Saturday, 22 December 2012

Medical applications I

Role of androgen receptor CAG to repeat polymorphism in prostate cancer, and spinal and bulbar muscular atrophy by Kumar
Androgens male sex hormones play a central role in development, differentiation and functional maturation of male reproductive and accessory sex tissues.  Most of its biological effects are mediated through an intracellular TF, androgen receptor (AR) at level of gene regulation. AR is in superfamily of nuclear hormone receptors. It passes signals from steroid/hormone to target genes by interacting with spec response element DNA sequences and coregulatory proteins which consist of activators and corepressors.

Mutations in AR gen e are linked to endocrine dysfunctions. Eg polymorphism involving expansion of CAG codon repeat, coding for polyglutamine (polyQ) tract in Nterminal domain, which has a powerful transactivation region.  PolyQ length variations are associated with prostate cancer, neurodegenerative disease, Kennedy's Disease.

AR consists of N-terminal domain, DNA bbinding domain and ligand binding domain. Between DBD and LBD are structurally flexible aa sequences, the hinge region. DBD specifies high affinity binding to DNA at certain short spec nt sequences or response elements. C-terminal domain has LBD and within it, a small activation function (AF2) fomain that regulates transcription.

Another activation domain, AF1 is in NTD. It is constitutively active in absence of ligand. Transactivation functions of AF1 are responsibly for recruiting coactivator/corepressor molcules to form active preinitiation complexes through TATA box and Pol II for regulation of target gene transcription.

Role of androgen receptor CAG repeats in diseases
Prostate cancer is most common nonskin malignancy and second leading cause of cancer deaths in American men.   Androgens acting through AR are important for normal function and structure of prostate. Androgens are strong tumour promoters. They augment effect of carcinogens and stimulate cell division.

Androgens control gene expression. Prostate tissue must maintain proper struictuer and functions of AR to avoid unwanted side effects of androgens. AR gene is on X chromosome. AR is expressed in nearly all prostate cancers. AR is a candidate gene for prostate cancer.

AR N-terminal region has polyQ, proline (polyP), and glycine homopolymeric sequences. This distinguishes AR from other members of the nuclear receptor superfamily.  AR size has variability due to polymorphic regions.

Normal PolQ lengths in Caucasian males is 22 repeats. Shorter polyQ associated with increase risk of prostate cancer. PolyQ lengths greater than 40 are related to Kennedy's disease.

Androgen receptor CAG repeat and prostate cancer
Prostate cancers can be either androgen-dependent or androgen-independent. For the former, progression, cell growth and survival depend on AR regulation. The latter relies on cellular pathways which may or may not involve AR.

Inverse relationship between CAG chain length and AR transcriptional activity.

Shorter polyQ length is associated with younger age at diagnosis. Higher risk of prostate cancer.  Short alleles may impose higher transactivation on receptor. Inverse relation between number of Q residues in polyQ tract and transcriptional activity.

SBMA is characterised by death of motor neurons expressing high levels of AR. Degeneration of motor neurons are in spinal cord and bulbar regions. Androgen insensitvity. AR polyQ chain is 40-62 in SBMA patients.

Extension of polyQ length in AR longer than 40 causes neurotoxicity. For pathogenic phenotype to be exhibited in Drosophila, AR must be able to bind ligand,. form active AF2 pocket, translcoate to nucleus, associate with DNA and interact with cofactors via AF2.

AR protein with elongated polyQ forms intracellular aggregates. Self association depends on androgen hormone binding AR.  PolyQ tract is exposed on ligand association as AR is released from heat chock proteins complex. This allows physical interaction with other polyQ ARs or production of aberrant conformational changes in AR. These lead to aggregation.

Extende polyQ length induces inclusion body formatoin in affected neurons which are often truncated. Proteolyitc cleavage  may cause enhanced toxicity of AR gene products.

Some studies show that transcriptional dysregulation of AR may underlie molecular mechanism of neuronal dysfunction in SBMA.  Certain AR coactivator are sequestered into nuclear inclusions in SBMA. SMA mouse model suggests nuclear inclusion induces transcriptional dysregulation of dynactin 1. GR, a physiological regulator of transcription, can modulate elongated polyQ mediated protein aggregation in AR and Huntington's protein.

