Publications by authors named "Russell S Lo"

8 Publications

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Exploiting the Autozygome to Support Previously Published Mendelian Gene-Disease Associations: An Update.

Front Genet 2020 31;11:580484. Epub 2020 Dec 31.

Department of Genetics, King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia.

There is a growing interest in standardizing gene-disease associations for the purpose of facilitating the proper classification of variants in the context of Mendelian diseases. One key line of evidence is the independent observation of pathogenic variants in unrelated individuals with similar phenotypes. Here, we expand on our previous effort to exploit the power of autozygosity to produce homozygous pathogenic variants that are otherwise very difficult to encounter in the homozygous state due to their rarity. The identification of such variants in genes with only tentative associations to Mendelian diseases can add to the existing evidence when observed in the context of compatible phenotypes. In this study, we report 20 homozygous variants in 18 genes (, and ) that satisfy the ACMG classification for pathogenic/likely pathogenic if the involved genes had confirmed rather than tentative links to diseases. These variants were selected because they were truncating, founder with compelling segregation or supported by robust functional assays as with the variant that we present its validation using yeast model. Our findings support the previously reported disease associations for these genes and represent a step toward their confirmation.
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http://dx.doi.org/10.3389/fgene.2020.580484DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7806527PMC
December 2020

A yeast-based complementation assay elucidates the functional impact of 200 missense variants in human PSAT1.

J Inherit Metab Dis 2020 07 27;43(4):758-769. Epub 2020 Feb 27.

Pacific Northwest Research Institute, Seattle, Washington.

Defects in serine biosynthesis resulting from loss of function mutations in PHGDH, PSAT1, and PSPH cause a set of rare, autosomal recessive diseases known as Neu-Laxova syndrome (NLS) or serine-deficiency disorders. The diseases present with a broad range of phenotypes including lethality, severe neurological manifestations, seizures, and intellectual disability. However, because L-serine supplementation, especially if started prenatally, can ameliorate and in some cases even prevent symptoms, knowledge of pathogenic variants is medically actionable. Here, we describe a functional assay that leverages the evolutionary conservation of an enzyme in the serine biosynthesis pathway, phosphoserine aminotransferase, and the ability of the human protein-coding sequence (PSAT1) to functionally replace its yeast ortholog (SER1). Results from our quantitative, yeast-based assay agree well with clinical annotations and expectations based on the disease literature. Using this assay, we have measured the functional impact of the 199 PSAT1 variants currently listed in ClinVar, gnomAD, and the literature. We anticipate that the assay could be used to comprehensively assess the functional impact of all SNP-accessible amino acid substitution mutations in PSAT1, a resource that could aid variant interpretation and identify potential NLS carriers.
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http://dx.doi.org/10.1002/jimd.12227DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7444316PMC
July 2020

A tetO Toolkit To Alter Expression of Genes in Saccharomyces cerevisiae.

ACS Synth Biol 2015 Jul 17;4(7):842-52. Epub 2015 Mar 17.

†Howard Hughes Medical Institute, ‡Department of Genome Sciences, §Department of Medicine, University of Washington, Seattle, Washington 98195, United States.

Strategies to optimize a metabolic pathway often involve building a large collection of strains, each containing different versions of sequences that regulate the expression of pathway genes. Here, we develop reagents and methods to carry out this process at high efficiency in the yeast Saccharomyces cerevisiae. We identify variants of the Escherichia coli tet operator (tetO) sequence that bind a TetR-VP16 activator with differential affinity and therefore result in different TetR-VP16 activator-driven expression. By recombining these variants upstream of the genes of a pathway, we generate unique combinations of expression levels. Here, we built a tetO toolkit, which includes the I-OnuI homing endonuclease to create double-strand breaks, which increases homologous recombination by 10(5); a plasmid carrying six variant tetO sequences flanked by I-OnuI sites, uncoupling transformation and recombination steps; an S. cerevisiae-optimized TetR-VP16 activator; and a vector to integrate constructs into the yeast genome. We introduce into the S. cerevisiae genome the three crt genes from Erwinia herbicola required for yeast to synthesize lycopene and carry out the recombination process to produce a population of cells with permutations of tetO variants regulating the three genes. We identify 0.7% of this population as making detectable lycopene, of which the vast majority have undergone recombination at all three crt genes. We estimate a rate of ∼20% recombination per targeted site, much higher than that obtained in other studies. Application of this toolkit to medically or industrially important end products could reduce the time and labor required to optimize the expression of a set of metabolic genes.
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http://dx.doi.org/10.1021/sb500363yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4506738PMC
July 2015

Massively parallel single-amino-acid mutagenesis.

Nat Methods 2015 Mar 5;12(3):203-6, 4 p following 206. Epub 2015 Jan 5.

Department of Genome Sciences, University of Washington, Seattle, Washington, USA.

Random mutagenesis methods only partially cover the mutational space and are constrained by DNA synthesis length limitations. Here we demonstrate programmed allelic series (PALS), a single-volume, site-directed mutagenesis approach using microarray-programmed oligonucleotides. We created libraries including nearly every missense mutation as singleton events for the yeast transcription factor Gal4 (99.9% coverage) and human tumor suppressor p53 (93.5%). PALS-based comprehensive missense mutational scans may aid structure-function studies, protein engineering, and the interpretation of variants identified by clinical sequencing.
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http://dx.doi.org/10.1038/nmeth.3223DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4344410PMC
March 2015

Protein interaction analysis of senataxin and the ALS4 L389S mutant yields insights into senataxin post-translational modification and uncovers mutant-specific binding with a brain cytoplasmic RNA-encoded peptide.

