Reduced pigmentation (rp), a mouse model of Hermansky-Pudlak syndrome, encodes a novel component of the BLOC-1 complex

Blood ◽  
2004 ◽  
Vol 104 (10) ◽  
pp. 3181-3189 ◽  
Author(s):  
Babette Gwynn ◽  
Jose A. Martina ◽  
Juan S. Bonifacino ◽  
Elena V. Sviderskaya ◽  
M. Lynn Lamoreux ◽  
...  
Keyword(s):  
Amino Acid ◽  
Protein Trafficking ◽  
Protein Complexes ◽  
Stop Codon ◽  
Premature Stop Codon ◽  
Amino Acid Identity ◽  
Organelle Biogenesis ◽  
Hermansky Pudlak Syndrome ◽  
Human Orthologue ◽  
Lysosome Related Organelles

Abstract Hermansky-Pudlak syndrome (HPS), a disorder of organelle biogenesis, affects lysosomes, melanosomes, and platelet dense bodies. Seven genes cause HPS in humans (HPS1-HPS7) and at least 15 nonallelic mutations cause HPS in mice. Where their function is known, the HPS proteins participate in protein trafficking and vesicle docking/fusion events during organelle biogenesis. HPS-associated genes participate in at least 4 distinct protein complexes: the adaptor complex AP-3; biogenesis of lysosome-related organelles complex 1 (BLOC-1), consisting of 4 HPS proteins (pallidin, muted, cappuccino, HPS7/sandy); BLOC-2, consisting of HPS6/ruby-eye, HPS5/ruby-eye-2, and HPS3/cocoa; and BLOC-3, consisting of HPS1/pale ear and HPS4/light ear. Here, we report the cloning of the mouse HPS mutation reduced pigmentation (rp). We show that the wild-type rp gene encodes a novel, widely expressed 195-amino acid protein that shares 87% amino acid identity with its human orthologue and localizes to punctate cytoplasmic structures. Further, we show that phosphorylated RP is part of the BLOC-1 complex. In mutant rp/rp mice, a premature stop codon truncates the protein after 79 amino acids. Defects in all the 5 known components of BLOC-1, including RP, cause severe HPS in mice, suggesting that the subunits are nonredundant and that BLOC-1 plays a key role in organelle biogenesis.

The pink gene encodes the Drosophila orthologue of the human Hermansky–Pudlak syndrome 5 (HPS5) gene

Genome ◽  
2007 ◽  
Vol 50 (6) ◽  
pp. 548-556 ◽  
Author(s):  
Monika Syrzycka ◽  
Lori A. McEachern ◽  
Jennifer Kinneard ◽  
Kristel Prabhu ◽  
Kathleen Fitzpatrick ◽  
...  
Keyword(s):  
Protein Trafficking ◽  
Protein Complexes ◽  
Model Organism ◽  
Autosomal Recessive Disorders ◽  
Dense Granules ◽  
Eye Colour ◽  
Hermansky Pudlak Syndrome ◽  
Lysosome Related Organelles ◽  
Key Participants

Hermansky–Pudlak syndrome (HPS) consists of a set of human autosomal recessive disorders, with symptoms resulting from defects in genes required for protein trafficking in lysosome-related organelles such as melanosomes and platelet dense granules. A number of human HPS genes and rodent orthologues have been identified whose protein products are key components of 1 of 4 different protein complexes (AP-3 or BLOC-1, -2, and -3) that are key participants in the process. Drosophila melanogaster has been a key model organism in demonstrating the in vivo significance of many genes involved in protein trafficking pathways; for example, mutations in the “granule group” genes lead to changes in eye colour arising from improper protein trafficking to pigment granules in the developing eye. An examination of the chromosomal positioning of Drosophila HPS gene orthologues suggested that CG9770, the Drosophila HPS5 orthologue, might correspond to the pink locus. Here we confirm this gene assignment, making pink the first eye colour gene in flies to be identified as a BLOC complex gene.

