BACHD rats expressing full-length mutant huntingtin exhibit differences in social behavior compared to wild-type littermates

Background: Huntington’s disease is a neurodegenerative disorder caused by a CAG expansion in the huntingtin gene, resulting in a polyglutamine expansion in the ubiquitously expressed mutant huntingtin protein. Objective: Here we set out to identify proteins interacting with the full-length wild-type and mutant huntingtin protein in the mice cortex brain region to understand affected biological processes in Huntington’s disease pathology. Methods: Full-length huntingtin with 20 and 140 polyQ repeats were formaldehyde-crosslinked and isolated via their N-terminal Flag-tag from 2-month-old mice brain cortex. Interacting proteins were identified and quantified by label-free liquid chromatography-mass spectrometry (LC-MS/MS). Results: We identified 30 interactors specific for wild-type huntingtin, 14 interactors specific for mutant huntingtin and 14 shared interactors that interacted with both wild-type and mutant huntingtin, including known interactors such as F8a1/Hap40. Syt1, Ykt6, and Snap47, involved in vesicle transport and exocytosis, were among the proteins that interacted specifically with wild-type huntingtin. Various other proteins involved in energy metabolism and mitochondria were also found to associate predominantly with wild-type huntingtin, whereas mutant huntingtin interacted with proteins involved in translation including Mapk3, Eif3h and Eef1a2. Conclusion: Here we identified both shared and specific interactors of wild-type and mutant huntingtin, which are involved in different biological processes including exocytosis, vesicle transport, translation and metabolism. These findings contribute to the understanding of the roles that wild-type and mutant huntingtin play in a variety of cellular processes both in healthy conditions and Huntington’s disease pathology.

Download Full-text

Reliable Resolution of Full-Length Huntingtin Alleles by Quantitative Immunoblotting

Journal of Huntington s Disease ◽

10.3233/jhd-200463 ◽

2021 ◽

pp. 1-11

Author(s):

Nicholas S. Caron ◽

Christine Anderson ◽

Hailey Findlay Black ◽

Shaun S. Sanders ◽

Fanny L. Lemarié ◽

...

Keyword(s):

Huntington's Disease ◽

Biological Samples ◽

Antisense Oligonucleotide ◽

Clinical Development ◽

Full Length ◽

Preclinical Studies ◽

Mutant Huntingtin ◽

Wild Type ◽

Wide Range ◽

Quantitative Immunoblotting

Background: Therapeutics that lower mutant huntingtin (mHTT) have shown promise in preclinical studies and are in clinical development for the treatment of Huntington’s disease (HD). Multiple assays have been developed that either quantify mHTT or total HTT but may not accurately measure levels of wild type HTT (wtHTT) in biological samples. Objective: To optimize a method that can be used to resolve, quantify and directly compare levels of full length wtHTT and mHTT in HD samples. Methods: We provide a detailed quantitative immunoblotting protocol to reproducibly resolve full length wtHTT and mHTT in multiple HD mouse and patient samples. Results: We show that this assay can be modified, depending on the sample, to resolve wtHTT and mHTT with a wide range of polyglutamine differences (ΔQs 22–179). We also demonstrate that this method can be used to quantify allele-selective lowering of mHTT using an antisense oligonucleotide in HD patient-derived cells. Conclusion: This quantitative immunoblotting method can be used to reliably resolve full length HTT alleles with ΔQs≥22 and allows for direct comparison of wtHTT and mHTT levels in HD samples.

Download Full-text

F01 Establishment of novel assays for quantification of full-length wild-type and mutant huntingtin by using the alphalisa technology

Journal of Neurology Neurosurgery & Psychiatry ◽

10.1136/jnnp-2012-303524.66 ◽

2012 ◽

Vol 83 (Suppl 1) ◽

pp. A21.2-A21

Author(s):

MU Sajjad ◽

G Pinard ◽

C Fafard ◽

C Illy ◽

Å Petersén ◽

...

Keyword(s):

Full Length ◽

Mutant Huntingtin ◽

Wild Type

Download Full-text

Expression and Functional Assessment of an Alternatively Spliced Extracellular Ca2+-Sensing Receptor in Growth Plate Chondrocytes

Endocrinology ◽

10.1210/en.2005-0256 ◽

2005 ◽

Vol 146 (12) ◽

pp. 5294-5303 ◽

Cited By ~ 55

Author(s):

Luis Rodriguez ◽

Chialing Tu ◽

Zhiqiang Cheng ◽

Tsui-Hua Chen ◽

Daniel Bikle ◽

...

