A functional analysis of the putative polymorphisms A91V and N252S and 22 missense perforin mutations associated with familial hemophagocytic lymphohistiocytosis

Abstract Up to 60% of cases of the autosomal recessive immunodeficiency hemophagocytic lymphohistiocytosis (HLH) are associated with mutations in the perforin (PRF1) gene. In this study, we expressed wild-type and mutated perforin in rat basophil leukemia cells to study the effect on lytic function of the substitutions A91V and N252S (commonly considered to be neutral polymorphisms) and 22 perforin missense substitutions first identified in HLH patients. Surprisingly, we found that A91V perforin was expressed at reduced levels compared with wild-type perforin, resulting in partial loss of lytic capacity. In contrast, expression and function of N252S-substituted perforin were normal. Most HLH-associated mutations resulted in protein degradation (probably due to misfolding) and complete loss of perforin activity, the exception being R232H, which retained approximately 30% wild-type activity. Several other mutated proteins (H222Q, C73R, F157V, and D313V) had no detectable lytic activity but were expressed at normal levels, suggesting that their functional defect might map downstream at the level of the target cell membrane. One further perforin substitution identified in an HLH patient (V183G) was normally expressed and displayed normal lysis. This report represents the first systematic functional analysis of HLH-associated missense mutations and the 2 most common perforin polymorphisms. (Blood. 2005;105:4700-4706)

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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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Abstract TP269: Distinct Molecular Mechanisms of Htra1 Mutants in Manifesting Heterozygotes With Carasil

Stroke ◽

10.1161/str.48.suppl_1.tp269 ◽

2017 ◽

Vol 48 (suppl_1) ◽

Author(s):

Hiroaki Nozaki ◽

Taisuke Kato ◽

Megumi Nihonmatsu ◽

Yohei Saito ◽

Ikuko Mizuta ◽

...

Keyword(s):

Autosomal Recessive ◽

Molecular Mechanisms ◽

Small Vessel Disease ◽

Gel Filtration ◽

Gene Mutations ◽

Missense Mutations ◽

Inherited Disease ◽

Wild Type ◽

Vessel Disease ◽

Activation Cascade

Introduction: Cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL), an autosomal recessive inherited cerebral small vessel disease (CSVD), involves severe leukoaraiosis, multiple lacunar infarcts, early-onset alopecia, and spondylosis deformans. High-temperature requirement serine peptidase A1 (HTRA1) gene mutations cause CARASIL by decreasing HTRA1 protease activity. Although CARASIL is a recessive inherited disease, heterozygous mutations in the HTRA1 gene were recently identified in 11 families with CSVD. Because CSVD is frequently observed in elderly individuals, it is unclear which mutants truly contribute to CSVD pathogenesis. Here, we found heterozygous mutations in the HTRA1 gene in individuals with CSVD and investigated the differences in biochemical characteristics between these mutant HTRA1s and mutant HTRA1s observed in homozygotes. Methods: We recruited 113 unrelated index patients with clinically diagnosed CSVD. The coding sequences of the HTRA1 gene were analyzed. We evaluated HTRA1 protease activities using casein assays and oligomeric HTRA1 formation using gel filtration chromatography. Results: We found 4 heterozygous missense mutations in the HTRA1 gene (p.G283E, p.P285L, p.R302Q, and p.T319I) in 6 patients from 113 unrelated index patients and in 2 siblings in 2 unrelated families with p.R302Q. These mutant HTRA1s showed markedly decreased protease activities and inhibited wild-type HTRA1 activity, whereas 2 of 3 mutant HTRA1s reported in CARASIL (A252T and V297M) did not inhibit wild- type HTRA1 activity. Wild-type HTRA1 forms trimers; however, G283E and T319I HTRA1, observed in manifesting heterozygotes, did not form trimers. P285L and R302Q HTRA1s formed trimers, but their mutations were located in domains that are important for trimer-associated HTRA1 activation; in contrast, A252T and V297M HTRA1s, which have been observed in CARASIL, also formed trimers but had mutations outside the domains important for trimer- associated HTRA1 activation. Conclusions: The mutant HTRA1s observed in manifesting heterozygotes might result in an impaired HTRA1 activation cascade of HTRA1 or be unable to form stable trimers.