scholarly journals Cappuccino, a mouse model of Hermansky-Pudlak syndrome, encodes a novel protein that is part of the pallidin-muted complex (BLOC-1)

Blood ◽  
2003 ◽  
Vol 101 (11) ◽  
pp. 4402-4407 ◽  
Author(s):  
Steven L. Ciciotte ◽  
Babette Gwynn ◽  
Kengo Moriyama ◽  
Marjan Huizing ◽  
William A. Gahl ◽  
...  
Keyword(s):  
Amino Acids ◽  
Protein Complexes ◽  
Mutation Screening ◽  
Organelle Biogenesis ◽  
Vesicle Docking ◽  
Dense Bodies ◽  
Associated Proteins ◽  
Hermansky Pudlak Syndrome ◽  
Lysosome Related Organelles ◽  
Novel Protein

Abstract Hermansky-Pudlak syndrome (HPS) is a disorder of organelle biogenesis affecting 3 related organelles—melanosomes, platelet dense bodies, and lysosomes. Four genes causing HPS in humans (HPS1-HPS4) are known, and at least 15 nonallelic mutations cause HPS in the mouse. Where their functions are known, the HPS-associated proteins are involved in some aspect of intracellular vesicular trafficking, that is, protein sorting and vesicle docking and fusion. Biochemical and genetic evidence indicates that the HPS-associated genes encode components of at least 3 distinct protein complexes: the adaptor complex AP-3; the HPS1/HPS4 complex; and BLOC-1 (biogenesis of lysosome-related organelles complex-1), consisting of the proteins encoded at 2 mouse HPS loci, pallid (pa) and muted (mu), and at least 3 other unidentified proteins. Here, we report the cloning of the mouse HPS mutation cappuccino (cno). We show that the wild-type cno gene encodes a novel, ubiquitously expressed cytoplasmic protein that coassembles with pallidin and the muted protein in the BLOC-1 complex. Further, we identify a frameshift mutation in mutant cno/cno mice. The C-terminal 81 amino acids are replaced with 72 different amino acids in the mutant CNO protein, and its ability to interact in BLOC-1 is abolished. We performed mutation screening of patients with HPS and failed to identify any CNO defects. Notably, although defects in components of the HPS1/HPS4 and the AP-3 complexes are associated with HPS in humans, no defects in the known components of BLOC-1 have been identified in 142 patients with HPS screened to date, suggesting that BLOC-1 function may be critical in humans.

scholarly journals A rare large duplication of MLH1 identified in Lynch syndrome

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Abhishek Kumar ◽  
Nagarajan Paramasivam ◽  
Obul Reddy Bandapalli ◽  
Matthias Schlesner ◽  
Tianhui Chen ◽  
...  
Keyword(s):  
Amino Acid ◽  
Lynch Syndrome ◽  
Splice Site ◽  
Stop Codon ◽  
Premature Stop Codon ◽  
Splice Donor Site ◽  
Laboratory Diagnostics ◽  
Mmr Genes ◽  
Base Pair Deletion ◽  
Splice Site Variant

Abstract Background The most frequently identified strong cancer predisposition mutations for colorectal cancer (CRC) are those in the mismatch repair (MMR) genes in Lynch syndrome. Laboratory diagnostics include testing tumors for immunohistochemical staining (IHC) of the Lynch syndrome-associated DNA MMR proteins and/or for microsatellite instability (MSI) followed by sequencing or other techniques, such as denaturing high performance liquid chromatography (DHPLC), to identify the mutation. Methods In an ongoing project focusing on finding Mendelian cancer syndromes we applied whole-exome/whole-genome sequencing (WES/WGS) to 19 CRC families. Results Three families were identified with a pathogenic/likely pathogenic germline variant in a MMR gene that had previously tested negative in DHPLC gene variant screening. All families had a history of CRC in several family members across multiple generations. Tumor analysis showed loss of the MMR protein IHC staining corresponding to the mutated genes, as well as MSI. In family A, a structural variant, a duplication of exons 4 to 13, was identified in MLH1. The duplication was predicted to lead to a frameshift at amino acid 520 and a premature stop codon at amino acid 539. In family B, a 1 base pair deletion was found in MLH1, resulting in a frameshift and a stop codon at amino acid 491. In family C, we identified a splice site variant in MSH2, which was predicted to lead loss of a splice donor site. Conclusions We identified altogether three pathogenic/likely pathogenic variants in the MMR genes in three of the 19 sequenced families. The MLH1 variants, a duplication of exons 4 to 13 and a frameshift variant, were novel, based on the InSiGHT and ClinVar databases; the MSH2 splice site variant was reported by a single submitter in ClinVar. As a variant class, duplications have rarely been reported in the MMR gene literature, particularly those covering several exons.

