The Possible Structural Models for Polyglutamine Aggregation: A Molecular Dynamics Simulations Study

Abstract

Huntington's disease is a neurodegenerative disorder caused by a polyglutamine (polyQ) expansion near the N-terminus of huntingtin. Previous studies have suggested that polyQ aggregation occurs only when the number of glutamine (Q) residues is more than 36-40, the disease threshold. However, the structural characteristics of polyQ nucleation in the very early stage of aggregation still remain elusive. In this study, we designed 18 simulation trials to determine the possible structural models for polyQ nucleation and aggregation with various shapes and sizes of initial β-helical structures, such as left-handed circular, right-handed rectangular, and left- and right-handed triangular. Our results show that the stability of these models significantly increases with increasing the number of rungs, while it is rather insensitive to the number of Qs in each rung. In particular, the 3-rung β-helical models are stable when they adopt the left-handed triangular and right-handed rectangular conformations due to the fact that they preserve high β-turn and β-sheet contents, respectively, during the simulation courses. Thus, we suggested that these two stable β-helical structures with at least 3 rungs might serve as the possible nucleation seeds for polyQ depending on how the structural elements of β-turn and β-sheet are sampled and preserved during the very early stage of aggregation.     

The Interaction of Polyglutamine Peptides with Lipid Membranes Is Regulated by Flanking Sequences Associated with Huntingtin

Huntington disease (HD) is caused by an expanded polyglutamine (poly(Q)) repeat near the N terminus of the huntingtin (htt) protein. Expanded poly(Q) facilitates formation of htt aggregates, eventually leading to deposition of cytoplasmic and intranuclear inclusion bodies containing htt. Flanking sequences directly adjacent to the poly(Q) domain, such as the first 17 amino acids on the N terminus (Nt17) and the polyproline (poly(P)) domain on the C-terminal side of the poly(Q) domain, heavily influence aggregation. Additionally, htt interacts with a variety of membraneous structures within the cell, and Nt17 is implicated in lipid binding. To investigate the interaction between htt exon1 and lipid membranes, a combination of in situ atomic force microscopy, Langmuir trough techniques, and vesicle permeability assays were used to directly monitor the interaction of a variety of synthetic poly(Q) peptides with different combinations of flanking sequences (KK-Q₃₅-KK, KK-Q₃₅-P₁₀-KK, Nt17-Q₃₅-KK, and Nt17-Q₃₅-P₁₀-KK) on model membranes and surfaces. Each peptide aggregated on mica, predominately forming extended, fibrillar aggregates. In contrast, poly(Q) peptides that lacked the Nt17 domain did not appreciably aggregate on or insert into lipid membranes. Nt17 facilitated the interaction of peptides with lipid surfaces, whereas the poly(P) region enhanced this interaction. The aggregation of Nt17-Q₃₅-P₁₀-KK on the lipid bilayer closely resembled that of a htt exon1 construct containing 35 repeat glutamines. Collectively, this data suggests that the Nt17 domain plays a critical role in htt binding and aggregation on lipid membranes, and this lipid/htt interaction can be further modulated by the presence of the poly(P) domain.     

Biochemical mechanisms for a possible involvement of the transglutaminase activity in the pathogenesis of the polyglutamine diseases: Minireview article

Summary. Transglutaminases are a family of enzymes which show the common capacity to catalyse the cross-linking of protein substrates. Some members of this family of enzymes are also capable to catalyse other chemical reactions for the cell life. The distribution and the role of these enzymes have been studied in numerous cell types and tissues, but only recently their expression and functions started to be investigated in the Nervous System. One of the main biochemical properties of the Transglutaminase enzymes is to form large protein aggregates that are insoluble in all known protein detergents. Recently, the Transglutaminase activity has been hypothesised to be involved in the pathogenetic mechanisms responsible for the formation of cellular inclusions present in the Corea Major and in other polyglutamine diseases. In this review we describe the biochemical mechanisms by which the Transglutaminases could play a critical role in the physiopathology of the polyglutamine diseases.     

Histone H3 is aberrantly phosphorylated in glutamine-repeat diseases

Double-labeling immunohistochemical studies staining with anti-ubiquitin and anti-phosphoserine antibodies and application of an enzymatic dephosphorylation technique reveal neuronal inclusions and affected nuclei to be aberrantly phosphorylated in brain tissues with patients with glutamine-repeat diseases. Regional distribution of the phosphorylated nuclei in neurons correlates with the pathology. To identify the target nuclear protein, transient expression of Huntington's disease exon 1 gene containing an expanded glutamine repeat was generated in a cell culture and nuclear inclusions were isolated with a fluorescence-activated cell sorting system. Immunoblotting studies of the aggregated nuclear proteins using anti-phosphoserine antibody demonstrate the protein of the aberrant phosphorylation as histone H3. The immunoblots of control and diseased brain tissues demonstrate that the phosphorylation of histone H3 is commonly increased in the diseased brains. Aberrant phosphorylation of histone H3 is surmised to be a shared pathological process in glutamine-repeat diseases.     