Polyglutamine diseases: emerging concepts in pathogenesis and therapy by Shao and Diamond
Proteolytic change
Several polyQ disease eg HD, SBMA and SCA3 are linked to proteolytic cleavage. This liberates toxic polyQ-containing fragments.

Conformational change
Expanded polyQ protein is aggregation prone in vitro. Expanded polyQ tract in target protein facilitates transition to a novel toxic conformation.

Conformal change in expanded Htt peptide precedes its aggregation.  α-helical state predominates after protein purification. After several days protein shifts to β-sheet rich conformation. More prone to aggregation. Causes cellular toxicity.

CBP has been found in nuclear inclusions formed by polyQ expanded proteins eg. Htt, AR, ataxin-1 and atrophin-1.  Depletion from its normal nuclear locations. Disrupts its regulation of target genes.

Sp1 associates with Htt in polyQ-dependent fashion. This interaction repressed Sp1 transcriptional activity.

In its soluble form, polyQ-expanded Htt is reduced in its cytoplasmic interaction with REST/NRSF.

Metabolism and mitochondrial dysfunction
HD patients show metabolic defects, weight loss despite adequate calorie intake. Linked to mitochondrial dysfunction. Htt protein might influence mitochondrial function.

Proteotoxic stress
Pathogenic polyQ length threshold that causes human disease matches that which predisposes polyQ proteins to aggregate in vitro. Protein misfolding may play a key role in pathogenesis. Autophagy (process where cell can degrade aggregated proteins) has been implicated in resistance to polyQ pathology in cells, Drosophila and mice. Loss of autophagy induces neurodegeneration in mice associated with accumulated misfolded proteins. Proteasome malfunction has been implicated in polyQ pagthogenesis. In cultured cells, large intracellular inclusions formed by Htt and cystic fibrosis TM conductance regulatory protein are associated with proteasome impairment. Problems might be acused by inability of proteasome to guest soluble expanded polyQ proteins.

Aggregation vs inclusion formation
Soluble polyQ oligomers were detected in a mouse model in SBMA They were composed of N-terminal fragments of AR. They appeared weeks before symptom onset, before detectable inclusions and disappeared with castration which halts disease progression. Misfolded protein may interfere with critical cellular events. It may challenge cell's ability to prevent more widespread misfolding and compromise its ability to keep up with protein degradation.

Alteration of normal protein function
PolyQ disease are dominantly inherited.  Dominant effect might be caused from perturbation of normal polyQ protein function. Eg HTt inactivation in mice causes neurodegeneration. Overexpression of wt Htt in transgenic mice can resue mutant Ntt tocivity.

Therapeutic strategies
Reversing cellular defects
PolyQ pathogenesis is a problem of transcriptional regulation. Mutant proteins disrupt acetyltransferase activbity. HDAC inhibitors have shown efficacy in disease models.

Targeting polyglutamine proteins
Gene therapy
Selectively reduce expression of expanded allele  Use siRNAs to knock down.

Caspase activation is implicated in pathogenesis of polyQ diseases  They cleave polyQ proteins and induce apoptosis. If specific proteases are found to cleave polyQ proteins to generate toxic fragments. Create protease inhibitors.

Protein clearance
Stimulate cellular degradation pathways that preferentially target misfolded disease proteins.

Protein aggregation
Direct targeting of polyQ aggregates. Induce molecular chpaerons eg Hsp70 that aid protein refolding and degradation.

Stabilising native conformation
Expanded polyQ protean can exist in multiple conformations. Influence equilibrium between toxic and nontoxic conformation. Target protein to stabilise native conformable  A QBP1 stabilised native conformation of a thioredoxin/polyQ fusion protein. Prevents it from converting into aggregation prone βsheet rich conformation.

Altered transcription in yeast expressing expanded polyglutamine by Hughes
To determine whether polyQ-mediated transcriptional dysregulation occurs in yeast, they expressed polyQ tracts in S. cerevisiae. Gene expression profiles were determined for strains expressing either a cytoplasmic or nuclear protein with 23 or 75 glutamines. These profiles were compared to profiles of mutant yeast strains.