scholarly journals CRISPR/Cas9-mediated mutation revealed cytoplasmic tail is dispensable for IZUMO1 function and male fertility

Reproduction ◽  
2016 ◽  
Vol 152 (6) ◽  
pp. 665-672 ◽  
Author(s):  
Samantha A M Young ◽  
Haruhiko Miyata ◽  
Yuhkoh Satouh ◽  
Masanaga Muto ◽  
Martin R Larsen ◽  
...  
Keyword(s):  
Amino Acid ◽  
Point Mutation ◽  
Male Fertility ◽  
Stop Codon ◽  
Premature Stop Codon ◽  
Cytoplasmic Tail ◽  
Binding Partner ◽  
C Terminus ◽  
Manipulation System

IZUMO1 is a protein found in the head of spermatozoa that has been identified as essential for sperm–egg fusion. Its binding partner in the egg has been discovered (JUNO); however, the roles of several domains within IZUMO1 remain unexplored. One such domain is the C-terminus, which undergoes major phosphorylation changes in the cytoplasmic portion of the protein during rat epididymal transit. However, the cytoplasmic tail of IZUMO1 in many species is highly variable, ranging from 55 to one amino acid. Therefore, to understand the role of the cytoplasmic tail of IZUMO1 in mouse, we utilised the gene manipulation system of CRISPR/Cas9 to generate a point mutation resulting in a premature stop codon, producing mice with truncated IZUMO1. Mice without the cytoplasmic tail of IZUMO1 showed normal fertility but decreased the amount of protein, indicating that whilst this region is important for the expression level of IZUMO1, it is dispensable for fertilisation in the mouse.

scholarly journals In VivoEffects of Leptin-Related Synthetic Peptides on Body Weight and Food Intake in Female ob/obMice: Localization of Leptin Activity to Domains Between Amino Acid Residues 106–140*

Endocrinology ◽  
1997 ◽  
Vol 138 (4) ◽  
pp. 1413-1418 ◽  
Author(s):  
Patricia Grasso ◽  
Matthew C. Leinung ◽  
Stacy P. Ingher ◽  
Daniel W. Lee
Keyword(s):  
Body Weight ◽  
Weight Gain ◽  
Amino Acid ◽  
Food Intake ◽  
Stop Codon ◽  
Premature Stop Codon ◽  
Amino Acid Residues ◽  
Inactive Form ◽  
In Vivo Effects