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Missense mutations in the human β fibrinogen gene cause congenital afibrinogenemia by impairing fibrinogen secretion

Blood ◽

10.1182/blood.v95.4.1336.004k16_1336_1341 ◽

2000 ◽

Vol 95 (4) ◽

pp. 1336-1341 ◽

Cited By ~ 75

Author(s):

Stefano Duga ◽

Rosanna Asselta ◽

Elena Santagostino ◽

Sirous Zeinali ◽

Tatjana Simonic ◽

...

Keyword(s):

Autosomal Recessive ◽

Autosomal Recessive Disorder ◽

Chain Gene ◽

Missense Mutations ◽

Wild Type ◽

Large Deletions ◽

Congenital Afibrinogenemia ◽

Low Levels ◽

A Chain ◽

Mutation Mechanisms

Congenital afibrinogenemia is a rare autosomal recessive disorder characterized by bleeding that varies from mild to severe and by complete absence or extremely low levels of plasma and platelet fibrinogen. Although several mutations in the fibrinogen genes associated with dysfibrinogenemia and hypofibrinogenemia have been described, the genetic defects of congenital afibrinogenemia are largely unknown, except for a recently reported 11-kb deletion of the fibrinogen A-chain gene. Nevertheless, mutation mechanisms other than the deletion of a fibrinogen gene are likely to exist because patients with afibrinogenemia showing no gross alteration within the fibrinogen cluster have been reported. We tested this hypothesis by studying the affected members of two families, one Italian and one Iranian, who had no evidence of large deletions in the fibrinogen genes. Sequencing of the fibrinogen genes in the 2 probands detected 2 different homozygous missense mutations in exons 7 and 8 of the Bβ-chain gene, leading to amino acid substitutions Leu353Arg and Gly400Asp, respectively. Transient transfection experiments with plasmids expressing wild-type and mutant fibrinogens demonstrated that the presence of either mutation was sufficient to abolish fibrinogen secretion. These findings demonstrated that missense mutations in the Bβ fibrinogen gene could cause congenital afibrinogenemia by impairing fibrinogen secretion.

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Trade-offs between enzyme fitness and solubility illuminated by deep mutational scanning

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1614437114 ◽

2017 ◽

Vol 114 (9) ◽

pp. 2265-2270 ◽

Cited By ~ 55

Author(s):

Justin R. Klesmith ◽

John-Paul Bacik ◽

Emily E. Wrenbeck ◽

Ryszard Michalczyk ◽

Timothy A. Whitehead

Keyword(s):

Surface Display ◽

Protein Solubility ◽

Fitness Landscapes ◽

Yeast Surface Display ◽

Crystallographic Structure ◽

Missense Mutations ◽

Classification Models ◽

Wild Type ◽

Trade Offs ◽

And Function

Proteins are marginally stable, and an understanding of the sequence determinants for improved protein solubility is highly desired. For enzymes, it is well known that many mutations that increase protein solubility decrease catalytic activity. These competing effects frustrate efforts to design and engineer stable, active enzymes without laborious high-throughput activity screens. To address the trade-off between enzyme solubility and activity, we performed deep mutational scanning using two different screens/selections that purport to gauge protein solubility for two full-length enzymes. We assayed a TEM-1 beta-lactamase variant and levoglucosan kinase (LGK) using yeast surface display (YSD) screening and a twin-arginine translocation pathway selection. We then compared these scans with published experimental fitness landscapes. Results from the YSD screen could explain 37% of the variance in the fitness landscapes for one enzyme. Five percent to 10% of all single missense mutations improve solubility, matching theoretical predictions of global protein stability. For a given solubility-enhancing mutation, the probability that it would retain wild-type fitness was correlated with evolutionary conservation and distance to active site, and anticorrelated with contact number. Hybrid classification models were developed that could predict solubility-enhancing mutations that maintain wild-type fitness with an accuracy of 90%. The downside of using such classification models is the removal of rare mutations that improve both fitness and solubility. To reveal the biophysical basis of enhanced protein solubility and function, we determined the crystallographic structure of one such LGK mutant. Beyond fundamental insights into trade-offs between stability and activity, these results have potential biotechnological applications.

