Dimensional SDS-PAGE Research Articles

Characterization of functional protein complexes from Alzheimer\u2019s disease and healthy brain by mass spectrometry-based proteome analysis

Alzheimer’s disease (AD) is a complex neurodegenerative disorder with impaired protein activities. Proteins in the form of complexes have a ubiquitous role in diverse range of cellular functions. The key challenge is to identify novel disease associated protein complexes and their potential role in the progression of AD pathology. Protein complexes were obtained from AD brain prefrontal cortex and age matched controls by Blue Native-Polyacrylamide Gel Electrophoresis. A proteomic analysis was performed using second dimension SDS-PAGE followed by nano LC–MS/MS. Differentially expressed proteins were mapped to existing biological networks by Ingenuity Pathway Analysis (IPA). A total of 13 protein complexes with their interacting proteins were resolved on SDS-PAGE. We identified 34 protein spots and found significant abundance difference between the two experimental samples. IPA analysis revealed degeneration of neurons and cell death as a major consequence of protein dysregulation. Furthermore, focused network analysis suggested an integrated regulation of the identified proteins through APP and MAPT dependent mechanisms. The interacting differentially expressed proteins in AD were found to be part of concomitant signaling cascades terminating in neuronal cell death. The identified protein networks and pathways warrant further research to study their actual contribution to AD pathology.

Does and duration of exposure time dependent regulation of proteins in the urinary bladder of hamsters exposed to sodium arsenite

Does and duration of exposure time dependent regulation of proteins in the urinary bladder of hamsters exposed to sodium arsenite

Regulation of transgelin and GST-pi proteins in the tissues of hamsters exposed to sodium arsenite

Regulation of transgelin and GST-pi proteins in the tissues of hamsters exposed to sodium arsenite

Salivary Protein Profiling of Head and Neck Cancer Patients at West Nusa Tenggara Province General Hospital: A preliminary study based on single dimension SDS-PAGE analysis

The initial aim of this study was to investigate the protein profiles found in the saliva of head and neck cancer (HNC) patients at the Regional General Hospital of West Nusa Tenggara Province, Indonesia. Saliva samples were collected from 21 patients with head and neck cancer (confirmed from the results of anatomical and pathology examination). Mainly the types of HNC are sinonasal and nasopharyngeal cancers. As the control, saliva samples from 10 healthy individuals were used. Each subject thoroughly rinsed their mouth with a commercial mineral water and around 2-3 mL of un-stimulated saliva was collected in a 10 mL disposable plastic container with screw cap and immediately placed on ice. Sodium azide and protease inhibitors were added into the collected saliva and then transported to the laboratory within 1 hour of sample collection and centrifuged. The supernatant was taken; the protein concentration was measured, and then lyophilized. Protein profiles were analyzed using 1D-SDS-PAGE at 12.5% running gel. The data obtained were analyzed descriptively. In this preliminary study protein bands with a molecular weight range of 35-63 kDa were observed in the saliva of HNC patients, which were not found in the saliva of healthy individuals. Whether the expression of the specific protein bands can be used as a non-invasive biomarker in patients with head and neck cancer; needs to be investigated further.

Mass Spectrometry Based Comparative Proteomics Using One Dimensional and Two Dimensional SDS-PAGE of Rat Atria Induced with Obstructive Sleep Apnea.

Proteomics involves large-scale comprehensive study of specific proteomes which have been widely used in the field of biomarker discovery, drug development, disease diagnosis and therapy. Comprehensive proteomics involve two or more proteomics approaches that are confirmatory, complementary, and/or synergistic. Obstructive sleep apnea (OSA) is a sleep disorder which causes respiratory cessation (due to upper airway collapse). Here we describe a comprehensive MS based label-free quantitative proteomic analysis of the OSA induced rat atria homogenates and matched controls by using 1 dimensional SDS PAGE (1-D PAGE) and 2 dimensional SDS PAGE (2-D PAGE) separation of the proteins, enzymatic digestion and analysis by nanoliquid chromatography tandem-mass spectrometry (LC-MS/MS). The outcomes from the 1D-PAGE and 2D-PAGE studies not only identified dysregulated proteins due to OSA, but also confirmed and complemented each other.