PLoS One 2013 11;8(11):e78837. Epub 2013 Nov 11.

Comparative Genomics Centre, School of Pharmacy and Molecular Sciences, James Cook University, Townsville, Queensland, Australia ; Department of Pediatrics, University of California San Diego, La Jolla, California, United States of America.

Senataxin is a large 303 kDa protein linked to neuron survival, as recessive mutations cause Ataxia with Oculomotor Apraxia type 2 (AOA2), and dominant mutations cause amyotrophic lateral sclerosis type 4 (ALS4). Senataxin contains an amino-terminal protein-interaction domain and a carboxy-terminal DNA/RNA helicase domain. In this study, we focused upon the common ALS4 mutation, L389S, by performing yeast two-hybrid screens of a human brain expression library with control senataxin or L389S senataxin as bait. Interacting clones identified from the two screens were collated, and redundant hits and false positives subtracted to yield a set of 13 protein interactors. Among these hits, we discovered a highly specific and reproducible interaction of L389S senataxin with a peptide encoded by the antisense sequence of a brain-specific non-coding RNA, known as BCYRN1. We further found that L389S senataxin interacts with other proteins containing regions of conserved homology with the BCYRN1 reverse complement-encoded peptide, suggesting that such aberrant protein interactions may contribute to L389S ALS4 disease pathogenesis. As the yeast two-hybrid screen also demonstrated senataxin self-association, we confirmed senataxin dimerization via its amino-terminal binding domain and determined that the L389S mutation does not abrogate senataxin self-association. Finally, based upon detection of interactions between senataxin and ubiquitin-SUMO pathway modification enzymes, we examined senataxin for the presence of ubiquitin and SUMO monomers, and observed this post-translational modification. Our senataxin protein interaction study reveals a number of features of senataxin biology that shed light on senataxin normal function and likely on senataxin molecular pathology in ALS4.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0078837PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3823977PMC
August 2014

Activity-enhancing mutations in an E3 ubiquitin ligase identified by high-throughput mutagenesis.

Proc Natl Acad Sci U S A 2013 Apr 18;110(14):E1263-72. Epub 2013 Mar 18.

Howard Hughes Medical Institute, Department of Genome Sciences, University of Washington, Seattle, WA 98195, USA.

Although ubiquitination plays a critical role in virtually all cellular processes, mechanistic details of ubiquitin (Ub) transfer are still being defined. To identify the molecular determinants within E3 ligases that modulate activity, we scored each member of a library of nearly 100,000 protein variants of the murine ubiquitination factor E4B (Ube4b) U-box domain for auto-ubiquitination activity in the presence of the E2 UbcH5c. This assay identified mutations that enhance activity both in vitro and in cellular p53 degradation assays. The activity-enhancing mutations fall into two distinct mechanistic classes: One increases the U-box:E2-binding affinity, and the other allosterically stimulates the formation of catalytically active conformations of the E2∼Ub conjugate. The same mutations enhance E3 activity in the presence of another E2, Ube2w, implying a common allosteric mechanism, and therefore the general applicability of our observations to other E3s. A comparison of the E3 activity with the two different E2s identified an additional variant that exhibits E3:E2 specificity. Our results highlight the general utility of high-throughput mutagenesis in delineating the molecular basis of enzyme activity.
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http://dx.doi.org/10.1073/pnas.1303309110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3619334PMC
April 2013

Sites of ubiquitin attachment in Saccharomyces cerevisiae.

Proteomics 2012 Jan 20;12(2):236-40. Epub 2011 Dec 20.

Howard Hughes Medical Institute, University of Washington, Seattle, WA, USA.

Sites of ubiquitin modification have been identified by mass spectrometry based on the increase in molecular mass of a tryptic peptide carrying two additional glycine residues from the ubiquitin moiety. However, such peptides with GG shifts have been difficult to discover. We identify 870 unique sites of ubiquitin attachment on 438 different proteins of the yeast Saccharomyces cerevisiae.
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http://dx.doi.org/10.1002/pmic.201100166DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3337332PMC
January 2012

Large-scale identification of yeast integral membrane protein interactions.

Proc Natl Acad Sci U S A 2005 Aug 10;102(34):12123-8. Epub 2005 Aug 10.

Department of Genome Sciences, Howard Hughes Medical Institute, University of Washington, Seattle, WA 98195, USA.

We carried out a large-scale screen to identify interactions between integral membrane proteins of Saccharomyces cerevisiae by using a modified split-ubiquitin technique. Among 705 proteins annotated as integral membrane, we identified 1,985 putative interactions involving 536 proteins. To ascribe confidence levels to the interactions, we used a support vector machine algorithm to classify interactions based on the assay results and protein data derived from the literature. Previously identified and computationally supported interactions were used to train the support vector machine, which identified 131 interactions of highest confidence, 209 of the next highest confidence, 468 of the next highest, and the remaining 1,085 of low confidence. This study provides numerous putative interactions among a class of proteins that have been difficult to analyze on a high-throughput basis by other approaches. The results identify potential previously undescribed components of established biological processes and roles for integral membrane proteins of ascribed functions.
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http://dx.doi.org/10.1073/pnas.0505482102DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1189342PMC
August 2005