Keyword(s):

Cell Surface ◽

Growth Plate ◽

Inositol Phosphate ◽

Intact Cell ◽

Chinese Hamster ◽

Full Length ◽

Wild Type ◽

Growth Plate Chondrocytes ◽

Matrix Gene ◽

Alternatively Spliced

The extracellular Ca2+-sensing receptor (CaR) plays an essential role in mineral homeostasis. Studies to generate CaR-knockout (CaR−/−) mice indicate that insertion of a neomycin cassette into exon 5 of the mouse CaR gene blocks the expression of full-length CaRs. This strategy, however, allows for the expression of alternatively spliced CaRs missing exon 5 [Exon5(−)CaRs]. These experiments addressed whether growth plate chondrocytes (GPCs) from CaR−/− mice express Exon5(−)CaRs and whether these receptors activate signaling. RT-PCR and immunocytochemistry confirmed the expression of Exon5(−)CaR in growth plates from CaR−/− mice. In Chinese hamster ovary or human embryonic kidney-293 cells, recombinant human Exon5(−)CaRs failed to activate phospholipase C likely due to their inability to reach the cell surface as assessed by intact-cell ELISA and immunocytochemistry. Human Exon5(−)CaRs, however, trafficked normally to the cell surface when overexpressed in wild-type or CaR−/− GPCs. Immunocytochemistry of growth plate sections and cultured GPCs from CaR−/− mice showed easily detectable cell-membrane expression of endogenous CaRs (presumably Exon5(−)CaRs), suggesting that trafficking of this receptor form to the membrane can occur in GPCs. In GPCs from CaR−/− mice, high extracellular [Ca2+] ([Ca2+]e) increased inositol phosphate production with a potency comparable with that of wild-type GPCs. Raising [Ca2+]e also promoted the differentiation of CaR−/− GPCs as indicated by changes in proteoglycan accumulation, mineral deposition, and matrix gene expression. Taken together, our data support the idea that expression of Exon5(−)CaRs may compensate for the loss of full-length CaRs and be responsible for sensing changes in [Ca2+]e in GPCs in CaR−/− mice.

Download Full-text

Yeast cells lacking 5'-->3' exoribonuclease 1 contain mRNA species that are poly(A) deficient and partially lack the 5' cap structure

Molecular and Cellular Biology ◽

10.1128/mcb.13.8.4826-4835.1993 ◽

1993 ◽

Vol 13 (8) ◽

pp. 4826-4835

Author(s):

C L Hsu ◽

A Stevens

Keyword(s):

Full Length ◽

Yeast Cells ◽

Mrna Turnover ◽

Turnover Rates ◽

Wild Type ◽

Mrna Species ◽

Turnover Process ◽

Hydrolysis Of ◽

Extension Analysis ◽

Wild Type Yeast

Analysis of the slowed turnover rates of several specific mRNA species and the higher cellular levels of some of these mRNAs in Saccharomyces cerevisiae lacking 5'-->3' exoribonuclease 1 (xrn1 cells) has led to the finding that these yeast contain higher amounts of essentially full-length mRNAs that do not bind to oligo(dT)-cellulose. On the other hand, the length of mRNA poly(A) chains found after pulse-labeling of cells lacking the exoribonuclease, the cellular rate of synthesis of oligo(dT)-bound mRNA, and the initial rate of its deadenylation appeared quite similar to the same measurements in wild-type yeast cells. Examination of the 5' cap structure status of the poly(A)-deficient mRNAs by comparative analysis of the m7G content of poly(A)- and poly(A)+ RNA fractions of wild-type and xrn1 cells suggested that the xrn1 poly(A)- mRNA fraction is low in cap structure content. Further analysis of the 5' termini by measurements of the rate of 5'-->3' exoribonuclease 1 hydrolysis of specific full-length mRNA species showed that approximately 50% of the xrn1 poly(A)-deficient mRNA species lack the cap structure. Primer extension analysis of the 5' terminus of ribosomal protein 51A (RP51A) mRNA showed that about 30% of the poly(A)-deficient molecules of the xrn1 cells are slightly shorter at the 5' end. The finding of some accumulation of poly(A)-deficient mRNA species partially lacking the cap structure together with the reduction of the rate of mRNA turnover in cells lacking the enzyme suggest a possible role for 5'-->3' exoribonuclease 1 in the mRNA turnover process.

