A substrate binding model for the KEOPS tRNA modifying complex

AbstractThe KEOPS complex, which is conserved across archaea and eukaryotes, is composed of four core subunits; Pcc1, Kae1, Bud32 and Cgi121. KEOPS is crucial for the fitness of all organisms examined. In humans, pathogenic mutations in KEOPS genes lead to Galloway–Mowat syndrome, an autosomal-recessive disease causing childhood lethality. Kae1 catalyzes the universal and essential tRNA modification N6-threonylcarbamoyl adenosine, but the precise roles of all other KEOPS subunits remain an enigma. Here we show using structure-guided studies that Cgi121 recruits tRNA to KEOPS by binding to its 3’ CCA tail. A composite model of KEOPS bound to tRNA reveals that all KEOPS subunits form an extended tRNA-binding surface that we have validated in vitro and in vivo to mediate the interaction with the tRNA substrate and its modification. These findings provide a framework for understanding the inner workings of KEOPS and delineate why all KEOPS subunits are essential.

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Structure-Function Analysis of Escherichia coli MnmG (GidA), a Highly Conserved tRNA-Modifying Enzyme

Journal of Bacteriology ◽

10.1128/jb.00650-09 ◽

2009 ◽

Vol 191 (24) ◽

pp. 7614-7619 ◽

Cited By ~ 32

Author(s):

Rong Shi ◽

Magda Villarroya ◽

Rafael Ruiz-Partida ◽

Yunge Li ◽

Ariane Proteau ◽

...

Keyword(s):

Escherichia Coli ◽

Conformational Changes ◽

Function Analysis ◽

Trna Modification ◽

Conserved Residues ◽

Limited Trypsinolysis ◽

Fad Binding ◽

Trna Binding

ABSTRACT The MnmE-MnmG complex is involved in tRNA modification. We have determined the crystal structure of Escherichia coli MnmG at 2.4-Å resolution, mutated highly conserved residues with putative roles in flavin adenine dinucleotide (FAD) or tRNA binding and MnmE interaction, and analyzed the effects of these mutations in vivo and in vitro. Limited trypsinolysis of MnmG suggests significant conformational changes upon FAD binding.

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DJ-1 regulates the integrity and function of ER-mitochondria association through interaction with IP3R3-Grp75-VDAC1

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1906565116 ◽

2019 ◽

Vol 116 (50) ◽

pp. 25322-25328 ◽

Cited By ~ 20

Author(s):

Yi Liu ◽

Xiaopin Ma ◽

Hisashi Fujioka ◽

Jun Liu ◽

Shengdi Chen ◽

...

Keyword(s):

Autosomal Recessive ◽

Cellular Mechanism ◽

Loss Of Function ◽

Wild Type ◽

Calcium Efflux ◽

And Function ◽

The Brain ◽

Early Onset Parkinson’S Disease

Loss-of-function mutations in DJ-1 are associated with autosomal recessive early onset Parkinson’s disease (PD), yet the underlying pathogenic mechanism remains elusive. Here we demonstrate that DJ-1 localized to the mitochondria-associated membrane (MAM) both in vitro and in vivo. In fact, DJ-1 physically interacts with and is an essential component of the IP3R3-Grp75-VDAC1 complexes at MAM. Loss of DJ-1 disrupted the IP3R3-Grp75-VDAC1 complex and led to reduced endoplasmic reticulum (ER)-mitochondria association and disturbed function of MAM and mitochondria in vitro. These deficits could be rescued by wild-type DJ-1 but not by the familial PD-associated L166P mutant which had demonstrated reduced interaction with IP3R3-Grp75. Furthermore, DJ-1 ablation disturbed calcium efflux-induced IP3R3 degradation after carbachol treatment and caused IP3R3 accumulation at the MAM in vitro. Importantly, similar deficits in IP3R3-Grp75-VDAC1 complexes and MAM were found in the brain of DJ-1 knockout mice in vivo. The DJ-1 level was reduced in the substantia nigra of sporadic PD patients, which was associated with reduced IP3R3-DJ-1 interaction and ER-mitochondria association. Together, these findings offer insights into the cellular mechanism in the involvement of DJ-1 in the regulation of the integrity and calcium cross-talk between ER and mitochondria and suggests that impaired ER-mitochondria association could contribute to the pathogenesis of PD.