Cellular and proteomic events associated with the localized formation of smut-gall during Zizania latifolia–Ustilago esculenta interaction

The perennial wild rice Zizania latifolia is confined in the swampy habitat and wetland of the Indo-Burma biodiversity hotspot of India and infection by the biotrophic fungus Ustilago esculenta is hallmarked by swellings that develop to form localized smut-gall at the topmost internodal region. The cellular and proteomic events involved in the non-systemic colonization of Z. latifolia by U. esculenta leading to smut-gall formation is poorly understood. Proteins were extracted from the smut-gall region at the topmost internodal region below the apical meristematic tissue from the infected and uninfected parts of Z. latifolia. By combining transmission electron microscopy (TEM) and fluorescent microscopy (FM), we showed that U. esculenta hyphal morphological transitions and movement occurred both intercellularly and intracellularly while sporulation occurred intracellularly in selective cells. Following proteome profiling using two dimensional SDS-PAGE at different phenological phases of smut-gall development and U. esculenta infection, differentially expressed proteins bands and their relative abundance were detected and subjected to liquid chromatography-tandem mass spectrometric (LC-MS/MS) analysis. Importantly, the fungus explores at least 7 metabolic pathways and 5 major biological processes to subdue the host defense and thrive successfully on Z. latifolia. The fungus U. esculenta produces proteases and energy acquisition proteins those enhance it's defensive and survival mode in the host. The identified differentially regulated proteins shed-light into why inflorescence is being replaced by bulbous smut-gall at late stages of the disease, as well as the development of resistance in some Z. latifolia plants against U. esculenta infection.

Protein Extraction Protocol from Musa sp. Shoots and Roots Tissue for Non-reducing One Dimensional SDS-PAGE Analysis

Background: Protein extraction from plant tissues is a great challenge since they contain low amount of proteins and rich in proteases, secondary metabolites and oxidative enzymes. Besides, dealing with limited amount of starting material would pose another challenge in getting high protein yield. The aim of this study is to determine the best protein extraction protocol for recalcitrant tissues of banana (Musa sp.) specific for non-reducing one dimensional SDS-PAGE analysis. Methods: Three protein extraction protocols were compared which include TCA-acetone, phenol and phosphate buffer saline (PBS) using small amount of starting material. Modifications were done to each protocol to suit non-reducing SDS-PAGE analysis and subsequent downstream processes. Results: Of the three protocols compared, TCA-acetone protocol gave the highest yield and quality of protein extract from only a small amount of starting material based on protein quantification and non-reducing one dimensional SDS-PAGE analysis compared to phenol and phosphate buffer saline (PBS) protocol. Conclusion: Even though various protein extraction protocols have been developed to date, there is no one-size-fits-all when it comes to different types of plant tissues from varieties of plant species. TCA-acetone protocol was found to be most effective in extracting protein from banana shoot and root tissues even for small amount of starting material in absence of reducing agent. Keywords: Protein extraction, recalcitrant tissue, Musa sp., non-reducing SDS-PAGE, TCA-acetone protocol, reducing agents.

Simple and rapid system for two-dimensional gel electrophoresis technique: A laboratory exercise for high school students.

