RTL8 promotes nuclear localization of UBQLN2 to subnuclear compartments associated with protein quality control.

The brain-expressed ubiquilins (UBQLNs) 1, 2 and 4 are a family of ubiquitin adaptor proteins that participate broadly in protein quality control (PQC) pathways, including the ubiquitin proteasome system (UPS). One family member, UBQLN2, has been implicated in numerous neurodegenerative diseases including ALS/FTD. UBQLN2 typically resides in the cytoplasm but in disease can translocate to the nucleus, as in Huntington's disease where it promotes the clearance of mutant Huntingtin. How UBQLN2 translocates to the nucleus and clears aberrant nuclear proteins, however, is not well understood. In a mass spectrometry screen to discover UBQLN2 interactors, we identified a family of small (13 kDa), highly homologous uncharacterized proteins, RTL8, and confirmed the interaction between UBQLN2 and RTL8 both in vitro using recombinant proteins and in vivo using mouse brain tissue. Under endogenous and overexpressed conditions, RTL8 localizes to nucleoli. When co-expressed with UBQLN2, RTL8 promotes nuclear translocation of UBQLN2. RTL8 also facilitates UBQLN2's nuclear translocation during heat shock. UBQLN2 and RTL8 colocalize within ubiquitin-enriched subnuclear structures containing PQC components. The robust effect of RTL8 on the nuclear translocation and subnuclear localization of UBQLN2 does not extend to the other brain-expressed ubiquilins, UBQLN1 and UBQLN4. Moreover, compared to UBQLN1 and UBQLN4, UBQLN2 preferentially stabilizes RTL8 levels in human cell lines and in mouse brain, supporting functional heterogeneity among UBQLNs. As a novel UBQLN2 interactor that recruits UBQLN2 to specific nuclear compartments, RTL8 may regulate UBQLN2 function in nuclear protein quality control.

Mutant UBQLN2 promotes toxicity by modulating intrinsic self-assembly.

UBQLN2 is one of a family of proteins implicated in ubiquitin-dependent protein quality control and integrally tied to human neurodegenerative disease. Whereas wild-type UBQLN2 accumulates in intraneuronal deposits in several common age-related neurodegenerative diseases, mutations in the gene encoding this protein result in X-linked amyotrophic lateral sclerosis/frontotemporal dementia associated with TDP43 accumulation. Using in vitro protein analysis, longitudinal fluorescence imaging and cellular, neuronal, and transgenic mouse models, we establish that UBQLN2 is intrinsically prone to self-assemble into higher-order complexes, including liquid-like droplets and amyloid aggregates. UBQLN2 self-assembly and solubility are reciprocally modulated by the protein's ubiquitin-like and ubiquitin-associated domains. Moreover, a pathogenic UBQLN2 missense mutation impairs droplet dynamics and favors amyloid-like aggregation associated with neurotoxicity. These data emphasize the critical link between UBQLN2's role in ubiquitin-dependent pathways and its propensity to self-assemble and aggregate in neurodegenerative diseases.

Influence of a curcumin derivative on hIAPP aggregation in the absence and presence of lipid membranes.

The deposition of aggregates of human islet amyloid polypeptide (hIAPP) has been correlated with the death of ß-cells in type II diabetes mellitus. The actual molecular mechanism of cell death remains largely unknown; however, it has been postulated that the process of aggregation from monomeric hIAPP is closely involved. A possible cause of cellular toxicity may be through the disruption of structural integrity of the cell membrane by IAPP. Herein, a water-soluble curcumin derivative, CurDAc, is used to investigate the mitigation of hIAPP aggregation in the absence and presence of lipid membrane.

Inhibition of IAPP Aggregation and Toxicity by Natural Products and Derivatives.

Fibrillar aggregates of human islet amyloid polypeptide, hIAPP, a pathological feature seen in some diabetes patients, are a likely causative agent for pancreatic beta-cell toxicity, leading to a transition from a state of insulin resistance to type II diabetes through the loss of insulin producing beta-cells by hIAPP induced toxicity. Because of the probable link between hIAPP and the development of type II diabetes, there has been strong interest in developing reagents to study the aggregation of hIAPP and possible therapeutics to block its toxic effects. Natural products are a class of compounds with interesting pharmacological properties against amyloids which have made them interesting targets to study hIAPP. Specifically, the ability of polyphenolic natural products, EGCG, curcumin, and resveratrol, to modulate the aggregation of hIAPP is discussed. Furthermore, we have outlined possible mechanistic discoveries of the interaction of these small molecules with the peptide and how they may mitigate toxicity associated with peptide aggregation. These abundantly found agents have been long used to combat diseases for many years and may serve as useful templates toward developing therapeutics against hIAPP aggregation and toxicity.