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Naturally occurring mutations in the thrombomodulin gene leading to impaired expression and function

Blood ◽

10.1182/blood.v99.10.3646 ◽

2002 ◽

Vol 99 (10) ◽

pp. 3646-3653 ◽

Cited By ~ 28

Author(s):

Gabriella Kunz ◽

Ann-Kristin Öhlin ◽

Antonella Adami ◽

Bengt Zöller ◽

Peter Svensson ◽

...

Keyword(s):

Cell Surface ◽

Protein C ◽

Thromboembolic Disease ◽

Missense Mutations ◽

Wild Type ◽

Michaelis Constant ◽

Complementary Dna ◽

Naturally Occurring ◽

Cofactor Activity ◽

And Function

Sporadic mutations in the thrombomodulin (TM) gene occur in patients with both arterial and venous thrombosis, but the effects of these mutations on expression and function are largely unexplored. Full-length wild-type TM complementary DNA (cDNA) was incorporated into vector pcDNA6 for transfection into COS-7 cells for transient expression. Mutagenesis was performed to create 7 TM mutants with natural mutations either previously identified (Ala25Thr, Gly61Ala, Asp468Tyr, Pro477Ser, Pro483Leu) or reported here (an 11-base pair [bp] deletion, del791-801, leading to STOP306, and a missense mutation, Arg385Ser). Four mutations were found to detrimentally affect the level of expression of the TM protein. Of the missense mutations, 3 had reduced expression compared to wild-type TM (100%), Arg385Ser (50.2% ± 5%, P < .001), Pro477Ser (76.8% ± 1%, P < .001), Pro483Leu (82.1% ± 8%, P < .007). No TM protein expression could be detected on the cell surface for mutation del791-801. The cofactor activity of TM in protein C activation was also evaluated. The Michaelis constant (Km) for wild-type thrombin-TM complex was 634 ± 6 nmol/L. Two mutants, with Arg385Ser and Pro477Ser, had increased (P < .0001) Km, 2967 ± 283 nM, and 2342 ± 219 nM, respectively, demonstrating impaired function of the thrombin-TM complex. This work presents biochemical evidence that certain (but not all) natural mutations in the TM gene reduce expression and impair function of the protein on the cell surface, and helps clarify the suggested contribution that these mutations might make to the risk of thromboembolic disease.

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A mutant p53/Hif1α/miR-30d axis reprograms the secretory pathway promoting the release of a prometastatic secretome

Cell Stress ◽

10.15698/cst2020.11.235 ◽

2020 ◽

Vol 4 (11) ◽

pp. 261-264

Author(s):

Valeria Capaci ◽

Fiamma Mantovani ◽

Giannino Del Sal

Keyword(s):