Polyacrylamide gel electrophoresis (PAGE) is the most classical technique favored worldwide for resolution of macromolecules in many biochemistry laboratories due to its incessant advanced developments and wide modifications. These ever-growing advancements in the basic laboratory equipments lead to emergence of many expensive, complex, and tricky laboratory equipments. Practical courses of biochemistry at high school or undergraduate levels are often affected by these complications. Two dimensional gel electrophoresis technique (2D-PAGE) used for resolving thousands of proteins in a gel is a combination of isoelectric focusing (first dimension gel electrophoresis technique) and sodium-dodecylsulphate PAGE (second dimension gel electrophoresis technique or SDS-PAGE). Two different laboratory equipments are needed to carry out effective 2D-PAGE technique, which also invites extra burden to the school laboratory. Here, we describe a low cost, time saving and simple gel cassette for protein 2D-PAGE technique that uses easily fabricated components and routine off-the-shelf materials. The performance of the apparatus was verified in a practical exercise by a group of high school students with positive outcomes. © 2018 by The International Union of Biochemistry and Molecular Biology, 46(3):237-244, 2018.

Peran Brush Border Membrane Vesicle (BBMV) dalam proses infeksi Baculovirus

The Role of Brush Border Membrane Vesicle (BBMV) in The Baculovirus Infection. Although baculovirus becoming recognized as potential important agents for control pests, there are several important obstacles for commercial use insecticide. One important obstacle is their narrow host. The other obstacle is due to its development resistance, the phenomenon whereby host larvae become progressively more resistant to fatal infection as they age within and among instars. From practical stand point, this phenomenon impacts the effectiveness of baculovirus control programs against agricultural and forest insect pests that is necessary to adjust application levels in the response to the demography of the target insect population. Purified columnar cells derived from midgut of armyworm Pseudaletia separata was used for fusion assay and observed by means of fluorescence assay based on self-quenching of octadecylrhodamine B chloride (R18). Confocal microscopy of the columnar cells that had been combined with labeled virus showed that the fluorescence appeared on the apical part in the brush border membrane, this indicated that there was fusion between virus and the cells. Two dimensional SDS-PAGE and immunobloting assay confirmed that the receptor exist within the columnar cells.

Overlapping Role of Respiratory Supercomplex Factor Rcf2 and Its N-terminal Homolog Rcf3 in Saccharomyces cerevisiae

The mitochondrial electron transport chain consists of individual protein complexes arranged into large macromolecular structures, termed respiratory chain supercomplexes or respirasomes. In the yeast Saccharomyces cerevisiae, respiratory chain supercomplexes form by association of the bc1 complex with the cytochrome c oxidase. Formation and maintenance of these assemblies are promoted by specific respiratory supercomplex factors, the Rcf proteins. For these proteins a regulatory function in bridging the electron transfer within supercomplexes has been proposed. Here we report on the maturation of Rcf2 into an N- and C-terminal peptide. We show that the previously uncharacterized Rcf3 (YBR255c-A) is a homolog of the N-terminal Rcf2 peptide, whereas Rcf1 is homologous to the C-terminal portion. Both Rcf3 and the C-terminal fragment of Rcf2 associate with monomeric cytochrome c oxidase and respiratory chain supercomplexes. A lack of Rcf2 and Rcf3 increases oxygen flux through the respiratory chain by up-regulation of the cytochrome c oxidase activity. A double gene deletion of RCF2 and RCF3 affects cellular survival under non-fermentable growth conditions, suggesting an overlapping role for both proteins in the regulation of the OXPHOS activity. Furthermore, our data suggest an association of all three Rcf proteins with the bc1 complex in the absence of a functional cytochrome c oxidase and identify a supercomplex independent interaction network of the Rcf proteins.

The identification of FERM domain protein in serum infected with Plasmodium berghei

Malaria continues to affect 500 million people worldwide every year. In this study, we compared the protein profile of uninfected and Plasmodium berghei-infected serum samples by one dimensional SDS-PAGE analysis, MALDI-TOF/TOF mass spectrometry and confirmed by semi-quantitative RT-PCR. Also the protein interacting networks were established using STRING protein⿿protein interaction analysis. We observed for the first time the upregulation of FERM domain during P. berghei infection. We predict that FRMD5 along with the other protein partners (identified by STRING analysis) are involved in the merozoites entry or protein trafficking where cell to cell interaction happens with the host erythrocyte; hence, upregulation.