The polyglycine and polyglutamine repeats in the androgen receptor gene in Japanese and Caucasian populations

Human androgen receptor (AR) gene contains two polymorphic trinucleotide repeats of CAG and GGC, which code for polyglutamine and polyglycine tracts in the N-terminal domain in which the receptor activity resides. Longer repeats induce decrease of transactivation function in the AR receptor, weaken an anti-proliferative effect on various steroid-related tissues, and may promote the carcinogenesis of these cancers, such as breast, endometrial, and ovarian cancers. However, the incidences of these steroid-related cancers are remarkably lower in Japanese than in Caucasians. We hypothesize that the GGC and CAG repeats in AR gene correspond to lower incidence of steroid-related cancers in the Japanese population. To test this hypothesis, these two polymorphic trinucleotide repeats in AR gene were genotyped in 221 Japanese and 177 Caucasians. The results of genotyping in these loci clearly show that the distribution of GGC repeat is significantly different between these populations (P<0.001). Japanese (73.7%) had 16 GGC repeats compared to 53.3% for Caucasians. Japanese (3.8%) also had 17 GGC repeats compared to 36.2% for Caucasians. No Japanese had more than 18 GGC repeats compared to 3.4% for Caucasians. The length of CAG repeats in the Japanese population was not significantly different than that of the Caucasian population, although the CAG repeats varied from 14 to 31 and 15 to 29 repeats in Japanese and German populations, respectively. This study demonstrates that the Japanese population has shorter GGC compared to the Caucasian population, which may explain the incidences of estrogen-related cancers in these populations.     

Sleeping Beauty-mediated down-regulation of huntingtin expression by RNA interference

Huntington disease (HD) is a devastating neurologic disorder that is characterized by abnormal expansion of a CAG nt repeat in the first exon of the huntingtin (htt) gene, producing a mutant protein with an elongated polyglutamine stretch. The presence of this mutant protein is correlated with the characteristic loss of striatal neurons and the clinical manifestation of HD. Currently there is no effective treatment for the associated cell death. The aim of this study was to evaluate an innovative strategy combining RNA interference (RNAi) and gene transfer via the nonviral Sleeping Beauty (SB) transposon system to down-regulate Htt expression. siRNA expression vectors were designed to target exons 1, 4, 6, and 62 of the human htt gene. Real-time RT-PCR and Western blot analysis were used to quantify Htt mRNA and protein levels, respectively, in human cell lines. The results indicated that selected siRNA constructs significantly decreased Htt mRNA and protein levels relative to controls. In addition, SB transposition of the siRNA constructs into the genome reduced long-term protein expression of Htt by approximately 90%. The combination of siRNA, the SB transposon, and an accurate transgenic mouse model may permit evaluation of this approach in preventing the pathogenesis associated with expression of mutant Htt.     

Interaction with Polyglutamine Aggregates Reveals a Q/N-rich Domain in TDP-43

The identification of pathologic TDP-43 aggregates in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration, followed by the discovery of dominantly inherited point mutations in TDP-43 in familial ALS, have been critical insights into the mechanism of these untreatable neurodegenerative diseases. However, the biochemical basis of TDP-43 aggregation and the mechanism of how mutations in TDP-43 lead to disease remain enigmatic. In efforts to understand how TDP-43 alters its cellular localization in response to proteotoxic stress, we found that TDP-43 is sequestered into polyglutamine aggregates. Furthermore, we found that binding to polyglutamine aggregates requires a previously uncharacterized glutamine/asparagine (Q/N)-rich region in the C-terminal domain of TDP-43. Sequestration into polyglutamine aggregates causes TDP-43 to be cleared from the nucleus and become detergent-insoluble. Finally, we observed that sequestration into polyglutamine aggregates led to loss of TDP-43-mediated splicing in the nucleus and that polyglutamine toxicity could be partially rescued by increasing expression of TDP-43. These data indicate pathologic sequestration into polyglutamine aggregates, and loss of nuclear TDP-43 function may play an unexpected role in polyglutamine disease pathogenesis. Furthermore, as Q/N domains have a strong tendency to self-aggregate and in some cases can function as prions, the identification of a Q/N domain in TDP-43 has important implications for the mechanism of pathologic aggregation of TDP-43 in ALS and other neurodegenerative diseases.     

Curcumin enhances the polyglutamine-expanded truncated N-terminal huntingtin-induced cell death by promoting proteasomal malfunction

Formation of neuronal intranuclear inclusions of the disease proteins that are ubiquitinated and often associated with various proteasome components is the major hallmark of the polyglutamine diseases. Curcumin is a polyphenolic compound having anti-inflammatory, anti-tumor, and anti-oxidative properties. Recently, curcumin has been reported to suppress the amyloid-beta accumulation, oxidative damage, and inflammation in the transgenic mice model of Alzheimer's disease. Here, we found that the treatment of curcumin increases the polyglutamine-expanded truncated N-terminal huntingtin (mutant huntingtin) aggregation and mutant huntingtin-dependent cell death. Curcumin also causes rapid proteasomal malfunction in the mutant huntingtin expressing cells in comparison with normal glutamine repeat expressing cells. Finally, we show that N-acetyl cysteine (NAC), a potent antioxidant, reverted the curcumin-induced mutant huntingtin aggregation and proteasomal malfunction in the mutant huntingtin expressing cells. NAC also protects curcumin-induced cell death. Our result suggests that curcumin promotes mutant huntingtin-induced cell death by mimicking proteasomal dysfunction.