Secretory Pathway ◽

Mutant P53 ◽

Missense Mutations ◽

Wild Type ◽

Metastatic Colonization ◽

Non Invasive ◽

Xenograft Models ◽

Non Coding Rnas ◽

Cellular Pathways ◽

And Function

TP53 missense mutations are frequent driver events during tumorigenesis. The majority of TP53 mutations are missense and occur within the DNA binding domain of p53, leading to expression of mutant p53 (mut-p53) proteins that not only lose the tumor suppressive functions of the wild-type (wt-p53) form, but can also acquire novel oncogenic features fostering tumor growth, metastasis and chemoresistance. Mut-p53 affects fundamental cellular pathways and functions through different mechanisms, a major one being the alteration of gene expression. In our recent work (Capaci et al., 2020, Nat Commun) we found that mut-p53, via miR-30d, modifies structure and function of the Golgi apparatus (GA) and induces increased rate of trafficking. This culminates in the release of a pro-malignant secretome, which is capable of remodeling the tumor microenvironment (TME), to increase stiffness of the extracellular matrix (ECM), favouring metastatic colonization, as shown by cell-based assays and experiments of metastatic niche preconditioning in mouse xenograft models. This study provides new insights into the mechanisms by which mut-p53, through induction of non-coding RNAs, can exert pro-tumorigenic functions in a non-cell-autonomous fashion, and highlights potential non-invasive biomarkers and therapeutic targets to treat tumors harboring mut-p53 (Figure 1).

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Novel loss-of-function mutation in HERC2 is associated with severe developmental delay and paediatric lethality

Journal of Medical Genetics ◽

10.1136/jmedgenet-2020-106873 ◽

2020 ◽

pp. jmedgenet-2020-106873

Author(s):

Marilena Elpidorou ◽

Sunayna Best ◽

James A Poulter ◽

Verity Hartill ◽

Emma Hobson ◽

...

Keyword(s):

Autosomal Recessive ◽

Stop Codon ◽

Neurodevelopmental Disorder ◽

Premature Stop Codon ◽

Homozygous Deletion ◽

Missense Mutations ◽

Loss Of Function ◽

Functional Characterisation ◽

Mitochondrial Functions ◽

And Function

BackgroundThe HERC2 gene encodes a 527 kDa E3 ubiquitin protein ligase that has key roles in cell cycle regulation, spindle formation during mitosis, mitochondrial functions and DNA damage responses. It has essential roles during embryonic development, particularly for neuronal and muscular functions. To date, missense mutations in HERC2 have been associated with an autosomal recessive neurodevelopmental disorder with some phenotypical similarities to Angelman syndrome, and a homozygous deletion spanning HERC2 and OCA2 causing a more severe neurodevelopmental phenotype.Methods and resultsWe ascertained a consanguineous family with a presumed autosomal recessive severe neurodevelopmental disorder that leads to paediatric lethality. In affected individuals, we identified a homozygous HERC2 frameshift variant that results in a premature stop codon and complete loss of HERC2 protein. Functional characterisation of this variant in fibroblasts, from one living affected individual, revealed impaired mitochondrial network and function as well as disrupted levels of known interacting proteins such as XPA.ConclusionThis study extends the genotype–phenotype correlation for HERC2 variants to include a distinct lethal neurodevelopmental disorder, highlighting the importance of further characterisation for HERC2-related disorders.

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5-HT2A receptors: location and functional analysis in intestines of wild-type and 5-HT2A knockout mice

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00435.2001 ◽

2002 ◽

Vol 282 (5) ◽

pp. G877-G893 ◽

Cited By ~ 65

Author(s):

Elena Fiorica-Howells ◽

Rene Hen ◽

Jay Gingrich ◽

Zhishan Li ◽

Michael D. Gershon

Keyword(s):