Differential Proteome Analysis of Different Brucella spp. by Two Dimensional Electrophoresis

Brucellosis is a major bacterial zoonosis affecting domestic livestock, wild mammals, as well as humans around the globe. In the present study, proteomes of three different species of Brucella were compared by two dimensional electrophoresis (2-DE). B. abortus, B. melitensis and B. suis, were cultured in Mueller–Hinton agar. The cells were harvested by centrifugation and lysed in lysis buffer. Protein fraction was purified by 2-DE cleanup kit and subjected to isoelectric focusing (IEF) using 3–10 pH gradient Immobiline dry strips. After IEF, the strips were equilibrated and subjected to 2nd dimension SDS PAGE. The gels were stained with Coomassie Brilliant blue stain, scanned and analysed using Image master platinum 7. A total of 381, 386 and 362 protein spots were detected for B. abortus, B. melitensis and B. suis, respectively. Out of them, 312 spots (proteins) were common to all the three species of the bacteria, the remaining spots were unique either to one species or two, but were not present in all the three species. Twenty-two proteins were significantly over-expressed in B. abortus as compared to B. melitensis and B. suis, while 12 proteins were significantly over-expressed in B. melitensis as compared to the rest of the species.

Study of the Protein Complex, Pore Diameter, and Pore-forming Activity of the Borrelia burgdorferi P13 Porin

P13 is one of the major outer membrane proteins of Borrelia burgdorferi. Previous studies described P13 as a porin. In the present study some structure and function aspects of P13 were studied. P13 showed according to lipid bilayer studies a channel-forming activity of 0.6 nanosiemens in 1 m KCl. Single channel and selectivity measurements demonstrated that P13 had no preference for either cations or anions and showed no voltage-gating up to ±100 mV. Blue native polyacrylamide gel electrophoresis was used to isolate and characterize the P13 protein complex in its native state. The complex had a high molecular mass of about 300 kDa and was only composed of P13 monomers. The channel size was investigated using non-electrolytes revealing an apparent diameter of about 1.4 nm with a 400-Da molecular mass cut-off. Multichannel titrations with different substrates reinforced the idea that P13 forms a general diffusion channel. The identity of P13 within the complex was confirmed by second dimension SDS-PAGE, Western blotting, mass spectrometry, and the use of a p13 deletion mutant strain. The results suggested that P13 is the protein responsible for the 0.6-nanosiemens pore-forming activity in the outer membrane of B. burgdorferi.

Proteomic characterization of the subpellicular cytoskeleton of Toxoplasma gondii tachyzoites

Toxoplasma, the causative agent of toxoplasmosis in animals and humans, has a subpellicular cytoskeleton that is involved in motility, cell shape and invasion. Knowledge of components of the cytoskeleton is necessary to understand the invasion mechanisms as well as for the identification of possible therapeutic targets. To date, most cytoskeletal components of Toxoplasma remain unidentified due mainly to the lack of reproducible methods for their isolation. Based on the successful isolation of the cytoskeleton, it was possible to report for the first time, the proteomic characterization of the subpellicular cytoskeleton of Toxoplasma formed by 95 cytoskeletal proteins through proteomic analysis by tandem mass spectrometry of one dimension SDS PAGE. By bioinformatic analysis of the data, proteins were classified as: 18 conventional cytoskeletal proteins; 10 inner membrane complex proteins, including 7 with alveolin repeats; 5 new proteins with alveolin like repeats; 37 proteins associated with other organelles and 25 novel proteins of unknown function. One of the alveolin like proteins not previously described in Toxoplasma named TgArticulin was partially characterized with a specific monoclonal antibody. Presence of TgArticulin was exclusively associated with the cytoskeleton fraction with a cortical distribution. Functions for the several molecules identified are proposed. This manuscript describes, for the first time, the proteome of the subpellicular cytoskeleton of Toxoplasma gondii. The importance of this study is related to the role of the cytoskeleton in the highly invasive capability of a parasite that causes abortion, blindness, and death by encephalitis in immunocompromised patients. Proteomic characterization of the cytoskeleton of T. gondii tachyzoites was possible by the development of a successful procedure for the isolation of the subpellicular cytoskeleton. Knowledge of the composition of the cytoskeleton of Toxoplasma is fundamental for the understanding of the motility and host cell invasion mechanisms, and for the future design and development of toxoplasmicidal drugs with effects against specific components of the cytoskeleton of this parasite that are absent in mammal host cells.

