human Genetics Unit, IGMM, University of Edinburgh, Edinburgh, UK.

DNA methylation and Polycomb are key factors in the establishment of vertebrate cellular identity and fate. Here we report de novo missense mutations in DNMT3A, which encodes the DNA methyltransferase DNMT3A. These mutations cause microcephalic dwarfism, a hypocellular disorder of extreme global growth failure. Substitutions in the PWWP domain abrogate binding to the histone modifications H3K36me2 and H3K36me3, and alter DNA methylation in patient cells. Polycomb-associated DNA methylation valleys, hypomethylated domains encompassing developmental genes, become methylated with concomitant depletion of H3K27me3 and H3K4me3 bivalent marks. Such de novo DNA methylation occurs during differentiation of Dnmt3a pluripotent cells in vitro, and is also evident in Dnmt3a dwarf mice. We therefore propose that the interaction of the DNMT3A PWWP domain with H3K36me2 and H3K36me3 normally limits DNA methylation of Polycomb-marked regions. Our findings implicate the interplay between DNA methylation and Polycomb at key developmental regulators as a determinant of organism size in mammals.
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http://dx.doi.org/10.1038/s41588-018-0274-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6520989PMC
January 2019

Mouse mutations cause retinal degeneration and reduced mitochondrial function.

Dis Model Mech 2018 12 18;11(12). Epub 2018 Dec 18.

MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine, University of Edinburgh, Crewe Road, Edinburgh EH4 2XU, UK

Isocitrate dehydrogenase (IDH) is an enzyme required for the production of α-ketoglutarate from isocitrate. IDH3 generates the NADH used in the mitochondria for ATP production, and is a tetramer made up of two α, one β and one γ subunit. Loss-of-function and missense mutations in both and have previously been implicated in families exhibiting retinal degeneration. Using mouse models, we investigated the role of IDH3 in retinal disease and mitochondrial function. We identified mice with late-onset retinal degeneration in a screen of ageing mice carrying an ENU-induced mutation, E229K, in Mice homozygous for this mutation exhibit signs of retinal stress, indicated by GFAP staining, as early as 3 months, but no other tissues appear to be affected. We produced a knockout of and found that homozygous mice do not survive past early embryogenesis. compound heterozygous mutants exhibit a more severe retinal degeneration compared with homozygous mutants. Analysis of mitochondrial function in mutant cell lines highlighted a reduction in mitochondrial maximal respiration and reserve capacity levels in both and cells. Loss-of-function mutants do not exhibit the same retinal degeneration phenotype, with no signs of retinal stress or reduction in mitochondrial respiration. It has previously been reported that the retina operates with a limited mitochondrial reserve capacity and we suggest that this, in combination with the reduced reserve capacity in mutants, explains the degenerative phenotype observed in mutant mice.This article has an associated First Person interview with the first author of the paper.
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http://dx.doi.org/10.1242/dmm.036426DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6307916PMC
December 2018

A Graph-Based Approach for Detecting Sequence Homology in Highly Diverged Repeat Protein Families.

Methods Mol Biol 2019 ;1851:251-261

MRC Human Genetics Unit, MRC Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK.

Reconstructing evolutionary relationships in repeat proteins is notoriously difficult due to the high degree of sequence divergence that typically occurs between duplicated repeats. This is complicated further by the fact that proteins with a large number of similar repeats are more likely to produce significant local sequence alignments than proteins with fewer copies of the repeat motif. Furthermore, biologically correct sequence alignments are sometimes impossible to achieve in cases where insertion or translocation events disrupt the order of repeats in one of the sequences being aligned. Combined, these attributes make traditional phylogenetic methods for studying protein families unreliable for repeat proteins, due to the dependence of such methods on accurate sequence alignment.We present here a practical solution to this problem, making use of graph clustering combined with the open-source software package HH-suite, which enables highly sensitive detection of sequence relationships. Carrying out multiple rounds of homology searches via alignment of profile hidden Markov models, large sets of related proteins are generated. By representing the relationships between proteins in these sets as graphs, subsequent clustering with the Markov cluster algorithm enables robust detection of repeat protein subfamilies.
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http://dx.doi.org/10.1007/978-1-4939-8736-8_13DOI Listing
May 2019

Editorial overview: Sequences and topology: Dynamic sequences and topologies of proteins.

