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1.
Lab Chip ; 22(24): 4822-4830, 2022 12 06.
Artigo em Inglês | MEDLINE | ID: mdl-36382608

RESUMO

High-throughput phenotypic cell sorting is critical to the development of cell-based therapies and cell screening discovery platforms. However, current cytometry platforms are limited by throughput, number of fractionated populations that can be isolated, cell viability, and cost. We present an ultrathroughput microfluidic cell sorter capable of processing hundreds of millions of live cells per hour per device based on protein expression. This device, a next-generation microfluidic cell sorter (NG-MICS), combines multiple technologies, including 3D printing, reversible clamp sealing, and superhydrophobic treatments to create a reusable and user-friendly platform ready for deployment. The utility of such a platform is demonstrated through the rapid isolation of mature natural killer cells from peripheral blood mononuclear cells, for use in CAR-NK therapies at clinically-relevant scale.


Assuntos
Leucócitos Mononucleares , Microfluídica
2.
Sci Adv ; 8(35): eabo7792, 2022 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-36054348

RESUMO

Circulating tumor cells (CTCs) break free from primary tumors and travel through the circulation system to seed metastatic tumors, which are the major cause of death from cancer. The identification of the major genetic factors that enhance production and persistence of CTCs in the bloodstream at a whole genome level would enable more comprehensive molecular mechanisms of metastasis to be elucidated and the identification of novel therapeutic targets, but this remains a challenging task due to the heterogeneity and extreme rarity of CTCs. Here, we describe an in vivo genome-wide CRISPR knockout screen using CTCs directly isolated from a mouse xenograft. This screen elucidated SLIT2-a gene encoding a secreted protein acting as a cellular migration cue-as the most significantly represented gene knockout in the CTC population. SLIT2 knockout cells are highly metastatic with hypermigratory and mesenchymal phenotype, resulting in enhanced cancer progression in xenograft models.


Assuntos
Células Neoplásicas Circulantes , Animais , Transição Epitelial-Mesenquimal , Xenoenxertos , Humanos , Camundongos , Metástase Neoplásica/patologia , Células Neoplásicas Circulantes/patologia
3.
ACS Cent Sci ; 8(1): 102-109, 2022 Jan 26.
Artigo em Inglês | MEDLINE | ID: mdl-35106377

RESUMO

Phage display is a critical tool for developing antibodies. However, existing approaches require many time-consuming rounds of biopanning and screening of potential candidates due to a high rate of failure during validation. Herein, we present a rapid on-cell phage display platform which recapitulates the complex in vivo binding environment to produce high-performance human antibodies in a short amount of time. Selection is performed in a highly stringent heterogeneous mixture of cells to quickly remove nonspecific binders. A microfluidic platform then separates antigen-presenting cells with high throughput and specificity. An unsupervised machine learning algorithm analyzes sequences of phage from all pools to identify the structural trends that contribute to affinity and proposes ideal candidates for validation. In a proof-of-concept screen against human Frizzled-7, a key ligand in the Wnt signaling pathway, antibodies with picomolar affinity were discovered in two rounds of selection that outperformed current gold-standard reagents. This approach, termed µCellect, is low cost, high throughput, and compatible with a wide variety of cell types, enabling widespread adoption for antibody development.

4.
ACS Cent Sci ; 8(12): 1618-1626, 2022 Dec 28.
Artigo em Inglês | MEDLINE | ID: mdl-36589880

RESUMO

Genome-wide loss-of-function screens are critical tools to identify novel genetic regulators of intracellular proteins. However, studying the changes in the organelle-specific expression profile of intracellular proteins can be challenging due to protein localization differences across the whole cell, hindering context-dependent protein expression and activity analyses. Here, we describe nuPRISM, a microfluidics chip specifically designed for large-scale isolated nuclei sorting. The new device enables rapid genome-wide loss-of-function phenotypic CRISPR-Cas9 screens directed at intranuclear targets. We deployed this technology to identify novel genetic regulators of ß-catenin nuclear accumulation, a phenotypic hallmark of APC-mutated colorectal cancer. nuPRISM expands our ability to capture aberrant nuclear morphological and functional traits associated with distinctive signal transduction and subcellular localization-driven functional processes with substantial resolution and high throughput.