Self-assembly of a nine-residue amyloid-forming peptide fragment of SARS corona virus E-protein: mechanism of self aggregation and amyloid-inhibition of hIAPP.

Molecular self-assembly, a phenomenon widely observed in nature, has been exploited through organic molecules, proteins, DNA, and peptides to study complex biological systems. These self-assembly systems may also be used in understanding the molecular and structural biology which can inspire the design and synthesis of increasingly complex biomaterials. Specifically, use of these building blocks to investigate protein folding and misfolding has been of particular value since it can provide tremendous insights into peptide aggregation related to a variety of protein misfolding diseases, or amyloid diseases (e.g., Alzheimer's disease, Parkinson's disease, type-II diabetes). Herein, the self-assembly of TK9, a nine-residue peptide of the extra membrane C-terminal tail of the SARS corona virus envelope, and its variants were characterized through biophysical, spectroscopic, and simulated studies, and it was confirmed that the structure of these peptides influences their aggregation propensity, hence, mimicking amyloid proteins. TK9, which forms a beta-sheet rich fibril, contains a key sequence motif that may be critical for beta-sheet formation, thus making it an interesting system to study amyloid fibrillation. TK9 aggregates were further examined through simulations to evaluate the possible intra- and interpeptide interactions at the molecular level. These self-assembly peptides can also serve as amyloid inhibitors through hydrophobic and electrophilic recognition interactions. Our results show that TK9 inhibits the fibrillation of hIAPP, a 37 amino acid peptide implicated in the pathology of type-II diabetes. Thus, biophysical and NMR experimental results have revealed a molecular level understanding of peptide folding events, as well as the inhibition of amyloid-protein aggregation are reported.

Kinetics and thermodynamics of metal-binding to histone deacetylase 8.

Histone deacetylase 8 (HDAC8) was originally classified as a Zn(II)-dependent deacetylase on the basis of Zn(II)-dependent HDAC8 activity in vitro and illumination of a Zn(II) bound to the active site. However, in vitro measurements demonstrated that HDAC8 has higher activity with a bound Fe(II) than Zn(II), although Fe(II)-HDAC8 rapidly loses activity under aerobic conditions. These data suggest that in the cell HDAC8 could be activated by either Zn(II) or Fe(II). Here we detail the kinetics, thermodynamics, and selectivity of Zn(II) and Fe(II) binding to HDAC8. To this end, we have developed a fluorescence anisotropy assay using fluorescein-labeled suberoylanilide hydroxamic acid (fl-SAHA). fl-SAHA binds specifically to metal-bound HDAC8 with affinities comparable to SAHA. To measure the metal affinity of HDAC, metal binding was coupled to fl-SAHA and assayed from the observed change in anisotropy. The metal KD values for HDAC8 are significantly different, ranging from picomolar to micromolar for Zn(II) and Fe(II), respectively. Unexpectedly, the Fe(II) and Zn(II) dissociation rate constants from HDAC8 are comparable, koff ∼0.0006 s(-1), suggesting that the apparent association rate constant for Fe(II) is slow (∼3 × 10(3) M(-1) s(-1)). Furthermore, monovalent cations (K(+) or Na(+)) that bind to HDAC8 decrease the dissociation rate constant of Zn(II) by ≥100-fold for K(+) and ≥10-fold for Na(+), suggesting a possible mechanism for regulating metal exchange in vivo. The HDAC8 metal affinities are comparable to the readily exchangeable Zn(II) and Fe(II) concentrations in cells, consistent with either or both metal cofactors activating HDAC8.

In Search of Aggregation Pathways of IAPP and Other Amyloidogenic Proteins: Finding Answers through NMR Spectroscopy.