Curr Opin Struct Biol 2018 06;50:vii-viii

Wellcome Trust Sanger Institute, Wellcome Genome Campus, Cambridge CB10 1SA, UK. Electronic address:

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http://dx.doi.org/10.1016/j.sbi.2018.07.005DOI Listing
June 2018

Interrogation of Mammalian Protein Complex Structure, Function, and Membership Using Genome-Scale Fitness Screens.

Cell Syst 2018 05 16;6(5):555-568.e7. Epub 2018 May 16.

Dana-Farber Cancer Institute, 450 Brookline Avenue, Boston, MA 02215, USA; Broad Institute of Harvard and MIT, 415 Main Street, Cambridge, MA 02142, USA; Harvard Medical School, Boston, MA 02115, USA. Electronic address:

Protein complexes are assemblies of subunits that have co-evolved to execute one or many coordinated functions in the cellular environment. Functional annotation of mammalian protein complexes is critical to understanding biological processes, as well as disease mechanisms. Here, we used genetic co-essentiality derived from genome-scale RNAi- and CRISPR-Cas9-based fitness screens performed across hundreds of human cancer cell lines to assign measures of functional similarity. From these measures, we systematically built and characterized functional similarity networks that recapitulate known structural and functional features of well-studied protein complexes and resolve novel functional modules within complexes lacking structural resolution, such as the mammalian SWI/SNF complex. Finally, by integrating functional networks with large protein-protein interaction networks, we discovered novel protein complexes involving recently evolved genes of unknown function. Taken together, these findings demonstrate the utility of genetic perturbation screens alone, and in combination with large-scale biophysical data, to enhance our understanding of mammalian protein complexes in normal and disease states.
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http://dx.doi.org/10.1016/j.cels.2018.04.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6152908PMC
May 2018

Computational Modelling of Protein Complex Structure and Assembly.

Methods Mol Biol 2018 ;1764:347-356

MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK.

Sequence and structure space are nowadays sufficiently large that we can use computational methods to model the structure of proteins based on sequence similarity alone. Not only useful as a standalone tool, homology modelling has also had a transformative effect on the ease with which we can solve crystal structures and electron density maps. Another technique-molecular dynamics-aims to model protein structures from first principles and, thanks to increases in computational power, is slowly becoming a viable tool for studying protein complexes. Finally, the prediction of protein assembly pathways from three-dimensional structures of complexes is also now becoming possible.
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http://dx.doi.org/10.1007/978-1-4939-7759-8_22DOI Listing
February 2019

Experimental Characterization of Protein Complex Structure, Dynamics, and Assembly.

Methods Mol Biol 2018 ;1764:3-27

MRC Human Genetics Unit, Institute of Genetics & Molecular Medicine, University of Edinburgh, Edinburgh, UK.

Experimental methods for the characterization of protein complexes have been instrumental in achieving our current understanding of the protein universe and continue to progress with each year that passes. In this chapter, we review some of the most important tools and techniques in the field, covering the important points in X-ray crystallography, cryo-electron microscopy, NMR spectroscopy, and mass spectrometry. Novel developments are making it possible to study large protein complexes at near-atomic resolutions, and we also now have the ability to study the dynamics and assembly pathways of protein complexes across a range of sizes.
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http://dx.doi.org/10.1007/978-1-4939-7759-8_1DOI Listing
February 2019

Ligand Binding Site Structure Influences the Evolution of Protein Complex Function and Topology.

Cell Rep 2018 03;22(12):3265-3276

MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine, University of Edinburgh, Crewe Road, Edinburgh EH4 2XU, UK.