Functional Analysis ◽

Smooth Muscle ◽

Plasma Membranes ◽

Colonic Transit ◽

Paneth Cells ◽

Wild Type ◽

Longitudinal Smooth Muscle ◽

Transit Times ◽

Deep Muscle ◽

And Function

The distribution and function of the 5-hydroxytryptamine (5-HT2A) receptor were investigated in the intestines of wild-type (5-HT2A +/+) and knockout (5-HT2A−/−) mice. In 5-HT2A +/+ mice, rats, and guinea pigs, 5-HT2A receptor immunoreactivity was found on circular and longitudinal smooth muscle cells, neurons, enterocytes, and Paneth cells. Muscular 5-HT2A receptors were concentrated in caveolae; neuronal 5-HT2A receptors were found intracellularly and on the plasma membranes of nerve cell bodies and axons. Neuronal 5-HT2A immunoreactivity was detected as early as E14 in ganglia, intravillus nerves, and the deep muscle plexus. The 5-HT2A −/− colon did not express 5-HT2A receptors and did not contract in response to exogenous 5-HT. 5-HT2A −/− enterocytes were smaller, Paneth cells fewer, and muscle layers thinner (and showed degeneration) compared with those of 5-HT2A +/+ littermates. The 5-HT2A receptor may thus be required for the maintenance and/or development of enteric neuroeffectors and other enteric functions, although gastrointestinal and colonic transit times in 5-HT2A −/− and +/+ mice did not differ significantly.

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A case report of novel mutation in PRF1 gene, which causes familial autosomal recessive hemophagocytic lymphohistiocytosis

BMC Medical Genetics ◽

10.1186/s12881-017-0404-9 ◽

2017 ◽

Vol 18 (1) ◽

Author(s):

Mohammad Reza Bordbar ◽

Farzaneh Modarresi ◽

Mohammad Ali Farazi Fard ◽

Hassan Dastsooz ◽

Nader Shakib Azad ◽

...

Keyword(s):

Case Report ◽

Hemophagocytic Lymphohistiocytosis ◽

Autosomal Recessive ◽

Novel Mutation ◽

Prf1 Gene

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Platelet Activation and Aggregation Induced by Recombinant von Willebrand Factors Reproducing Four Type 2B von Willebrand Disease Missense Mutations

Thrombosis and Haemostasis ◽

10.1055/s-0037-1614241 ◽

1998 ◽

Vol 79 (01) ◽

pp. 211-216 ◽

Cited By ~ 13

Author(s):

Lysiane Hilbert ◽

Claudine Mazurier ◽

Christophe de Romeuf

Keyword(s):

Platelet Aggregation ◽

Platelet Activation ◽

Von Willebrand Disease ◽

Platelet Glycoprotein ◽

Missense Mutations ◽

Inhibit Platelet Aggregation ◽

Wild Type ◽

A1 Domain ◽

Von Willebrand ◽

Willebrand Factor

SummaryType 2B of von Willebrand disease (vWD) refers to qualitative variants with increased affinity of von Willebrand factor (vWF) for platelet glycoprotein Ib (GPIb). All the mutations responsible for type 2B vWD have been located in the A1 domain of vWF. In this study, various recombinant von Willebrand factors (rvWF) reproducing four type 2B vWD missense mutations were compared to wild-type rvWF (WT-rvWF) for their spontaneous binding to platelets and their capacity to induce platelet activation and aggregation. Our data show that the multimeric pattern of each mutated rvWF is similar to that of WT-rvWF but the extent of spontaneous binding and the capacity to induce platelet activation and aggregation are more important for the R543Q and V553M mutations than for the L697V and A698V mutations. Both the binding of mutated rvWFs to platelets and platelet aggregation induced by type 2B rvWFs are inhibited by monoclonal anti-GPIb and anti-vWF antibodies, inhibitors of vWF binding to platelets in the presence of ristocetin, as well as by aurin tricarboxylic acid. On the other hand, EDTA and a monoclonal antibody directed against GPIIb/IIIa only inhibit platelet aggregation. Furthermore, the incubation of type 2B rvWFs with platelets, under stirring conditions, results in the decrease in high molecular weight vWF multimers in solution, the extent of which appears correlated with that of plasma vWF from type 2B vWD patients harboring the corresponding missense mutation. This study supports that the binding of different mutated type 2B vWFs onto platelet GPIb induces various degrees of platelet activation and aggregation and thus suggests that the phenotypic heterogeneity of type 2B vWD may be related to the nature and/or location of the causative point mutation.

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