The deposition of aggregates of human islet amyloid peptide (hIAPP) has been correlated with the death of insulin-producing beta (ß) cells in type II diabetes mellitus. The actual molecular mechanism of cell death remains unknown; however, it has been postulated that the process of aggregation and amyloid fibril growth from monomeric hIAPP is closely involved. Intermediate IAPP aggregates are highly toxic to islet cells, but lack of structural knowledge of these oligomers and complications in applying biophysical techniques to their study have been the main obstacles in designing structure-based therapeutics. Furthermore, the involvement of metal ions (Cu(2+) and Zn(2+)) associated with hIAPP has demonstrated an effect on the aggregation pathway. In the absence of well-defined targets, research attempting to attenuate amyloid-linked toxicity has been substantially slowed. Therefore, obtaining high-resolution structural insights on these intermediates through NMR techniques can provide information on preventing IAPP aggregation. In this Perspective, a review of avenues to obtain fundamental new insights into the aggregation pathway of IAPP and other amyloidogenic proteins through NMR and other techniques is presented.

Tuning reactivity of diphenylpropynone derivatives with metal-associated amyloid-ß species via structural modifications.

A diphenylpropynone derivative, DPP2, has been recently demonstrated to target metal-associated amyloid-ß (metal-Aß) species implicated in Alzheimer's disease (AD). DPP2 was shown to interact with metal-Aß species and subsequently control Aß aggregation (reactivity) in vitro; however, its cytotoxicity has limited further biological applications. In order to improve reactivity toward Aß species and lower cytotoxicity, along with gaining an understanding of a structure-reactivity-cytotoxicity relationship, we designed, prepared, and characterized a series of small molecules (C1/C2, P1/P2, and PA1/PA2) as structurally modified DPP2 analogues. A similar metal binding site to that of DPP2 was contained in these compounds while their structures were varied to afford different interactions and reactivities with metal ions, Aß species, and metal-Aß species. Distinct reactivities of our chemical family toward in vitro Aß aggregation in the absence and presence of metal ions were observed. Among our chemical series, the compound (C2) with a relatively rigid backbone and a dimethylamino group was observed to noticeably regulate both metal-free and metal-mediated Aß aggregation to different extents. Using our compounds, cell viability was significantly improved, compared to that with DPP2. Lastly, modifications on the DPP framework maintained the structural properties for potential blood-brain barrier (BBB) permeability. Overall, our studies demonstrated that structural variations adjacent to the metal binding site of DPP2 could govern different metal binding properties, interactions with Aß and metal-Aß species, reactivity toward metal-free and metal-induced Aß aggregation, and cytotoxicity of the compounds, establishing a structure-reactivity-cytotoxicity relationship. This information could help gain insight into structural optimization for developing nontoxic chemical reagents toward targeting metal-Aß species and modulating their reactivity in biological systems.

Reactivity of diphenylpropynone derivatives toward metal-associated amyloid-ß species.

In Alzheimer's disease (AD), metal-associated amyloid-ß (metal-Aß) species have been suggested to be involved in neurotoxicity; however, their role in disease development is still unclear. To elucidate this aspect, chemical reagents have been developed as valuable tools for targeting metal-Aß species, modulating the interaction between the metal and Aß, and subsequently altering metal-Aß reactivity. Herein, we report the design, preparation, characterization, and reactivity of two diphenylpropynone derivatives (DPP1 and DPP2) composed of structural moieties for metal chelation and Aß interaction (bifunctionality). The interactions of these compounds with metal ions and Aß species were confirmed by UV-vis, NMR, mass spectrometry, and docking studies. The effects of these bifunctional molecules on the control of in vitro metal-free and metal-induced Aß aggregation were investigated and monitored by gel electrophoresis and transmission electron microscopy (TEM). Both DPP1 and DPP2 showed reactivity toward metal-Aß species over metal-free Aß species to different extents. In particular, DPP2, which contains a dimethylamino group, exhibited greater reactivity with metal-Aß species than DPP1, suggesting a structure-reactivity relationship. Overall, our studies present a new bifunctional scaffold that could be utilized to develop chemical reagents for investigating metal-Aß species in AD.

Metal-associated amyloid-ß species in Alzheimer's disease.

Highly concentrated metals such as Cu, Zn, and Fe are found in amyloid-ß (Aß) plaques within the brain of Alzheimer's disease (AD). In vitro and in vivo studies have suggested that metal binding to Aß could facilitate Aß aggregation and generate reactive oxygen species (ROS), which could contribute to the neuropathogenesis of AD. The connection between metal-Aß interaction/reactivity and AD development, however, has not been clearly revealed owing to the complexity of the disease. In this review, metal-Aß interaction/reactivity and its relation to neurotoxicity are briefly discussed. Additionally, our review illustrates the recent progress of small molecules, capable of targeting metal-Aß species and modulating their interaction/reactivity, which could offer a promising approach to interrogate their role in AD.