Transcriptional induction of genes encoding chaperones and heat-shock factors was caused by expression of expanded polyQ in either nucleus or cytoplasm. Transcriptional repression was most prominent in yeast expressing nuclear expanded polyQ and similar to profiles of yeast strains deleted for components of histone acetyl transferase complex SAGA. Promoter from one affected gene, PHO84 was repressed by expanded polyQ in a reporter gene assay. This effect was mitigated by histone deacetylase inhibitor, Trichostin A.

Nuclear expanded Q enhanced toxicity of a deletion in SAGA component SPT3.

Aggregation of polyQ in yeast is affected by nuclear localisation
In a mammalian cell model, nuclear localisation of expanded huntingtin fragment enhances its toxicity. To examine effects of nuclear localisation in yeast, they fused simian virus 40NLS to 23Q and 75Q-GFP fusions. 23Q fusion shows diffuse staining throughout cell.  NLS-23Q fusion localised to discrete foci in yeast nucleus. Same pattern of localisation for NLS-75Q fusion. This indicates that transport into nucleus can facilitate aggregation of nonexpanded polyQ repeats.

Expanded nuclear polyQ caused altered transcription in yeast
They grew yeast cells expressing either vector 23-GFP, 75Q-GFP, NLS-23Q-GFP, or NLS-75Q-GFP in parallel. poly(A)+ RNA was prepared fluorescently labelled and hybridised to yeast ORF DNA arrays. RNA from cells expressing  a form of polyQ was cohybridised with RNA prepared from vector-containing yeast.  Experiments done in duplicate with dye orientations reversed.

Expanded polyQ induced genes involved in protein folding and chaperone function. Of 35 genes induced in NLS-75Q-GFP yeast 14 are involved in protein folding.  Data indicates yeast constitutively expressing expanded polyQ mount a mild heatshock response.  PolyQ-mediated induction of heat shock factors has been observed in C.elegans and chaperone overexpression suppressed polyQ toxicity in Drosophila models. Of chaperone genes, HSP104 was induced to greatest degree in N75Q and NLS-N75Q strains. HSP104 is required for prion propagation and for cytoplasmic aggregation of expanded polyQ in yeast.

Comparing data from NLS75Q-expressing strain with gene expression profiles from several mutant strains showed overlap with several strains deficient in SAGA complex proteins. Among genes repressed in yeast containing mutations in components of histone acetyltransferase complex SAGA, 15 are repressed in common with SAGA mutants gcn5, spt3 and spt 20.

Of these core 15 genes, 10 are reoressed in NLS-75Q-containing yeast. Repression appears senmsitive to polyQ length and nuclear localisation.

Expanded nuclear polyglutamine induces transcriptional changes that parallel those in SAGA complex mutants. (A) An existing database ( was used to identify genes that were decreased in gcn5spt3, and spt20 mutants. Genes that were decreased in one or more strains are enumerated in the Venn diagram. (B) Of the 26 genes that were decreased in the strain expressing NLS75Q, 10 were also decreased in all three SAGA complex mutants. (C) List of the 10 genes common to SAGA complex mutants and NLS75Q.

Expanded nuclear polyQ enhances toxicity of a spt3 deletion
Transcript profile of NLS75Q strain indicates SAGA defect. Test for possible genetic interactions between polyQ and mutations in gene encoding components

Transform strains with deletions in SAGA components with equal amounts of either vector DNA, DNA encoding a nonexpanded polyQ tract or DNA encoding n expanded nuclear polyQtract. (NLS-75Q).  Strains were deleted for GCN5, SPT3 or SPT7 (SAGA components), GAL11 (component of RNAPII mediator complex), or SNF5 (component of SWF/SNF complex). Latter 2 genes contains Q tracts. Selected due to genetic interaction with SAGA.

Score toxicity by comparing transformation efficiencies of polyQ constructs to each other and vector. Significant toxicity when nuclear expanded polyQ was expressed in a strain deleted for SPT3. spt3 strain alone has slow growth and lower transformation efficiency than wild-type. However, NLS-75Q expression reduced number of colonies by 75% and decreased average colony size. These effects not seen with 23Q expression. Indicates that NLS-75Q interferes with SPT3-related SAGA function.