Abstract In C57BL/6J ob/ob mice, a single base mutation of the ob gene in codon 105 results in the replacement of arginine by a premature stop codon and production of a truncated inactive form of leptin. These observations suggest that leptin activity may be localized, at least in part, to domains distal to amino acid residue 104. To investigate this possibility, we synthesized six overlapping peptide amides corresponding to residues 106–167 of leptin, and examined their effects on body weight and food intake in female C57BL/6J ob/ob mice. When compared with vehicle-injected control mice, weight gain by mice receiving 28 daily 1-mg ip injections of LEP-(106–120), LEP-(116–130), or LEP-(126–140) was significantly (P < 0.01) reduced with no apparent toxicity. Weight gain by mice receiving LEP-(136–150), LEP-(146–160), or LEP-(156–167) was not significantly different from that of vehicle-injected control mice. The effects of LEP-(106–120), LEP-(116–130), or LEP-(126–140) were most pronounced during the first week of peptide treatment. Within 7 days, mice receiving these peptides lost 12.3%, 13.8%, and 9.8%, respectively, of their initial body weights. After 28 days, mice given vehicle alone, LEP-(136–150), LEP-(146–160), or LEP-(156–167) were 14.7%, 20.3%, 25.0%, and 24.8% heavier, respectively, than they were at the beginning of the study. Mice given LEP-(106–120) or LEP-(126–140) were only 1.8% and 4.2% heavier, respectively, whereas mice given LEP-(116–130) were 3.4% lighter. Food intake by mice receiving LEP-(106–120), LEP-(116–130), or LEP-(126–140), but not by mice receiving LEP-(136–150), LEP-(146–160), or LEP-(156–167), was reduced by 15%. The results of this study indicate 1) that leptin activity is localized, at least in part, in domains between residues 106–140; 2) that leptin-related peptides have in vivo effects similar to those of native leptin; and 3) offer hope for development of peptide analogs of leptin having potential application in human or veterinary medicine.

A G-to-A mutation in IVS-3 of the human gamma fibrinogen gene causing afibrinogenemia due to abnormal RNA splicing

Blood ◽  
2000 ◽  
Vol 96 (7) ◽  
pp. 2501-2505 ◽  
Author(s):  
Maurizio Margaglione ◽  
Rosa Santacroce ◽  
Donatella Colaizzo ◽  
Davide Seripa ◽  
Gennaro Vecchione ◽  
...  
Keyword(s):  
Amino Acid ◽  
Rna Splicing ◽  
Messenger Rna ◽  
Stop Codon ◽  
Premature Stop Codon ◽  
Autosomal Recessive Disorder ◽  
Chain Gene ◽  
Hemorrhagic Diathesis ◽  
Reading Frame ◽  
Congenital Afibrinogenemia

Abstract Congenital afibrinogenemia is a rare autosomal recessive disorder characterized by a hemorrhagic diathesis of variable severity. Although more than 100 families with this disorder have been described, genetic defects have been characterized in few cases. An investigation of a young propositus, offspring of a consanguineous marriage, with undetectable levels of functional and quantitative fibrinogen, was conducted. Sequence analysis of the fibrinogen genes showed a homozygous G-to-A mutation at the fifth nucleotide (nt 2395) of the third intervening sequence (IVS) of the γ-chain gene. Her first-degree relatives, who had approximately half the normal fibrinogen values and showed concordance between functional and immunologic levels, were heterozygtes. The G-to-A change predicts the disappearance of a donor splice site. After transfection with a construct, containing either the wild-type or the mutated sequence, cells with the mutant construct showed an aberrant messenger RNA (mRNA), consistent with skipping of exon 3, but not the expected mRNA. Sequencing of the abnormal mRNA showed the complete absence of exon 3. Skipping of exon 3 predicts the deletion of amino acid sequence from residue 16 to residue 75 and shifting of reading frame at amino acid 76 with a premature stop codon within exon 4 at position 77. Thus, the truncated γ-chain gene product would not interact with other chains to form the mature fibrinogen molecule. The current findings show that mutations within highly conserved IVS regions of fibrinogen genes could affect the efficiency of normal splicing, giving rise to congenital afibrinogenemia.