Age-Dependent Effect of Cyclic Feeding Regimen on the Level of Serum Apolipoproteins in Young and Old Rats

Aims: the aim of this study was to investigate the influence of intermittent fasting (IF) on the content of blood serum apolipoproteins in young and old animals and to ascertain whether there exist adaptation mechanisms to this dietary regimen. Study design: young (3-month) and old (20-month) rats were individually housed and randomly assigned to one of five groups (with 10 rats per group): (AL) fed ad libitum; (IF1) provided access to a limited amount of food (4g/100g and 2g/100g of food/body weight for young and old rats, respectively) every other day for 10 days; (R1) refeeded ad libitum for 20 days after IF1; (IF2) provided the same regimen as for IF1, but after successive IF1 and R1; (R2) refeeded ad libitum for 10 days after IF2. Methodology: plasma proteins were separated by one dimensional SDS-PAGE using a 7.5-15% gradient separating gel. Results: IF1 with 30% weight loss resulted in ~28% and ~24% decrease and ~121% increase in serum apolipoprotein A-I (apoA-I), apolipoprotein B-100 (apoB-100), and apolipoprotein E (apoE) levels of young rats, respectively, and after R1 the level of these Original Research Article International Journal of Biochemistry Research & Review, 4(6): 593-606, 2014 594 proteins was characterized by ~37%, ~66%, ~22% increase in comparison with control. IF1 and R1 in old animals were followed by ~53% increase in apoE content and ~13% decrease in the amount of apoB. IF2 was coupled with ~32% decrease in apoA-I level and ~133% increase in apoE concentration in young animals and ~39% and ~38% decrease in apoA-I and apoB-100 level in old animals, respectively. R2 produced ~48% and ~49% increase in apoA-I and apoE levels of young rats, respectively, and to ~31% increase in apoA-I content of old rats. Conclusion: the main outcome reached is the identification of differences in the effects of the dietary regimen during reapplication on apoE and apoB-100 serum levels in old animals and on apoA-I level in the case of young animals.

Proteomic analysis of rat proximal tubule cells following stretch-induced apoptosis in an in vitro model of kidney obstruction

Urinary tract obstruction (UTO) is a commonly noted disorder on prenatal ultrasound that has the potential to lead to permanent loss of renal function. To study the molecular processes of the disease, an in vitro model has been developed which involves mechanical stretch of proximal tubule cells grown on flexible plates which mimics the physiological conditions during UTO. This study employs a one dimensional SDS-PAGE fractionation procedure, followed by in-gel digest and LC-MS/MS analysis in a semi-quantitative experiment using spectral counting to relatively quantify changes in protein expression following the established model of UTO. Quantitative analysis shows 317 of the 1630 identified proteins express altered abundance, with 135 increased and 182 decreased in abundance as a result of stretch. Gene ontology (GO) and KEGG annotations implicate a number of physiological processes that have been previously shown in addition to some potentially novel processes in UTO. The quantitative proteomic analysis we performed here provides a more complete characterization of changes in protein abundance as a result of stretch than previous studies, and provides a number of previously undescribed proteins in proximal tubule cells that may play a role in UTO. Urinary tract obstruction (UTO) is a commonly noted abnormality on prenatal ultrasound that can either resolve spontaneously or require surgical intervention to prevent permanent renal damage or loss of function. While targeted studies of UTO have shown a number of pathological responses in proximal tubule cells, there are currently no large-scale quantitative studies that aim to elucidate a global cellular response. This study uses a semi-quantitative approach and applies a well characterized model of UTO to determine a number of cellular processes affected by UTO simulation and identifies a number of proteins with altered abundance that have not been noted previously in UTO. This article is part of a Special Issue entitled: Can Proteomics Fill the Gap Between Genomics and Phenotypes?