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R51Q SNX10 induces osteopetrosis by promoting uncontrolled fusion of monocytes to form giant, non-functional osteoclasts

10.1101/332551 ◽

2018 ◽

Author(s):

Maayan Barnea ◽

Merle Stein ◽

Sabina Winograd-Katz ◽

Moran Shalev ◽

Esther Arman ◽

...

Keyword(s):

Bone Resorption ◽

Mouse Model ◽

Autosomal Recessive ◽

Molecular Mechanisms ◽

Precursor Cell ◽

Unique Combination ◽

Sorting Nexin ◽

Autosomal Recessive Osteopetrosis

SummaryThe molecular mechanisms that regulate fusion of monocytes into functional osteoclasts are virtually unknown. We describe a knock-in mouse model for the R51Q mutation in sorting nexin 10 (SNX10) that exhibits osteopetrosis and related symptoms of patients of autosomal recessive osteopetrosis linked to this mutation. Osteopetrosis arises in homozygous R51Q SNX10 mice due to a unique combination of reduced numbers of osteoclasts that are non-functional. Fusion of mutant monocytes is deregulated and occurs rapidly and continuously to form giant, non-functional osteoclasts. Mutant osteoclasts mature quickly and survive poorly in vitro, possibly accounting for their scarcity in vivo. These cells also exhibit impaired ruffled borders, which are required for bone resorption, providing an additional basis for the osteopetrotic phenotype. More broadly, we propose that the maximal size of osteoclasts is actively determined by a genetically-regulated, cell-autonomous mechanism that limits precursor cell fusion, and for which SNX10 is required.

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Interaction between p53 N terminus and core domain regulates specific and nonspecific DNA binding

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1903077116 ◽

2019 ◽

Vol 116 (18) ◽

pp. 8859-8868 ◽

Cited By ~ 10

Author(s):

Fan He ◽

Wade Borcherds ◽

Tanjing Song ◽

Xi Wei ◽

Mousumi Das ◽

...

Keyword(s):

Dna Binding ◽

Posttranslational Modifications ◽

Dynamic Control ◽

Transactivation Domains ◽

Binding Surface ◽

N Terminus ◽

Response To Stress ◽

Stress Signals

The p53 tumor suppressor is a sequence-specific DNA binding protein that activates gene transcription to regulate cell survival and proliferation. Dynamic control of p53 degradation and DNA binding in response to stress signals are critical for tumor suppression. The p53 N terminus (NT) contains two transactivation domains (TAD1 and TAD2), a proline-rich region (PRR), and multiple phosphorylation sites. Previous work revealed the p53 NT reduced DNA binding in vitro. Here, we show that TAD2 and the PRR inhibit DNA binding by directly interacting with the sequence-specific DNA binding domain (DBD). NMR spectroscopy revealed that TAD2 and the PRR interact with the DBD at or near the DNA binding surface, possibly acting as a nucleic acid mimetic to competitively block DNA binding. In vitro and in vivo DNA binding analyses showed that the NT reduced p53 DNA binding affinity but improved the ability of p53 to distinguish between specific and nonspecific sequences. MDMX inhibits p53 binding to specific target promoters but stimulates binding to nonspecific chromatin sites. The results suggest that the p53 NT regulates the affinity and specificity of DNA binding by the DBD. The p53 NT-interacting proteins and posttranslational modifications may regulate DNA binding, partly by modulating the NT–DBD interaction.

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A Novel Homozygous Deletion within the FRY Gene Associated with Nonsyndromic Developmental Delay

Cytogenetic and Genome Research ◽

10.1159/000502598 ◽

2019 ◽

Vol 159 (1) ◽

pp. 19-25 ◽

Cited By ~ 1

Author(s):

Prabakaran Paulraj ◽

Michelle Bosworth ◽

Maria Longhurst ◽

Callie Hornbuckle ◽

Garrett Gotway ◽

...