Defects in the cappuccino (cno) gene on mouse chromosome 5 and human 4p cause Hermansky-Pudlak syndrome by an AP-3–independent mechanism

Blood ◽  
2000 ◽  
Vol 96 (13) ◽  
pp. 4227-4235 ◽  
Author(s):  
Babette Gwynn ◽  
Steven L. Ciciotte ◽  
Susan J. Hunter ◽  
Linda L. Washburn ◽  
Richard S. Smith ◽  
...  
Keyword(s):  
Mouse Chromosome ◽  
Protein Trafficking ◽  
Dense Body ◽  
Spontaneous Mutation ◽  
Oculocutaneous Albinism ◽  
Chromosome 5 ◽  
Prolonged Bleeding ◽  
Independent Mechanism ◽  
Hermansky Pudlak Syndrome ◽  
Lysosome Related Organelles

Defects in a triad of organelles (melanosomes, platelet granules, and lysosomes) result in albinism, prolonged bleeding, and lysosome abnormalities in Hermansky-Pudlak syndrome (HPS). Defects in HPS1, a protein of unknown function, and in components of the AP-3 complex cause some, but not all, cases of HPS in humans. There have been 15 inherited models of HPS described in the mouse, underscoring its marked genetic heterogeneity. Here we characterize a new spontaneous mutation in the mouse, cappuccino (cno), that maps to mouse chromosome 5 in a region conserved with human 4p15-p16. Melanosomes ofcno/cno mice are immature and dramatically decreased in number in the eye and skin, resulting in severe oculocutaneous albinism. Platelet dense body contents (adenosine triphosphate, serotonin) are markedly deficient, leading to defective aggregation and prolonged bleeding. Lysosomal enzyme concentrations are significantly elevated in the kidney and liver. Genetic, immunofluorescence microscopy, and lysosomal protein trafficking studies indicate that the AP-3 complex is intact in cno/cno mice. It was concluded that the cappuccino gene encodes a product involved in an AP-3–independent mechanism critical to the biogenesis of lysosome-related organelles.

scholarly journals YspM, a Newly Identified Ysa Type III Secreted Protein of Yersinia enterocolitica

2008 ◽  
Vol 190 (22) ◽  
pp. 7315-7325 ◽  
Author(s):  
Sarah E. Witowski ◽  
Kimberly A. Walker ◽  
Virginia L. Miller
Keyword(s):  
Amino Acid ◽  
Yersinia Enterocolitica ◽  
Premature Stop Codon ◽  
Amino Acid Identity ◽  
Host Cells ◽  
Secreted Protein ◽  
Functional Difference ◽  
Secretion Systems ◽  
Acid Identity ◽  
Type Iii

ABSTRACT Yersinia enterocolitica has three type three secretion systems, the flagellar, the plasmid Ysc type III secretion system (T3SS), and the chromosomal Ysa T3SS. The Ysc T3SS, through the proteins it secretes (Yops), prevents phagocytosis of Y. enterocolitica and is required for disease processes in the mouse host. Recent data demonstrate a role for the Ysa T3SS during initial colonization of the mouse via secretion of Ysps (Yersinia secreted proteins). This work characterizes the discovery of a newly identified Ysa type III secreted protein, YspM. Expression of yspM is regulated by temperature, NaCl concentration, and other known regulators of the ysa system. In addition, YspM is translocated into host cells via the Ysa T3SS. YspM is homologous to proteins classified as GDSL bacterial lipases, which possess a catalytic triad of amino acids (Ser, Asp, and His) located in three of five blocks of amino acid identity. Sequence analysis of the JB580v strain of Y. enterocolitica shows that, due to a premature stop codon, it no longer encodes the fifth block of amino acid identity containing the predicted catalytic histidine. However, seven other biotype 1B strains sequenced did possess the domain. A functional difference between the forms was revealed when YspM was expressed in Saccharomyces cerevisiae. Yeast growth was uninhibited when YspM from JB580v was expressed but greatly inhibited when YspM from Y295 (YspMY295) was expressed. Site-directed mutagenesis of the histidine of YspMY295 ablated the toxic effects. These results indicate that YspM is secreted by the Ysa T3SS and that, possibly due to lipase activity, it targets eukaryotic cellular component(s).

scholarly journals BLOC-1 Interacts with BLOC-2 and the AP-3 Complex to Facilitate Protein Trafficking on Endosomes