Use of Nonelectrolytes Reveals the Channel Size and Oligomeric Constitution of the Borrelia burgdorferi P66 Porin

In the Lyme disease spirochete Borrelia burgdorferi, the outer membrane protein P66 is capable of pore formation with an atypical high single-channel conductance of 11 nS in 1 M KCl, which suggested that it could have a larger diameter than ‘normal’ Gram-negative bacterial porins. We studied the diameter of the P66 channel by analyzing its single-channel conductance in black lipid bilayers in the presence of different nonelectrolytes with known hydrodynamic radii. We calculated the filling of the channel with these nonelectrolytes and the results suggested that nonelectrolytes (NEs) with hydrodynamic radii of 0.34 nm or smaller pass through the pore, whereas neutral molecules with greater radii only partially filled the channel or were not able to enter it at all. The diameter of the entrance of the P66 channel was determined to be ≤1.9 nm and the channel has a central constriction of about 0.8 nm. The size of the channel appeared to be symmetrical as judged from one-sidedness of addition of NEs. Furthermore, the P66-induced membrane conductance could be blocked by 80–90% by the addition of the nonelectrolytes PEG 400, PEG 600 and maltohexaose to the aqueous phase in the low millimolar range. The analysis of the power density spectra of ion current through P66 after blockage with these NEs revealed no chemical reaction responsible for channel block. Interestingly, the blockage of the single-channel conductance of P66 by these NEs occurred in about eight subconductance states, indicating that the P66 channel could be an oligomer of about eight individual channels. The organization of P66 as a possible octamer was confirmed by Blue Native PAGE and immunoblot analysis, which both demonstrated that P66 forms a complex with a mass of approximately 460 kDa. Two dimension SDS PAGE revealed that P66 is the only polypeptide in the complex.

Two dimensional SDS PAGE analysis of epididymal tissue proteins of normal and castrated bulls (Bos taurus) and identification of an androgen dependant protein

A study was undertaken with an objective of two dimensional SDS-PAGE analysis of protein profile in tissues of all caput, corpus and cauda of epididymis in both castrated and normal bulls and identification of androgen dependent protein/s in the epididymal tissues of bull (Bos taurus). Two dimensional SDS-PAGE analysis for protein spots of different molecular weight (MW) with pI 3.5 to 7.35 and pI > 7.35 to 9.3 between the three regions in the normal and castrated bull as well as between normal and castrated bull of a particular region revealed significant differences. Similarly, comparison between the three regions of the normal and castrated bull epididymis for the number of protein spots irrespective of the MW in pI range of 3.5 to 7.35 and pI range > 7.35 to 9.3 revealed significant differences. The number of protein spots found to be significantly higher in caput, corpus and cauda epididymis of the normal bull when compared to similar region of the castrated bull epididymis. Most of the proteins, which are secreted, are having MW between 20 and 85 kDa. Six proteins, which are known to be highly dependent on androgens, are also of acidic in nature except for one protein having basic pH. A protein spot (pI: 6.55–6.85, and MW: <20 kDa) which appeared at the same site in all the three regions of the epididymis in the normal bull but was absent at the same site in all the three regions of the castrated bull was subjected for identification of the protein by MALDI-MS. The results revealed that this protein is an interferon-stimulated protein and it could be ISG15/UCRP.