5.
ACS Nano ; 15(12): 19202-19210, 2021 12 28.
Artigo em Inglês | MEDLINE | ID: mdl-34813293

RESUMO

Advances in single-cell level profiling of the proteome require quantitative and versatile platforms, especially for rare cell analyses such as circulating tumor cell (CTC) profiling. Here we demonstrate an integrated microfluidic chip that uses magnetic nanoparticles to capture single tumor cells with high efficiency, permits on-chip incubation, and facilitates in situ cell-surface protein expression analysis. Combined with phage-based barcoding and next-generation sequencing technology, we were able to monitor changes in the expression of multiple surface markers stimulated in response to CTC adherence. Interestingly, we found fluctuations in the expression of Frizzled2 (FZD2) that reflected the microenvironment of the single cells. This platform has a high potential for in-depth screening of multiple surface antigens simultaneously in rare cells with single-cell resolution, which will provide further insights regarding biological heterogeneity and human disease.


Assuntos
Bacteriófagos , Nanopartículas , Células Neoplásicas Circulantes , Linhagem Celular Tumoral , Separação Celular , Humanos , Microfluídica , Microambiente Tumoral
6.
Nat Biomed Eng ; 3(10): 796-805, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31548591

RESUMO

Genome-scale functional genetic screens are used to identify key genetic regulators of a phenotype of interest. However, the identification of genetic modifications that lead to a phenotypic change requires sorting large numbers of cells, which increases operational times and costs and limits cell viability. Here, we introduce immunomagnetic cell sorting facilitated by a microfluidic chip as a rapid and scalable high-throughput method for loss-of-function phenotypic screening using CRISPR-Cas9. We used the method to process an entire genome-wide screen containing more than 108 cells in less than 1 h-considerably surpassing the throughput achieved by fluorescence-activated cell sorting, the gold-standard technique for phenotypic cell sorting-while maintaining high levels of cell viability. We identified modulators of the display of CD47, which is a negative regulator of phagocytosis and an important cell-surface target for immuno-oncology drugs. The top hit of the screen, the glutaminyl cyclase QPCTL, was validated and shown to modify the N-terminal glutamine of CD47. The method presented could bridge the gap between fluorescence-activated cell sorting and less flexible yet higher-throughput systems such as magnetic-activated cell sorting.


Assuntos
Genoma , Ensaios de Triagem em Larga Escala/métodos , Separação Imunomagnética/métodos , Fenótipo , Antígeno CD47/metabolismo , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Citometria de Fluxo , Edição de Genes , Humanos , Imunoterapia , Dispositivos Lab-On-A-Chip , Neoplasias/terapia
7.
Elife ; 5: e11184, 2016 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-27003594

RESUMO

The polyglutamine expansion in huntingtin protein causes Huntington's disease. Here, we investigated structural and biochemical properties of huntingtin and the effect of the polyglutamine expansion using various biophysical experiments including circular dichroism, single-particle electron microscopy and cross-linking mass spectrometry. Huntingtin is likely composed of five distinct domains and adopts a spherical α-helical solenoid where the amino-terminal and carboxyl-terminal regions fold to contain a circumscribed central cavity. Interestingly, we showed that the polyglutamine expansion increases α-helical properties of huntingtin and affects the intramolecular interactions among the domains. Our work delineates the structural characteristics of full-length huntingtin, which are affected by the polyglutamine expansion, and provides an elegant solution to the apparent conundrum of how the extreme amino-terminal polyglutamine tract confers a novel property on huntingtin, causing the disease.


Assuntos
Proteína Huntingtina/química , Proteína Huntingtina/metabolismo , Peptídeos/metabolismo , Fenômenos Biofísicos , Dicroísmo Circular , Espectrometria de Massas , Microscopia Eletrônica , Conformação Proteica
8.
Hum Mol Genet ; 23(9): 2324-38, 2014 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-24334607

RESUMO

Huntington's disease (HD) is an autosomal dominant, neurodegenerative disorder that can be characterized by the presence of protein inclusions containing mutant huntingtin within a subset of neurons in the brain. Since their discovery, the relevance of inclusions to disease pathology has been controversial. We show using super-resolution fluorescence imaging and Förster resonance energy transfer (FRET) in live cells, that mutant huntingtin fragments can form two morphologically and conformationally distinct inclusion types. Using fluorescence recovery after photobleaching (FRAP), we demonstrate that the two huntingtin inclusion types have unique dynamic properties. The ability to form one or the other type of inclusion can be influenced by the phosphorylation state of serine residues at amino acid positions 13 and 16 within the huntingtin protein. We can define two types of inclusions: fibrillar, which are tightly packed, do not exchange protein with the soluble phase, and result from phospho-modification at serines 13 and 16 of the N17 domain, and globular, which are loosely packed, can readily exchange with the soluble phase, and are not phosphorylated in N17. We hypothesize that the protective effect of N17 phosphorylation or phospho-mimicry seen in animal models, at the level of protein inclusions with elevated huntingtin levels, is to induce a conformation of the huntingtin amino-terminus that causes fragments to form tightly packed inclusions that do not exit the insoluble phase, and hence exert less toxicity. The identification of these sub-types of huntingtin inclusions could allow for drug discovery to promote protective inclusions of mutant huntingtin protein in HD.


