A cellular feature of Parkinson’s disease is cytosolic accumulation and amyloid formation of α-synuclein (α-syn), implicating a misregulation or impairment of protein degradation pathways involving ...the proteasome and lysosome. Within lysosomes, cathepsin D (CtsD), an aspartyl protease, is suggested to be the main protease for α-syn clearance; however, the protease alone only generates amyloidogenic C terminal-truncated species (e.g., 1–94, 5–94), implying that other proteases and/or environmental factors are needed to facilitate degradation and to avoid α-syn aggregation in vivo. Using liquid chromatography–mass spectrometry, to our knowledge, we report the first peptide cleavage map of the lysosomal degradation process of α-syn. Studies of purified mouse brain and liver lysosomal extracts and individual human cathepsins demonstrate a direct involvement of cysteine cathepsin B (CtsB) and L (CtsL). Both CtsB and CtsL cleave α-syn within its amyloid region and circumvent fibril formation. For CtsD, only in the presence of anionic phospholipids can this protease cleave throughout the α-syn sequence, suggesting that phospholipids are crucial for its activity. Taken together, an interplay exists between α-syn conformation and cathepsin activity with CtsL as the most efficient under the conditions examined. Notably, we discovered that CtsL efficiently degrades α-syn amyloid fibrils, which by definition are resistant to broad spectrum proteases. This work implicates CtsB and CtsL as essential in α-syn lysosomal degradation, establishing groundwork to explore mechanisms to enhance their cellular activity and levels as a potential strategy for clearance of α-syn.
Lewy bodies, hallmarks of Parkinson's disease, contain C-terminally truncated (ΔC) α-synuclein (α-syn). Here, we report fibril structures of three N-terminally acetylated (Ac) α-syn constructs, ...Ac1–140, Ac1–122, and Ac1–103, solved by cryoelectron microscopy. Both ΔC-α-syn variants exhibited faster aggregation kinetics, and Ac1–103 fibrils efficiently seeded the full-length protein, highlighting their importance in pathogenesis. Interestingly, fibril helical twists increased upon the removal of C-terminal residues and can be propagated through cross-seeding. Compared to that of Ac1–140, increased electron densities were seen in the N-terminus of Ac1–103, whereas the C-terminus of Ac1–122 appeared more structured. In accord, the respective termini of ΔC-α-syn exhibited increased protease resistance. Despite similar amyloid core residues, distinctive features were seen for both Ac1–122 and Ac1–103. Particularly, Ac1–103 has the tightest packed core with an additional turn, likely attributable to conformational changes in the N-terminal region. These molecular differences offer insights into the effect of C-terminal truncations on α-syn fibril polymorphism.
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•Effects of C-terminal truncations on α-syn fibril formation were studied.•Fibril helical twists increased upon the removal of C-terminal residues.•Fibril structures of full length and ΔC-α-syn were solved by cryoEM.•Conformational differences were revealed at N- and C-termini.•Structural insights were gained on α-syn fibril polymorphism.
Studies on aminopolymer/oxide composite materials for direct CO2 capture from air have often focused on the prototypical poly(ethylenimine) (PEI) as the aminopolymer. However, it is known that PEI ...will oxidatively degrade at elevated temperatures. This degradation has been ascribed to the presence of secondary amines, which, when oxidized, lose their CO2 capture capacity. Here, we demonstrate the use of small molecule poly(propylenimine) (PPI) in linear and dendritic architectures supported in silica as adsorbent materials for direct CO2 capture from air. Regardless of amine loading or aminopolymer architecture, the PPI-based sorbents are found to be more efficient for CO2 capture than PEI-based sorbents. Moreover, PPI is found to be more resistant to oxidative degradation than PEI, even while containing secondary amines, as supported by FTIR, NMR, and ESI-MS studies. These results suggest that PPI-based CO2 sorbents may allow for longer sorbent working lifetimes due to an increased tolerance to sorbent regeneration conditions and suggest that the presence of secondary amines may not mean that all aminopolymers will oxidatively degrade.
One of the current challenges facing biomedical researchers is the need to develop new approaches in preventing amyloid formation that is associated with disease. While amyloid is generally ...considered detrimental to the cell, examples of amyloids that maintain a benign nature and serve a specific function exist. Here, we review our work on the repeat domain (RPT) of the functional amyloid Pmel17. Specifically, the RPT domain contributes in generating amyloid fibrils in melanosomes upon which melanin biosynthesis occurs. Amyloid formation of RPT was shown to be pH sensitive, aggregating only under acidic conditions associated with melanosomal pH. Furthermore, preformed fibrils rapidly dissolved at neutral pH to generate benign monomeric species. From a biological perspective, this unique reversible aggregation/disaggregation is a safeguard against an event of releasing RPT fibrils in the cytosol, resulting in rapid fibril unfolding and circumventing cytotoxicity. Understanding how melanosomes preserve a safe environment will address vital questions that remain unanswered with pathological amyloids.