Download Full-text

Agrobacterium-Mediated Transformation of Aspergillus awamori in the Absence of Full-Length VirD2, VirC2, or VirE2 Leads to Insertion of Aberrant T-DNA Structures

Journal of Bacteriology ◽

10.1128/jb.186.7.2038-2045.2004 ◽

2004 ◽

Vol 186 (7) ◽

pp. 2038-2045 ◽

Cited By ~ 21

Author(s):

Caroline B. Michielse ◽

Arthur F. J. Ram ◽

Paul J. J. Hooykaas ◽

Cees A. M. J. J. van den Hondel

Keyword(s):

Single Copy ◽

Full Length ◽

Aspergillus Awamori ◽

Wild Type ◽

Dna Structures ◽

Dna Integration ◽

Virulence Proteins ◽

Type Transformation ◽

The Right ◽

Transposon Insertions

ABSTRACT Reductions to 2, 5, and 42% of the wild-type transformation efficiency were found when Agrobacterium mutants carrying transposon insertions in virD2, virC2, and virE2, respectively, were used to transform Aspergillus awamori. The structures of the T-DNAs integrated into the host genome by these mutants were analyzed by Southern and sequence analyses. The T-DNAs of transformants obtained with the virE2 mutant had left-border truncations, whereas those obtained with the virD2 mutant had truncated right ends. From this analysis, it was concluded that the virulence proteins VirD2 and VirE2 are required for full-length T-DNA integration and that these proteins play a role in protecting the right and left T-DNA borders, respectively. Multicopy and truncated T-DNA structures were detected in the majority of the transformants obtained with the virC2 mutant, indicating that VirC2 plays a role in correct T-DNA processing and is required for single-copy T-DNA integration.

Download Full-text

Mutational activation of c-raf-1 and definition of the minimal transforming sequence.

Molecular and Cellular Biology ◽

10.1128/mcb.10.6.2503 ◽

1990 ◽

Vol 10 (6) ◽

pp. 2503-2512 ◽

Cited By ~ 144

Author(s):

G Heidecker ◽

M Huleihel ◽

J L Cleveland ◽

W Kolch ◽

T W Beck ◽

...

Keyword(s):

Structural Features ◽

Kinase Domain ◽

Full Length ◽

Wild Type ◽

Amino Terminal ◽

Raf Kinase ◽

Rich Domain ◽

Binding Residue ◽

Transforming Activity ◽

Mutational Activation

A series of wild-type and mutant raf genes was transfected into NIH 3T3 cells and analyzed for transforming activity. Full-length wild-type c-raf did not show transforming activity. Two types of mutations resulted in oncogenic activity similar to that of v-raf: truncation of the amino-terminal half of the protein and fusion of the full-length molecule to gag sequences. A lower level of activation was observed for a mutant with a tetrapeptide insertion mapping to conserved region 2 (CR2), a serine- and threonine-rich domain located 100 residues amino-terminal of the kinase domain. To determine essential structural features of the transforming region of raf, we analyzed point and deletion mutants of v-raf. Substitutions of Lys-56 modulated the transforming activity, whereas mutation of Lys-53, a putative ATP binding residue, abolished it. Deletion analysis established that the minimal transforming sequence coincided precisely with CR3, the conserved Raf kinase domain. Thus, oncogenic activation of the Raf kinase can be achieved by removal of CR1 and CR2 or by steric distortion and requires retention of an active kinase domain. These findings are consistent with a protein structure model for the nonstimulated enzyme in which the active site is buried within the protein.

Download Full-text

Genetic variation in the social environment affects behavioral phenotypes of oxytocin receptor mutants in zebrafish

10.1101/2020.03.23.002873 ◽

2020 ◽

Author(s):

D. Ribeiro ◽

A.R. Nunes ◽

M.C. Teles ◽

S. Anbalagan ◽

J. Blechman ◽

...

Keyword(s):

Social Behavior ◽

Social Environment ◽

Oxytocin Receptor ◽

The Other ◽

Wild Type ◽

Knock Out ◽

The Social ◽

Wide Range ◽

Knockout Line ◽

Genetically Modified Animals

AbstractOxytocin-like peptides have been implicated in the regulation of a wide range of social behaviors across taxa. On the other hand, the social environment, which is composed of conspecifics genotypes, is also known to influence the development of social behavior, creating the possibility for indirect genetic effects. Here we used a knockout line for the oxytocin receptor in zebrafish to investigate how the genotypic composition of the social environment (Es) interacts with the oxytocin genotype (G) of the focal individual in the regulation of its social behavior. For this purpose, we have raised wild-type or knock-out zebrafish in either wild-type or knock-out shoals and tested different components of social behavior in adults. GxEs effects were detected in some behaviors, highlighting the need to control for GxEs effects when interpreting results of experiments using genetically modified animals, since the social environment can either rescue or promote phenotypes associated with specific genes.