Assuntos
Doença de Huntington/metabolismo , Corpos de Inclusão/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/metabolismo , Animais , Recuperação de Fluorescência Após Fotodegradação , Transferência Ressonante de Energia de Fluorescência , Proteína Huntingtina , Camundongos , Fosforilação , Técnicas de Cultura de Tecidos
9.
J Biol Chem ; 287(47): 39626-33, 2012 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-23012356

RESUMO

Among the known pathways of protein nuclear import, the karyopherin ß2/transportin pathway is only the second to have a defined nuclear localization signal (NLS) consensus. Huntingtin, a 350-kDa protein, has defined roles in the nucleus, as well as a CRM1/exportin-dependent nuclear export signal; however, the NLS and exact pathway of import have remained elusive. Here, using a live cell assay and affinity chromatography, we show that huntingtin has a karyopherin ß2-dependent proline-tyrosine (PY)-NLS in the amino terminus of the protein. This NLS comprises three consensus components: a basic charged sequence, a downstream conserved arginine, and a PY sequence. Unlike the classic PY-NLS, which has an unstructured intervening sequence between the consensus components, we show that a ß sheet structured region separating the consensus elements is critical for huntingtin NLS function. The huntingtin PY-NLS is also capable of import through the importin/karyopherin ß1 pathway but was not functional in all cell types tested. We propose that this huntingtin PY-NLS may comprise a new class of multiple import factor-dependent NLSs with an internal structural component that may regulate NLS activity.


Assuntos
Proteínas do Tecido Nervoso/genética , Sinais de Localização Nuclear/fisiologia , Proteínas Nucleares/genética , beta Carioferinas/genética , Animais , Humanos , Proteína Huntingtina , Camundongos , Camundongos Transgênicos , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/metabolismo , Mapeamento de Peptídeos/métodos , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , beta Carioferinas/metabolismo
10.
Proc Natl Acad Sci U S A ; 109(9): 3528-33, 2012 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-22331905

RESUMO

Huntington disease (HD) is a progressive neurodegenerative monogenic disorder caused by expansion of a polyglutamine stretch in the huntingtin (Htt) protein. Mutant huntingtin triggers neural dysfunction and death, mainly in the corpus striatum and cerebral cortex, resulting in pathognomonic motor symptoms, as well as cognitive and psychiatric decline. Currently, there is no effective treatment for HD. We report that intraventricular infusion of ganglioside GM1 induces phosphorylation of mutant huntingtin at specific serine amino acid residues that attenuate huntingtin toxicity, and restores normal motor function in already symptomatic HD mice. Thus, our studies have identified a potential therapy for HD that targets a posttranslational modification of mutant huntingtin with critical effects on disease pathogenesis.


Assuntos
Gangliosídeo G(M1)/uso terapêutico , Atividade Motora/efeitos dos fármacos , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/metabolismo , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Animais , Códon/efeitos dos fármacos , Corpo Estriado/metabolismo , Dimerização , Modelos Animais de Doenças , Fosfoproteína 32 Regulada por cAMP e Dopamina/biossíntese , Fosfoproteína 32 Regulada por cAMP e Dopamina/genética , Avaliação Pré-Clínica de Medicamentos , Gangliosídeo G(M1)/administração & dosagem , Proteína Huntingtina , Bombas de Infusão Implantáveis , Infusões Parenterais , Camundongos , Camundongos Mutantes Neurológicos , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/genética , Neurônios/metabolismo , Fosforilação/efeitos dos fármacos , Fosfosserina/análise , Desempenho Psicomotor/efeitos dos fármacos
11.
Nat Chem Biol ; 7(7): 453-60, 2011 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-21623356