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•The repeat domain (RPT) of human Pmel17 is a functional amyloid that sequesters and safely permits successful synthesis of melanin.•RPT forms amyloid fibrils only at the physiological pH (4–5.5) of melanosomes and rapidly disassembles at neutral pH.•The molecular origin of this pH dependence is largely attributed to the protonation of one glutamic acid.•Aggregation of RPT is strongly influenced by negatively charged lysolipids, an abundant melanosomal lipid.•RPT parallels other biological systems, suggesting that a common mechanism is shared among some functional amyloids.
Retinoblastoma (Rb) is the most common primary intraocular tumor in children. Local treatment of the intraocular disease is usually effective if diagnosed early; however advanced Rb can metastasize ...through routes that involve invasion of the choroid, sclera and optic nerve or more broadly via the ocular vasculature. Metastatic Rb patients have very high mortality rates. While current therapy for Rb is directed toward blocking tumor cell division and tumor growth, there are no specific treatments targeted to block Rb metastasis. Two such targets are matrix metalloproteinases-2 and -9 (MMP-2, -9), which degrade extracellular matrix as a prerequisite for cellular invasion and have been shown to be involved in other types of cancer metastasis. Cancer Clinical Trials with an anti-MMP-9 therapeutic antibody were recently initiated, prompting us to investigate the role of MMP-2, -9 in Rb metastasis.
We compare MMP-2, -9 activity in two well-studied Rb cell lines: Y79, which exhibits high metastatic potential and Weri-1, which has low metastatic potential. The effects of inhibitors of MMP-2 (ARP100) and MMP-9 (AG-L-66085) on migration, angiogenesis, and production of immunomodulatory cytokines were determined in both cell lines using qPCR, and ELISA. Cellular migration and potential for invasion were evaluated by the classic wound-healing assay and a Boyden Chamber assay.
Our results showed that both inhibitors had differential effects on the two cell lines, significantly reducing migration in the metastatic Y79 cell line and greatly affecting the viability of Weri-1 cells. The MMP-9 inhibitor (MMP9I) AG-L-66085, diminished the Y79 angiogenic response. In Weri-1 cells, VEGF was significantly reduced and cell viability was decreased by both MMP-2 and MMP-9 inhibitors. Furthermore, inhibition of MMP-2 significantly reduced secretion of TGF-β1 in both Rb models.
Collectively, our data indicates MMP-2 and MMP-9 drive metastatic pathways, including migration, viability and secretion of angiogenic factors in Rb cells. These two subtypes of matrix metalloproteinases represent new potential candidates for targeted anti-metastatic therapy for Rb.
The results are presented of a detailed combined experimental and theoretical investigation of the influence of coadsorbed electron-donating alkali atoms and the prototypical electron acceptor ...molecule 7,7,8,8-tetracyanoquinodimethane (TCNQ) on the Ag(100) surface. Several coadsorption phases were characterized by scanning tunneling microscopy, low-energy electron diffraction, and soft X-ray photoelectron spectroscopy. Quantitative structural data were obtained using normal-incidence X-ray standing wave (NIXSW) measurements and compared with the results of density functional theory (DFT) calculations using several different methods of dispersion correction. Generally, good agreement between theory and experiment was achieved for the quantitative structures, albeit with the prediction of the alkali atom heights being challenging for some methods. The adsorption structures depend sensitively on the interplay of molecule–metal charge transfer and long-range dispersion forces, which are controlled by the composition ratio between alkali atoms and TCNQ. The large difference in atomic size between K and Cs has negligible effects on stability, whereas increasing the ratio of K/TCNQ from 1:4 to 1:1 leads to a weakening of molecule–metal interaction strength in favor of stronger ionic bonds within the two-dimensional alkali–organic network. A strong dependence of the work function on the alkali donor–TCNQ acceptor coadsorption ratio is predicted.
Bacteria residing in the human gastrointestinal tract has a symbiotic relationship with its host. Animal models have demonstrated a relationship between exercise and gut microbiota composition. This ...was the first study to explore the relationship between cardiorespiratory fitness (maximal oxygen consumption, VO
max) and relative gut microbiota composition (Firmicutes to Bacteroidetes ratio F/B) in healthy young adults in a free-living environment. Twenty males and 17 females (25.7 ± 2.2 years), who did not take antibiotics in the last 6 months, volunteered for this study. VO
max was measured using a symptom-limited graded treadmill test. Relative microbiota composition was determined by analyzing DNA extracted from stool samples using a quantitative polymerase chain reaction that specifically measured the quantity of a target gene (16S rRNA) found in Firmicutes and Bacteroidetes. Relationships between F/B and potentially related dietary, anthropometric, and fitness variables were assessed using correlation analyses with an appropriate Bonferroni adjustment (
< .004). The average F/B ratio in all participants was 0.94 ± 0.03. The F/B ratio was significantly correlated to VO
max (
= .48,
< .003), but no other fitness, nutritional intake, or anthropometric variables (
> .004). VO
max explained ∼22% of the variance of an individual's relative gut bacteria as determined by the F/B ratio. These data support animal findings, demonstrating a relationship between relative human gut microbiota composition and cardiorespiratory fitness in healthy young adults. Gastrointestinal bacteria is integral in regulating a myriad of physiological processes, and greater insight regarding ramifications of exercise and nutrition on gut microbial composition may help guide therapies to promote human health.
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