Turning acetylation levels with HAT activators: therapeutic strategy in neurodegenerative diseases by Selvi et al
Acetylation loosens chromatin and facilitates transcription by neutralising positive charge of lysines. HAT action on gene transcription could affect global gene expression levels and can be gene specific (when histone acetylation is targeted to promoters).

Fig. 1. Mechanisms of p300 HAT activation. p300 acetyltransferase is subjected to several regulatory mechanisms which influence its activation and thereby its acetyltransferase activity. The first step in the activation of the enzyme is the acetylation of p300 itself, which subsequently brings about histone acetylation. This process is referred to as autoacetylation, which occurs as a transacetylation process. Several regulatory signals within the physiological system influence this process. A second mode of HAT induction is by the regulatory proteins; SIRT2 is a negative regulator, whereas nuclear receptors are positive regulators. The third mechanism involves the different post-translational modifications such as phosphorylation and methylation. Finally, exogenous addition of small molecules such as CTB, CTPB and nemorosone can also activate p300 HAT.
Huntington's disease is associated with transcriptional dysregulation. HDAC inhibitors may restore histone acetylation and transcriptional activation.

A new therapeutic option for neurological disease: activating HAT function

Preventing neuronal death: targeting CBP
Abnoram huntingtin binds acetyltransferase domains of CBP and PCAF. CBP redistributes in nuclear or cytoplasmic inclusions on overexpression of expanded HD constructs in cellular models. HAT activity is inhibited. Causes global histone deacetylation and cell death. Ubiquitylation and degradation of CBP could be selctively enhanced by mutated polyQ-expanded huntingtin.

CBP depletion and histone hypoacetylation was reported in models of mutant htt-induced neurotoxicity.

Histone deacetylation inhibitors arrest polyQ-dependent neurodegeneration in Drosophila by Steffan

Histone deacetylation inhibitors: pvreview and perspectives by Dokmanovic
Changes in expression of different HDACs have been reported in cancers. HDAC2 and 3 proteins are increased in colon cancer samples. HDAC1 is increased in gastric cancer.

HDACs, HDACi and gene expression
HATs and HDACs determine pattern of histone acetylation.

HDACs and HATs do not bind DNA directly. They interact with DNA through multiprotein complexes that include corepressors and coactivators.

HDACi  selectively alter a small proportion of expressed genes (2-10%) in transformed cells.  HDACi-induced selective alteration of gene transcription may be determined by composition and configuration of proteins in transcription factor complex icluding HDACs.

HDACi treatment inhibited induction of IFN-stumulated gene expression. HDACi inhibits catalytic sites on HDACs.

Altered acetylation in polyglutamic disease: an opportunity for therapeutic intervention? by Taylor

Fig. 2. (a) Dynamic regulation of histone acetylation involves balancing the activities of histone acetyltransferases and deacetylases. The acetylation status of the core histones influences chromatin structure and transcriptional activity. (b) The sequestration of acetylase activity by mutant polyglutamine is associated with a deficiency in histone acetylation. (c) The use of deacetylase inhibitors might tip the balance towards normal histone acetylation and restore transcriptional activity.

Hughes et al showed that expanded polyQ enhances toxicity of mutants in components of histone acetylation machinery. Repression of a target gene, Pho84, is reversed by treatment with trichostatin A (TSA), a histone deacetylase inhibitor.

Selvi (2010) says that HATs can be used as therapy in neurodegenerative diseases.

Polyglutamine-expanded ataxin-3 causes cerebellar dysfunction of SCA3 transgenic mice by inducing transcriptional dysregulation by Chou et al
SCA3 belongs to family of polyQ neurodegenerative disorders caused by expansion of unstable CAG repeat in coding region of gene.

Ataxin-3-Q79 mice showed altered mRNA expressions of proteins involved in glutamatergic neurotransmission, intracellular calcium signalling, MAP kinase pathways or regulating neuronal survival or differentiation compared to wt ataxin-3-Q22 mice.

This is supported by a study dhowing that nuclear localisation of polyQ-expanded Ataxin 3 ir required for manifestation of neurological symptoms in SCA3 transgenic mice.