RESUMO

Two serine residues within the first 17 amino acid residues of huntingtin (N17) are crucial for modulation of mutant huntingtin toxicity in cell and mouse genetic models of Huntington's disease. Here we show that the stress-dependent phosphorylation of huntingtin at Ser13 and Ser16 affects N17 conformation and targets full-length huntingtin to chromatin-dependent subregions of the nucleus, the mitotic spindle and cleavage furrow during cell division. Polyglutamine-expanded mutant huntingtin is hypophosphorylated in N17 in both homozygous and heterozygous cell contexts. By high-content screening in live cells, we identified kinase inhibitors that modulated N17 phosphorylation and hence huntingtin subcellular localization. N17 phosphorylation was reduced by casein kinase-2 inhibitors. Paradoxically, IKKß kinase inhibition increased N17 phosphorylation, affecting huntingtin nuclear and subnuclear localization. These data indicate that huntingtin phosphorylation at Ser13 and Ser16 can be modulated by small-molecule drugs, which may have therapeutic potential in Huntington's disease.


Assuntos
Caseína Quinase II/antagonistas & inibidores , Doença de Huntington/metabolismo , Quinase I-kappa B/antagonistas & inibidores , Proteínas do Tecido Nervoso/genética , Proteínas Nucleares/genética , Inibidores de Proteínas Quinases/farmacologia , Animais , Western Blotting , Linhagem Celular , Núcleo Celular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Retículo Endoplasmático/metabolismo , Imunofluorescência , Proteína Huntingtina , Doença de Huntington/enzimologia , Doença de Huntington/genética , Camundongos , Mutação , Fosforilação , Serina/genética , Fuso Acromático/metabolismo , Transfecção
12.
Hum Mol Genet ; 20(10): 1937-51, 2011 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-21355047

RESUMO

Huntington's disease (HD) is caused by an expanded CAG tract in the Interesting transcript 15 (IT15) gene encoding the 350 kDa huntingtin protein. Cellular stresses can trigger the release of huntingtin from the endoplasmic reticulum, allowing huntingtin nuclear entry. Here, we show that endogenous, full-length huntingtin localizes to nuclear cofilin-actin rods during stress and is required for the proper stress response involving actin remodeling. Mutant huntingtin induces a dominant, persistent nuclear rod phenotype similar to that described in Alzheimer's disease for cytoplasmic cofilin-actin rods. Using live cell temporal studies, we show that this stress response is similarly impaired when mutant huntingtin is present, or when normal huntingtin levels are reduced. In clinical lymphocyte samples from HD patients, we have quantitatively detected cross-linked complexes of actin and cofilin with complex formation varying in correlation with disease progression. By live cell fluorescence lifetime imaging measurement-Förster resonant energy transfer studies and western blot assays, we quantitatively observed that stress-activated tissue transglutaminase 2 (TG2) is responsible for the actin-cofilin covalent cross-linking observed in HD. These data support a direct role for huntingtin in nuclear actin re-organization, and describe a new pathogenic mechanism for aberrant TG2 enzymatic hyperactivity in neurodegenerative diseases.


Assuntos
Actinas/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Resposta ao Choque Térmico/genética , Doença de Huntington/enzimologia , Doença de Huntington/genética , Mutação/genética , Proteínas do Tecido Nervoso/genética , Proteínas Nucleares/genética , Transglutaminases/metabolismo , Fatores de Despolimerização de Actina/metabolismo , Animais , Linhagem Celular , Proteínas do Citoesqueleto/metabolismo , Proteínas de Ligação ao GTP/genética , Expressão Gênica/genética , Temperatura Alta , Humanos , Proteína Huntingtina , Espaço Intracelular/metabolismo , Linfócitos/metabolismo , Camundongos , Modelos Biológicos , Células NIH 3T3 , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/metabolismo , Ligação Proteica , Proteína 2 Glutamina gama-Glutamiltransferase , Transporte Proteico , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transglutaminases/genética
13.
FEBS J ; 275(17): 4252-62, 2008 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-18637947

RESUMO

After the successful cloning of the first gene for a polyglutamine disease in 1991, the expanded polyglutamine tract in the nine polyglutamine disease proteins became an obvious therapeutic target. Early hypotheses were that misfolded, precipitated protein could be a universal pathogenic mechanism. However, new data are accumulating on Huntington's disease and other polyglutamine diseases that appear to contradict the toxic aggregate hypothesis. Recent data suggest that the toxic species of protein in these diseases may be soluble mutant conformers, and that the protein context of expanded polyglutamine is critical to understanding disease specificity. Here we discuss recent publications that define other important therapeutic targets for polyglutamine-mediated neurodegeneration related to the context of the expanded polyglutamine tract in the disease protein.