Download Full-text

Increased metal tolerance and bioaccumulation of zinc and cadmium in Chlamydomonas reinhardtii expressing a AtHMA4 C-terminal domain protein

10.1101/2020.02.13.948307 ◽

2020 ◽

Author(s):

Aniefon Ibuot ◽

Rachel E. Webster ◽

Lorraine E. Williams ◽

Jon K. Pittman

Keyword(s):

Chlamydomonas Reinhardtii ◽

Metal Binding ◽

Metal Tolerance ◽

Alginate Beads ◽

Full Length ◽

Wild Type ◽

Microalgal Biomass ◽

Terminal Domain ◽

Metal Biosorption ◽

Transgenic Cells

AbstractThe use of microalgal biomass for metal pollutant bioremediation might be improved by genetic engineering to modify the selectivity or capacity of metal biosorption. A plant cadmium (Cd) and zinc (Zn) transporter (AtHMA4) was used as a transgene to increase the ability of Chlamydomonas reinhardtii to tolerate 0.2 mM Cd and 0.3 mM Zn exposure. The transgenic cells showed increased accumulation and internalisation of both metals compared to wild type. AtHMA4 was expressed either as the full-length protein or just the C-terminal tail, which is known to have metal binding sites. Similar Cd and Zn tolerance and accumulation was observed with expression of either the full-length protein or C-terminal domain, suggesting that enhanced metal tolerance was mainly due to increased metal binding rather than metal transport. The effectiveness of the transgenic cells was further examined by immobilisation in calcium alginate to generate microalgal beads that could be added to a metal contaminated solution. Immobilisation maintained metal tolerance, while AtHMA4-expressing cells in alginate showed a concentration-dependent increase in metal biosorption that was significantly greater than alginate beads composed of wild type cells. This demonstrates that expressing AtHMA4 full-length or C-terminus has great potential as a strategy for bioremediation using microalgal biomass.

Download Full-text

The Ubiquitin-associated Domain of hPLIC-2 Interacts with the Proteasome

Molecular Biology of the Cell ◽

10.1091/mbc.e02-11-0766 ◽

2003 ◽

Vol 14 (9) ◽

pp. 3868-3875 ◽

Cited By ~ 72

Author(s):

Maurits F. Kleijnen ◽

Rodolfo M. Alarcón ◽

Peter M. Howley

Keyword(s):

Proteasomal Degradation ◽

Binding Domain ◽

Full Length ◽

Degradation Pathways ◽

Wild Type ◽

Amino Terminal ◽

Uba Domain ◽

Carboxyl Terminal ◽

Protein Ligases

The ubiquitin-like hPLIC proteins can associate with proteasomes, and hPLIC overexpression can specifically interfere with ubiquitin-mediated proteolysis ( Kleijnen et al., 2000 ). Because the hPLIC proteins can also interact with certain E3 ubiquitin protein ligases, they may provide a link between the ubiquitination and proteasomal degradation machineries. The amino-terminal ubiquitin-like (ubl) domain is a proteasome-binding domain. Herein, we report that there is a second proteasome-binding domain in hPLIC-2: the carboxyl-terminal ubiquitin-associated (uba) domain. Coimmunoprecipitation experiments of wild-type and mutant hPLIC proteins revealed that the ubl and uba domains each contribute independently to hPLIC-2–proteasome binding. There is specificity for the interaction of the hPLIC-2 uba domain with proteasomes, because uba domains from several other proteins failed to bind proteasomes. Furthermore, the binding of uba domains to polyubiquitinated proteins does not seem to be sufficient for the proteasome binding. Finally, the uba domain is necessary for the ability of full-length hPLIC-2 to interfere with the ubiquitin-mediated proteolysis of p53. The PLIC uba domain has been reported to bind and affect the functions of proteins such as GABAAreceptor and presenilins. It is possible that the function of these proteins may be regulated or mediated through proteasomal degradation pathways.

Download Full-text