PolyQ-expanded ataxin3-Q79 could cause transcriptional dysregulation and alter cerebellar gene expressions  PolyQ disease proteins including mutant ataxin-3-Q79 interact with polyQ-rich nuclear TFs or cofactors. Ataxin-3 functions as a transcriptional repressor. It recruits HDAC 3 or inhibits HAT activity of transcriptional co-activators including CBP and p300.

Estrogen receptor mutations and changes in downstream gene expression and signalling by Barone et al
Estrogens regulate growth and differentiation of breast cancers. 2/3 breast  cancers express estrogen receptor alpha (ERα). 2 isoforms have been described, ERα and ERβ. Each is encoded by unique genes. Share common structural and functional organisation.

Classical ER (ERα) has an amino-terminal region that contains ligand-independent activation function (AF-1), a central DNA-binding domain (DBD), and a carboxy-terminal hormone binding domain (HBD), which contains ligand-dependent activation function (AF-2). Binding of hormone to ERα facilitates classic genomic activities of receptor. Its binding to estrogen response elements in target genes activate or repress gene expression.

Variant ERα protein isoforms
Erα splice variants have been identified in normal tissue and tumours. These mRNA variants are usually coexpressed with wt repector. Splice variants can confer dominant-positive or dominant-negative effecst on cancer cells.

A variant is ERα exon 3. It misses part of DBD. Shows most significant increase in levels in breast cancer tissue. Isoform functions as dominant negative receptor. It can suppress estrogen-induced transcriptional activity reduce anchorage-dependent growth  soft-agar colony forming ability and intro invasion when transfected in breast cancer cells. This reduction in estrogen receptor signalling may cause unchecked estrogen stimulation encouraging carcinogenic events.

ERα mutations in tumours
Y537N mutation was discovered in metastatic breast tumour. Eliminates a C-terminal tyr residue considered to be a phosphorylation site. Mutant shows constitutive transactivation activity. ACtivity was slightly affected by estradiol, tamoxifen.

A yeast-based bioassay for determination of functional and nonfunctional estrogen receptros by Balmelli-Galacchi
To characterise activities of ER in breast tumour, RNA isolated from breast cancer cells and one breast cancer specimen was reverse tramscribed. ER cDNA was amplified by PCR. Products wer ecloned into an expression vector by in vivo homologous recombination in yeast.  Yeast strain carries a reporter gene, ADE2 coupled to an estrogen response element. Activation of reporter by ER yielded white colonies. Lack of ER activity yielded red colonies. Cells were lysed and wester-blotted.  Yeast colonies were picked from plates and grown. Plasmids were isolated and separated by agarose gel electrophoresis. Sequence analysis. This allows discrimination between wt ER, constitutively active ER and inactive ER.

Gap repair assay for functional screening of ER
RNA was isoalted from samples and reverse transcribed.  With cDNA PCR was performed of ER coding sequence. Unpurified PCR products were transformed into yeast cells with linearised gap repair expression vector. ER coding sequences were integrated into vector by homologous recom in yeast-generating ciruclar plasmids.

Yeast colonies growing on selective medium repaired ER expression plasmid, causing ER protein expression in yeast. By transferring transformed colonies from medioum lackin estrogen to estrogen-containing medium, 3 diff phenotypes. Colonies red on medium lacking estrogen and turn white in estrogen are wt ER. Colonies white without estrogen and stay white in etrsogen contain constitutively active ER. Colonies red in presence and absence of estrogen contain either nonfunctional EWR or self-ligated empty vectors.

A comprehensive study of TP53 mutations in chronic lymphocytic leukemia: analyse of 1287 diagnostic and 1148 follow-up CLL samples by Pekova

TP53 is a TF that plays pivotal role in process of DNA repair and apoptosis. TP53 mutations status was analysed.

RNA was isolated from patients' cells and DNA was prepared.  TP53 underwent PCR and sequencing.

FASAY was performed.  ADE2- yeast strain yIG397 was grown in YPDA++ medium overnight, reinoculated into fresh YPDA++ and transformed. TP53 gene were PCR amplified with primers FASAY forward and reverse. PCR products were recombined into linearised vector. Transformants were plated onto selection media with low adenine. Transformants with wt TP53 were distinguished from mutant TP53 on colour. Mutant had small red colonies. Colonies with wt TP53 were white and large.

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