It has been suggested that the evolution of protein complexes is significantly influenced by stochastic, non-adaptive processes. Using ligand binding as a proxy of function, we show that the structure of ligand-binding sites significantly influences the evolution of protein complexes. We show that homomers with multi-chain binding sites (MBSs) evolve new functions slower than monomers or other homomers, and those binding cofactors and metals have more conserved quaternary structure than other homomers. Moreover, the ligands and ligand-binding pockets of homologous MBS homomers are more similar than monomers and other homomers. Our results suggest strong evolutionary selection for quaternary structure in cofactor-binding MBS homomers, whereas neutral processes are more important in complexes with single-chain binding sites. They also have pharmacological implications, suggesting that complexes with single-chain binding sites are better targets for selective drugs, whereas MBS homomers are good candidates for broad-spectrum antibiotic and multitarget drug design.
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http://dx.doi.org/10.1016/j.celrep.2018.02.085DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5873459PMC
March 2018

Cotranslational protein assembly imposes evolutionary constraints on homomeric proteins.

Nat Struct Mol Biol 2018 03 12;25(3):279-288. Epub 2018 Feb 12.

Wellcome Trust Sanger Institute, Wellcome Genome Campus, Cambridge, UK.

Cotranslational protein folding can facilitate rapid formation of functional structures. However, it can also cause premature assembly of protein complexes, if two interacting nascent chains are in close proximity. By analyzing known protein structures, we show that homomeric protein contacts are enriched toward the C termini of polypeptide chains across diverse proteomes. We hypothesize that this is the result of evolutionary constraints for folding to occur before assembly. Using high-throughput imaging of protein homomers in Escherichia coli and engineered protein constructs with N- and C-terminal oligomerization domains, we show that, indeed, proteins with C-terminal homomeric interface residues consistently assemble more efficiently than those with N-terminal interface residues. Using in vivo, in vitro and in silico experiments, we identify features that govern successful assembly of homomers, which have implications for protein design and expression optimization.
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http://dx.doi.org/10.1038/s41594-018-0029-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5995306PMC
March 2018

Characterization of a novel RP2-OSTF1 interaction and its implication for actin remodelling.

J Cell Sci 2018 02 20;131(4). Epub 2018 Feb 20.

MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine, University of Edinburgh, Western General Hospital, Crewe Road, Edinburgh EH4 2XU, UK

Retinitis pigmentosa 2 () is the causative gene for a form of X-linked retinal degeneration. RP2 was previously shown to have GTPase-activating protein (GAP) activity towards the small GTPase ARL3 via its N-terminus, but the function of the C-terminus remains elusive. Here, we report a novel interaction between RP2 and osteoclast-stimulating factor 1 (OSTF1), an intracellular protein that indirectly enhances osteoclast formation and activity and is a negative regulator of cell motility. Moreover, this interaction is abolished by a human pathogenic mutation in RP2. We utilized a structure-based approach to pinpoint the binding interface to a strictly conserved cluster of residues on the surface of RP2 that spans both the C- and N-terminal domains of the protein, and which is structurally distinct from the ARL3-binding site. In addition, we show that RP2 is a positive regulator of cell motility , recruiting OSTF1 to the cell membrane and preventing its interaction with the migration regulator Myo1E.
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http://dx.doi.org/10.1242/jcs.211748DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5868953PMC
February 2018

The genetic basis and evolution of red blood cell sickling in deer.

Nat Ecol Evol 2018 02 18;2(2):367-376. Epub 2017 Dec 18.

Molecular Systems Group, Medical Research Council London Institute of Medical Sciences, Du Cane Road, London, United Kingdom.

Crescent-shaped red blood cells, the hallmark of sickle-cell disease, present a striking departure from the biconcave disc shape normally found in mammals. Characterized by increased mechanical fragility, sickled cells promote haemolytic anaemia and vaso-occlusions and contribute directly to disease in humans. Remarkably, a similar sickle-shaped morphology has been observed in erythrocytes from several deer species, without obvious pathological consequences. The genetic basis of erythrocyte sickling in deer, however, remains unknown. Here, we determine the sequences of human β-globin orthologues in 15 deer species and use protein structural modelling to identify a sickling mechanism distinct from the human disease, coordinated by a derived valine (E22V) that is unique to sickling deer. Evidence for long-term maintenance of a trans-species sickling/non-sickling polymorphism suggests that sickling in deer is adaptive. Our results have implications for understanding the ecological regimes and molecular architectures that have promoted convergent evolution of sickling erythrocytes across vertebrates.
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http://dx.doi.org/10.1038/s41559-017-0420-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5777626PMC
February 2018