Abstract 511: Characterization of humoral response profiles of ovarian cancer patients as diagnostic biomarkers

Abstract Ovarian cancer is the 5th leading cause of cancer death among women and is the leading cause of death among gynecologic malignancies as a result of limitations in early diagnosis. It is associated with the induction of humoral immunity, characterized by the generation of IgG against a wide range of tumor-associated antigens. Although the role of circulating tumor-reactive IgG is unclear, it has been demonstrated to correlate with poor prognosis and survival. The use of tumor-reactive IgG as a potential diagnostic and/or prognostic tool has been limited since most studies have identified components with shared expression in non-neoplastic tissue and failed to define targets exhibiting ubiquitous expression in ovarian cancers. A number of these limitations result from the use of recombinant proteins (translated in non-mammalian cells) as targets to define immunoreactivity. The power in utilizing the autologous humoral response is its ability to define alterations which might lead to the altered appearance or aberrant association of antigen with other cellular components, thereby resulting in the induction of humoral responses. We therefore hypothesize that the ovarian patient humoral response to specific tumor antigens can serve as diagnostic biomarkers of ovarian cancer. To investigate patients’ humoral response to “natural” tumor antigens, ovarian tumor cell lysates and tumor-derived exosomes were separated into antigenic proteins by first dimension isoelectric focusing via an immobilized pH gradient and/or rotofor fractionation. Reactive proteins were further separated by second dimension SDS-PAGE, followed by western immunoblotting using autologous or heterologous patient sera. Circulating tumor-reactive IgG was found to recognize shared (MW 37-75 kD, pI 3.0-5.5) and distinct (MWexo ∼50 kD, pI 9.0-10.5; MWlys∼15 kD, pI 4.3-6.5) ovarian tumor cell proteins. Recognition of these antigenic proteins was not observed using antibodies derived from normal control patients. Collectively, our data supports that the circulating IgG produced in the ovarian cancer humoral response recognizes specific tumor antigens (many of which are shared among cancer patients) and will thus have utility in diagnosing these patients. Further identification of these antigenic proteins by mass spectrometry will produce specific diagnostic targets and may provide insights on common driver mechanisms in ovarian cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 511. doi:1538-7445.AM2012-511

Native Mutant Huntingtin in Human Brain: EVIDENCE FOR PREVALENCE OF FULL-LENGTH MONOMER

Huntington disease (HD) is caused by polyglutamine expansion in the N terminus of huntingtin (htt). Analysis of human postmortem brain lysates by SDS-PAGE and Western blot reveals htt as full-length and fragmented. Here we used Blue Native PAGE (BNP) and Western blots to study native htt in human postmortem brain. Antisera against htt detected a single band broadly migrating at 575-850 kDa in control brain and at 650-885 kDa in heterozygous and Venezuelan homozygous HD brains. Anti-polyglutamine antisera detected full-length mutant htt in HD brain. There was little htt cleavage even if lysates were pretreated with trypsin, indicating a property of native htt to resist protease cleavage. A soluble mutant htt fragment of about 180 kDa was detected with anti-htt antibody Ab1 (htt-(1-17)) and increased when lysates were treated with denaturants (SDS, 8 M urea, DTT, or trypsin) before BNP. Wild-type htt was more resistant to denaturants. Based on migration of in vitro translated htt fragments, the 180-kDa segment terminated ≈htt 670-880 amino acids. If second dimension SDS-PAGE followed BNP, the 180-kDa mutant htt was absent, and 43-50 kDa htt fragments appeared. Brain lysates from two HD mouse models expressed native full-length htt; a mutant fragment formed if lysates were pretreated with 8 M urea + DTT. Native full-length mutant htt in embryonic HD(140Q/140Q) mouse primary neurons was intact during cell death and when cell lysates were exposed to denaturants before BNP. Thus, native mutant htt occurs in brain and primary neurons as a soluble full-length monomer.