Assuntos
Doença de Huntington/patologia , Humanos , Doença de Huntington/genética , Modelos Teóricos , Peptídeos/genética
14.
Autophagy ; 4(1): 91-3, 2008 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-17986868

RESUMO

We have recently published the precise definition of an aminoterminal membrane association domain in huntingtin, capable of targeting to the endoplasmic reticulum and late endosomes as well as autophagic vesicles. In response to ER stress induced by several pathways, huntingtin releases from membranes and rapidly translocates into the nucleus. Huntingtin is then capable of nuclear export and re-association with the ER in the absence of stress. This release is inhibited when huntingtin contains the polyglutamine expansion seen in Huntington's disease. As a result, mutant huntingtin expressing cells have a perturbed ER and an increase in autophagic vesicles. Here, we discuss the potential function of the huntingtin protein as an ER sentinel, potentially regulating autophagy in response to ER stress. We compare these recent findings to the well characterized mammalian target of rapamycin, mTor, a protein described over a decade ago as related to huntingtin structurally by leucine-rich, repetitive HEAT sequences. Since then, the described functional similarities between Huntingtin and mTor are striking, and this new information about huntingtin's direct association with autophagic vesicles indicates that this structural similarity may extend to functional similarities having an impact upon ER functionality and autophagy.


Assuntos
Autofagia/fisiologia , Retículo Endoplasmático/ultraestrutura , Membranas Intracelulares/metabolismo , Estresse Oxidativo , Animais , Retículo Endoplasmático/metabolismo , Humanos , Proteína Huntingtina , Doença de Huntington/metabolismo , Doença de Huntington/fisiopatologia , Camundongos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo
15.
Hum Mol Genet ; 16(21): 2600-15, 2007 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-17704510

RESUMO

Huntington's disease is caused by an expanded polyglutamine tract in huntingtin protein, leading to accumulation of huntingtin in the nuclei of striatal neurons. The 18 amino-acid amino-terminus of huntingtin is an amphipathic alpha helical membrane-binding domain that can reversibly target to vesicles and the endoplasmic reticulum (ER). The association of huntingtin to the ER is affected by ER stress. A single point mutation in huntingtin 1-18 predicted to disrupt this helical structure displayed striking phenotypes of complete inhibition of polyglutamine-mediated aggregation, increased huntingtin nuclear accumulation and greatly increased mutant huntingtin toxicity in a striatal-derived mouse cell line. Huntingtin vesicular interaction mediated by 1-18 is specific to late endosomes and autophagic vesicles. We propose that huntingtin has a normal biological function as an ER-associated protein that can translocate to the nucleus and back out in response to ER stress or other events. The increased nuclear entry of mutant huntingtin due to loss of ER-targeting results in increased toxicity.


Assuntos
Núcleo Celular/metabolismo , Doença de Huntington/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Sinais de Localização Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Sequência de Aminoácidos , Animais , Autofagia , Linhagem Celular , Núcleo Celular/química , Sequência Conservada , Retículo Endoplasmático/química , Retículo Endoplasmático/metabolismo , Endossomos/química , Endossomos/metabolismo , Proteína Huntingtina , Camundongos , Dados de Sequência Molecular , Mutação , Proteínas do Tecido Nervoso/análise , Proteínas do Tecido Nervoso/genética , Sinais de Localização Nuclear/genética , Proteínas Nucleares/análise , Proteínas Nucleares/genética , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína
16.
Prog Neurobiol ; 83(4): 211-27, 2007 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-17240517

RESUMO

There are nine genetic neurodegenerative diseases caused by a similar genetic defect, a CAG DNA triplet-repeat expansion in the disease gene's open reading frame resulting in a polyglutamine expansion in the disease proteins. Despite the commonality of polyglutamine expansion, each of the polyglutamine diseases manifest as unique diseases, with some similarities, but important differences. These differences suggest that the context of the polyglutamine expansion is important to the mechanism of pathology of the disease proteins. Therefore, it is becoming increasingly paramount to understand the normal functions of these polyglutamine disease proteins, which include huntingtin, the polyglutamine-expanded protein in Huntington's disease (HD). Transcriptional dysregulation is seen in HD. Here we discuss the role of normal huntingtin in transcriptional regulation and misregulation in Huntington's disease in relation to potentially analogous model systems, and to other polyglutamine disease proteins. Huntingtin has functional roles in both the cytoplasm and the nucleus. One commonality of activity of polyglutamine disease proteins is at the level of protein dynamics and ability to import and export to and from the nucleus. Knowing the temporal location of huntingtin protein in response to signaling and neuronal communication could lead to valuable insights into an important trigger of HD pathology.