Keyword(s):

Developmental Delay ◽

Autosomal Recessive ◽

Pediatric Population ◽

Homozygous Deletion ◽

Homozygous Mutation ◽

Biallelic Mutation ◽

Snp Microarray ◽

Low Level

The role of autosomal recessive (AR) variants in clinically heterogeneous conditions such as intellectual disability and developmental delay (ID/DD) has been difficult to uncover. Implication of causative pathogenic AR variants often requires investigation within large and consanguineous families, and/or identifying rare biallelic variants in affected individuals. Furthermore, detection of homozygous gene-level copy number variants during first-line genomic microarray testing in the pediatric population is a rare finding. We describe a 6.7-year-old male patient with ID/DD and a novel homozygous deletion involving the FRY gene identified by genomic SNP microarray. This deletion was observed within a large region of homozygosity on the long arm of chromosome 13 and in a background of increased low-level (2.6%) autosomal homozygosity, consistent with a reported common ancestry in the family. FRY encodes a protein that regulates cell cytoskeletal dynamics, functions in chromosomal alignment in mitosis in vitro, and has been shown to function in the nervous system in vivo. Homozygous mutation of FRY has been previously reported in 2 consanguineous families from studies of autosomal recessive ID in Middle Eastern and Northern African populations. This report provides additional supportive evidence that deleterious biallelic mutation of FRY is associated with ID/DD and illustrates the utility of genomic SNP microarray detection of low-level homozygosity.

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The Hemoglobin-Deficit Mouse: Analysis of Phenotype and Hematopoiesis in the Transplant Model

Blood ◽

10.1182/blood.v90.5.2062 ◽

1997 ◽

Vol 90 (5) ◽

pp. 2062-2067 ◽

Cited By ~ 8

Author(s):

Michael L. Bloom ◽

Karen L. Simon-Stoos

Keyword(s):

Autosomal Recessive ◽

Significant Contribution ◽

Erythroid Differentiation ◽

Microcytic Anemia ◽

Mouse Mutant ◽

In Vivo Assays ◽

Transplant Model ◽

Autosomal Recessive Manner

Abstract The mouse mutant hemoglobin deficit (gene symbol hbd ) is characterized by a severe microcytic anemia that is inherited in an autosomal-recessive manner. To assess the mutation's effect on hematopoiesis, unfractionated bone marrow (BM) from either a mutant C57BL6/J-hbd/hbd, Gpi1b/Gpi1b (phenotype symbol HBD), or normal C57BL6/J -+hbd/+hbd, Gpi1b/Gpi1b mouse was injected intravenously into irradiated congenic C57BL6/J-+hbd/+hbd, Gpi1a/Gpi1a, Igha/Igha, Thy1a/Thy1a mice. The congenic recipients of mutant or normal marrow obtained complete red blood cell (RBC) and leukocyte reconstitution, with the exception of one recipient of HBD marrow. After 24 weeks posttransplantation, the normal recipients of HBD marrow obtained a microcytic anemia similar to the donor. These results suggest that the HBD phenotype is caused by a BM defect. We observed that the erythroid lineage derived from donor HBD marrow repopulated more slowly than the normal marrow at 4 weeks posttransplantation. To determine if this difference was a result of an erythropoietic defect, competitive repopulation was performed using either mutant or normal marrow competed against normal congenic marrow. For the erythroid lineage, no significant contribution from HBD marrow was observed. To assess if the RBC block was based on a deficiency of myeloid progenitors, both in vitro and in vivo assays were performed: absolute numbers of bone progenitors were increased, suggesting that the defect results in a late block to erythroid differentiation.

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Molecular basis for t6A modification in human mitochondria

Nucleic Acids Research ◽

10.1093/nar/gkaa093 ◽

2020 ◽

Vol 48 (6) ◽

pp. 3181-3194 ◽

Cited By ~ 2

Author(s):

Jing-Bo Zhou ◽

Yong Wang ◽

Qi-Yu Zeng ◽

Shi-Xin Meng ◽

En-Duo Wang ◽

...