2006 ◽  
Vol 17 (9) ◽  
pp. 4027-4038 ◽  
Author(s):  
Santiago M. Di Pietro ◽  
Juan M. Falcón-Pérez ◽  
Danièle Tenza ◽  
Subba R.G. Setty ◽  
Michael S. Marks ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Protein Trafficking ◽  
Adaptor Protein ◽  
Early Endosome ◽  
Related Protein ◽  
Genes Encoding ◽  
Cellular Machinery ◽  
Hermansky Pudlak Syndrome ◽  
Lysosome Related Organelles ◽  
Tyrosinase Related Protein

The adaptor protein (AP)-3 complex is a component of the cellular machinery that controls protein sorting from endosomes to lysosomes and specialized related organelles such as melanosomes. Mutations in an AP-3 subunit underlie a form of Hermansky-Pudlak syndrome (HPS), a disorder characterized by abnormalities in lysosome-related organelles. HPS in humans can also be caused by mutations in genes encoding subunits of three complexes of unclear function, named biogenesis of lysosome-related organelles complex (BLOC)-1, -2, and -3. Here, we report that BLOC-1 interacts physically and functionally with AP-3 to facilitate the trafficking of a known AP-3 cargo, CD63, and of tyrosinase-related protein 1 (Tyrp1), a melanosomal membrane protein previously thought to traffic only independently of AP-3. BLOC-1 also interacts with BLOC-2 to facilitate Tyrp1 trafficking by a mechanism apparently independent of AP-3 function. Both BLOC-1 and -2 localize mainly to early endosome-associated tubules as determined by immunoelectron microscopy. These findings support the idea that BLOC-1 and -2 represent hitherto unknown components of the endosomal protein trafficking machinery.

A G-to-A mutation in IVS-3 of the human gamma fibrinogen gene causing afibrinogenemia due to abnormal RNA splicing

Blood ◽  
2000 ◽  
Vol 96 (7) ◽  
pp. 2501-2505
Author(s):  
Maurizio Margaglione ◽  
Rosa Santacroce ◽  
Donatella Colaizzo ◽  
Davide Seripa ◽  
Gennaro Vecchione ◽  
...  
Keyword(s):  
Amino Acid ◽  
Rna Splicing ◽  
Messenger Rna ◽  
Stop Codon ◽  
Premature Stop Codon ◽  
Autosomal Recessive Disorder ◽  
Chain Gene ◽  
Hemorrhagic Diathesis ◽  
Reading Frame ◽  
Congenital Afibrinogenemia

Congenital afibrinogenemia is a rare autosomal recessive disorder characterized by a hemorrhagic diathesis of variable severity. Although more than 100 families with this disorder have been described, genetic defects have been characterized in few cases. An investigation of a young propositus, offspring of a consanguineous marriage, with undetectable levels of functional and quantitative fibrinogen, was conducted. Sequence analysis of the fibrinogen genes showed a homozygous G-to-A mutation at the fifth nucleotide (nt 2395) of the third intervening sequence (IVS) of the γ-chain gene. Her first-degree relatives, who had approximately half the normal fibrinogen values and showed concordance between functional and immunologic levels, were heterozygtes. The G-to-A change predicts the disappearance of a donor splice site. After transfection with a construct, containing either the wild-type or the mutated sequence, cells with the mutant construct showed an aberrant messenger RNA (mRNA), consistent with skipping of exon 3, but not the expected mRNA. Sequencing of the abnormal mRNA showed the complete absence of exon 3. Skipping of exon 3 predicts the deletion of amino acid sequence from residue 16 to residue 75 and shifting of reading frame at amino acid 76 with a premature stop codon within exon 4 at position 77. Thus, the truncated γ-chain gene product would not interact with other chains to form the mature fibrinogen molecule. The current findings show that mutations within highly conserved IVS regions of fibrinogen genes could affect the efficiency of normal splicing, giving rise to congenital afibrinogenemia.

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