Assuntos
Núcleo Celular/metabolismo , Doença de Huntington/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/metabolismo , Transcrição Gênica/fisiologia , Transporte Ativo do Núcleo Celular , Glutamina/metabolismo , Humanos , Proteína Huntingtina , Doença de Huntington/genética , Proteínas do Tecido Nervoso/genética , Proteínas Nucleares/genética , Peptídeos/metabolismo
17.
Can J Neurol Sci ; 33(3): 278-91, 2006 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-17001815

RESUMO

Since the early 1990s, DNA triplet repeat expansions have been found to be the cause in an ever increasing number of genetic neurologic diseases. A subset of this large family of genetic diseases has the expansion of a CAG DNA triplet in the open reading frame of a coding exon. The result of this DNA expansion is the expression of expanded glutamine amino acid repeat tracts in the affected proteins, leading to the term, Polyglutamine Diseases, which is applied to this sub-family of diseases. To date, nine distinct genes are known to be linked to polyglutamine diseases, including Huntington's disease, Machado-Joseph Disease and spinobulbar muscular atrophy or Kennedy's disease. Most of the polyglutamine diseases are characterized clinically as spinocerebellar ataxias. Here we discuss recent successes and advancements in polyglutamine disease research, comparing these different diseases with a common genetic flaw at the level of molecular biology and early drug design for a family of diseases where many new research tools for these genetic disorders have been developed. Polyglutamine disease research has successfully used interdisciplinary collaborative efforts, informative multiple mouse genetic models and advanced tools of pharmaceutical industry research to potentially serve as the prototype model of therapeutic research and development for rare neurodegenerative diseases.


Assuntos
Expansão das Repetições de DNA , Doenças Neurodegenerativas/genética , Peptídeos/genética , Animais , Ataxina-1 , Ataxinas , Sequência de Bases , Canadá , Humanos , Proteína Huntingtina , Modelos Moleculares , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Doenças Neurodegenerativas/patologia , Doenças Neurodegenerativas/fisiopatologia , Doenças Neurodegenerativas/terapia , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Conformação de Ácido Nucleico , Complexo de Endopeptidases do Proteassoma/metabolismo , Conformação Proteica , Receptores Androgênicos/metabolismo , Transdução de Sinais/fisiologia
18.
Biochem Cell Biol ; 84(6): 912-7, 2006 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-17215878

RESUMO

Huntington's disease is a progressive neurodegenerative genetic disorder that is caused by a CAG triplet-repeat expansion in the first exon of the IT15 gene. This CAG expansion results in polyglutamine expansion in the 350 kDa huntingtin protein. The exact function of huntingtin is unknown. Understanding the pathological triggers of mutant huntingtin, and distinguishing the cause of disease from downstream effects, is critical to designing therapeutic strategies and defining long- and short-term goals of therapy. Many studies that have sought to determine the functions of huntingtin by determining huntingtin's protein-protein interactions have been published. Through these studies, huntingtin has been seen to interact with a large number of proteins, and is likely a scaffolding protein for protein-protein interactions. Recently, using imaging, integrative proteomics, and cell biology, huntingtin has been defined as a membrane-associated protein, with activities related to axonal trafficking of vesicles and mitochondria. These functions have also been attributed to some huntingtin-interacting proteins. Additionally, discoveries of a membrane association domain and a palmitoylation site in huntingtin reinforce the fact that huntingtin is membrane associated. In Huntington's disease mouse and fly models, axonal vesicle trafficking is inhibited, and lack of proper uptake of neurotrophic factors may be an important pathological trigger leading to striatal cell death in Huntington's disease. Here we discuss recent advances from many independent groups and methodologies that are starting to resolve the elusive function of huntingtin in vesicle transport, and evidence that suggests that huntingtin may be directly involved in membrane interactions.


Assuntos
Membrana Celular/metabolismo , Doença de Huntington/metabolismo , Modelos Neurológicos , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/metabolismo , Vesículas Transportadoras/metabolismo , Humanos , Proteína Huntingtina , Doença de Huntington/etiologia , Doença de Huntington/genética , Doença de Huntington/patologia , Proteínas do Tecido Nervoso/genética , Proteínas Nucleares/genética , Vesículas Transportadoras/patologia
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