Keyword(s):

Base Pair ◽

Molecular Basis ◽

Protein Modification ◽

Trna Modification ◽

Anticodon Loop ◽

Recognition Mechanism ◽

Trna Recognition ◽

Translational Accuracy

Abstract N 6-Threonylcarbamoyladenosine (t6A) is a universal tRNA modification essential for translational accuracy and fidelity. In human mitochondria, YrdC synthesises an l-threonylcarbamoyl adenylate (TC-AMP) intermediate, and OSGEPL1 transfers the TC-moiety to five tRNAs, including human mitochondrial tRNAThr (hmtRNAThr). Mutation of hmtRNAs, YrdC and OSGEPL1, affecting efficient t6A modification, has been implicated in various human diseases. However, little is known about the tRNA recognition mechanism in t6A formation in human mitochondria. Herein, we showed that OSGEPL1 is a monomer and is unique in utilising C34 as an anti-determinant by studying the contributions of individual bases in the anticodon loop of hmtRNAThr to t6A modification. OSGEPL1 activity was greatly enhanced by introducing G38A in hmtRNAIle or the A28:U42 base pair in a chimeric tRNA containing the anticodon stem of hmtRNASer(AGY), suggesting that sequences of specific hmtRNAs are fine-tuned for different modification levels. Moreover, using purified OSGEPL1, we identified multiple acetylation sites, and OSGEPL1 activity was readily affected by acetylation via multiple mechanisms in vitro and in vivo. Collectively, we systematically elucidated the nucleotide requirement in the anticodon loop of hmtRNAs, and revealed mechanisms involving tRNA sequence optimisation and post-translational protein modification that determine t6A modification levels.

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A Human TATA Binding Protein-Related Protein with Altered DNA Binding Specificity Inhibits Transcription from Multiple Promoters and Activators

Molecular and Cellular Biology ◽

10.1128/mcb.19.11.7610 ◽

1999 ◽

Vol 19 (11) ◽

pp. 7610-7620 ◽

Cited By ~ 66

Author(s):

Paul A. Moore ◽

Josef Ozer ◽

Moreh Salunek ◽

Gwenael Jan ◽

Dennis Zerby ◽

...

Keyword(s):

Dna Binding ◽

Transcriptional Repression ◽

Transcription Initiation ◽

Binding Protein ◽

Tata Binding Protein ◽

Related Protein ◽

Multiple Promoters ◽

Binding Surface

ABSTRACT The TATA binding protein (TBP) plays a central role in eukaryotic and archael transcription initiation. We describe the isolation of a novel 23-kDa human protein that displays 41% identity to TBP and is expressed in most human tissue. Recombinant TBP-related protein (TRP) displayed barely detectable binding to consensus TATA box sequences but bound with slightly higher affinities to nonconsensus TATA sequences. TRP did not substitute for TBP in transcription reactions in vitro. However, addition of TRP potently inhibited basal and activated transcription from multiple promoters in vitro and in vivo. General transcription factors TFIIA and TFIIB bound glutathioneS-transferase–TRP in solution but failed to stimulate TRP binding to DNA. Preincubation of TRP with TFIIA inhibited TBP-TFIIA-DNA complex formation and addition of TFIIA overcame TRP-mediated transcription repression. TRP transcriptional repression activity was specifically reduced by mutations in TRP that disrupt the TFIIA binding surface but not by mutations that disrupt the TFIIB or DNA binding surface of TRP. These results suggest that TFIIA is a primary target of TRP transcription inhibition and that TRP may modulate transcription by a novel mechanism involving the partial mimicry of TBP functions.

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Global ubiquitylation analysis of mitochondria in primary neurons identifies physiological Parkin targets following activation of PINK1

10.1101/2021.04.01.438131 ◽

2021 ◽

Author(s):

Odetta Antico ◽

Alban Ordureau ◽

Michael Stevens ◽

Francois Singh ◽

Marek Gierlinski ◽

...

Keyword(s):

Autosomal Recessive ◽

Mitochondrial Outer Membrane ◽

Culture Studies ◽

Parkinsons Disease ◽

Recessive Mutations ◽

Ubiquitin E3 Ligase ◽

Cytoplasmic Proteins ◽

Online Resource

Autosomal recessive mutations in PINK1 and Parkin cause Parkinsons disease. How activation of PINK1 and Parkin leads to elimination of damaged mitochondria by mitophagy is largely based on cell culture studies with few molecular studies in neurons. Herein we have undertaken a global proteomic- analysis of mitochondria from mouse neurons to identify ubiquitylated substrates of endogenous Parkin activation. Comparative analysis with human iNeuron datasets revealed a subset of 49 PINK1-dependent diGLY sites upregulated upon mitochondrial depolarisation in 22 proteins conserved across mouse and human systems. These proteins were exclusively localised at the mitochondrial outer membrane (MOM) including, CISD1, CPT1A, ACSL1, and FAM213A. We demonstrate that these proteins can be directly ubiquitylated by Parkin in vitro. We also provide evidence for a subset of cytoplasmic proteins recruited to mitochondria that undergo PINK1 and Parkin independent ubiquitylation including SNX3, CAMK2A; and CAMK2B; indicating the presence of alternate ubiquitin E3 ligase pathways that are activated by mitochondrial depolarisation in neurons. Finally we have developed an online resource to visualise mitochondrial ubiquitin sites in neurons and search for ubiquitin components recruited to mitochondria upon mitochondrial depolarisation, MitoNUb. This analysis defines the ubiquitin architecture of damaged mitochondria and will aid in future studies to understand Parkin activation in neuronal subtypes. Our findings also suggest that monitoring ubiquitylation status of the 22 identified MOM proteins may represent robust biomarkers for PINK1 and Parkin activity in vivo.

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Reconstitution of defective respiratory burst activity with partially purified human neutrophil cytochrome B in two genetic forms of chronic granulomatous disease: possible role of Rap1A

Blood ◽

10.1182/blood.v79.9.2438.2438 ◽

1992 ◽

Vol 79 (9) ◽

pp. 2438-2445 ◽

Cited By ~ 9

Author(s):

MT Quinn ◽

JT Curnutte ◽

CA Parkos ◽

ML Mullen ◽

PJ Scott ◽

...

Keyword(s):

Chronic Granulomatous Disease ◽

Cytochrome B ◽

Human Neutrophil ◽

Autosomal Recessive ◽

Plasma Membranes ◽

Close Association ◽

Granulomatous Disease ◽

Detergent System

Abstract Neutrophil plasma membranes from patients with the X-linked and autosomal recessive forms of chronic granulomatous disease (CGD) that lack cytochrome b are incapable of generating superoxide anion (O2-) in vivo and in vitro. The O2- generating activity of these defective membranes was reconstituted with the addition of partially purified human neutrophil cytochrome b in a detergent-based, cell-free activation system. Depending on the detergent system used, 50% to 100% of the activity of control membranes was recovered, and this activity was directly dependent on the cytochrome b concentration. However, when cytochrome b was purified to 99% homogeneity, the reconstitutive capacity of the cytochrome was lost, possibly because of subtle denaturation of the cytochrome or the removal of an additional required cofactor. Examination of the latter possibility with respect to a protein known to coassociate with the cytochrome, ie, Rap1A, indicated that this ras-like protein was present in the partially purified cytochrome preparation used to reconstitute activity in CGD membranes, but was missing in the highly purified preparation. However, the finding that Rap1A was present in normal amounts in the neutrophil membranes from all four major types of CGD (including those missing cytochrome b) suggested that the conditions required of the reconstitution assay did not favor the reassociation of the membrane- derived Rap1A with exogenously added cytochrome b or that another unidentified membrane component was lost during the final purification step. The normal expression of Rap1A in CGD cell membranes also indicates that this protein is not responsible for the absence of O2- production in the X-linked and autosomal recessive cytochrome b- negative forms of CGD. Finally, these results show that the expression of Rap1A in the plasma membrane is not dependent on the coordinate expression of cytochrome b, despite the close association shown for these two proteins in the normal cell membrane.

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