Active Site Environment and Reactivity of Copper-Aß in Membrane Mimetic SDS Micellar Environment.

Alzheimer's disease (AD) is characterized by the abnormal aggregation of amyloid ß (Aß) peptide in extracellular deposits generated upon proteolysis of Amyloid Precursor Protein (APP). While copper (Cu2+) binds to Aß in soluble oligomeric and aggregated forms, its interaction with membrane-bound Aß remains elusive. Investigating these interactions is crucial for understanding AD pathogenesis. Here, utilizing SDS micelles as a simplified membrane mimic, we focus on elucidating the interplay between membrane-anchored Aß and copper, given their pivotal roles in AD. We employed spectroscopic techniques including UV, CD, and EPR to characterize the active site of Cu-Aß complexes. Our findings demonstrate that copper interacts with Aß peptides in membrane-mimicking micellar environments similarly to aqueous buffer solutions. Cu-Aß complexes in this medium also induce higher hydrogen peroxide (H2O2) production, potentially contributing to AD-related oxidative stress. Moreover, we observe an increased oxidation rate of neurotransmitter such as dopamine by Cu-Aß complexes. These results enhance our understanding of Cu-Aß interactions in AD pathology and offer insights into potential therapeutic interventions targeting this interaction.

Second Sphere Interactions in Amyloidogenic Diseases.

Amyloids are protein aggregates bearing a highly ordered cross ß structural motif, which may be functional but are mostly pathogenic. Their formation, deposition in tissues and consequent organ dysfunction is the central event in amyloidogenic diseases. Such protein aggregation may be brought about by conformational changes, and much attention has been directed toward factors like metal binding, post-translational modifications, mutations of protein etc., which eventually affect the reactivity and cytotoxicity of the associated proteins. Over the past decade, a global effort from different groups working on these misfolded/unfolded proteins/peptides has revealed that the amino acid residues in the second coordination sphere of the active sites of amyloidogenic proteins/peptides cause changes in H-bonding pattern or protein-protein interactions, which dramatically alter the structure and reactivity of these proteins/peptides. These second sphere effects not only determine the binding of transition metals and cofactors, which define the pathology of some of these diseases, but also change the mechanism of redox reactions catalyzed by these proteins/peptides and form the basis of oxidative damage associated with these amyloidogenic diseases. The present review seeks to discuss such second sphere modifications and their ramifications in the etiopathology of some representative amyloidogenic diseases like Alzheimer's disease (AD), type 2 diabetes mellitus (T2Dm), Parkinson's disease (PD), Huntington's disease (HD), and prion diseases.

Active-site environment of Cu bound amyloid ß and amylin peptides.

Alzheimer's disease (AD) and Type 2 Diabetes mellitus (T2Dm), two of the most common amyloidogenic diseases. They share a common pathological symptom, i.e., the formation of amyloid deposits comprised of amyloid ß and amylin peptides, respectively. Autopsy of brains of AD-affected patients shows the presence of abnormally high concentrations of Cu in the deposited amyloid ß plaques, while a significantly higher level of Cu is found in the serum of patients suffering from T2Dm. These invoke that Cu might play a crucial role in the onset of both AD and T2Dm. In fact, Cu is found to bind amyloid ß as well as amylin relevant to AD and T2Dm, respectively. Cu-Aß and Cu-amylin in their reduced states can generate partially reduced oxygen species (PROS) on reaction with O2 which leads to oxidative stress in the brain and in the pancreas, respectively. However, the pathway of O2 reduction is quite different for the two complexes. Moreover, the use of various spectroscopic techniques such as absorption, EPR, and CD involving native and site-directed mutants of the peptides show that their active-site environments are also dissimilar. Here, we have discussed the different aspects of Cu-Aß and Cu-amylin complexes including their pH-dependent coordination environments and their reactivity towards O2 which may be responsible for the oxidative stress associated with the two diseases. This depicts the significance of the Cu bound peptide complexes in the context of AD and T2Dm. Graphic abstract.

Parkin suppresses Drp1-independent mitochondrial division.

The cycle of mitochondrial division and fusion disconnect and reconnect individual mitochondria in cells to remodel this energy-producing organelle. Although dynamin-related protein 1 (Drp1) plays a major role in mitochondrial division in cells, a reduced level of mitochondrial division still persists even in the absence of Drp1. It is unknown how much Drp1-mediated mitochondrial division accounts for the connectivity of mitochondria. The role of a Parkinson's disease-associated protein-parkin, which biochemically and genetically interacts with Drp1-in mitochondrial connectivity also remains poorly understood. Here, we quantified the number and connectivity of mitochondria using mitochondria-targeted photoactivatable GFP in cells. We show that the loss of Drp1 increases the connectivity of mitochondria by 15-fold in mouse embryonic fibroblasts (MEFs). While a single loss of parkin does not affect the connectivity of mitochondria, the connectivity of mitochondria significantly decreased compared with a single loss of Drp1 when parkin was lost in the absence of Drp1. Furthermore, the loss of parkin decreased the frequency of depolarization of the mitochondrial inner membrane that is caused by increased mitochondrial connectivity in Drp1-knockout MEFs. Therefore, our data suggest that parkin negatively regulates Drp1-indendent mitochondrial division.

Cbl-associated protein regulates assembly and function of two tension-sensing structures in Drosophila.

Cbl-associated protein (CAP) localizes to focal adhesions and associates with numerous cytoskeletal proteins; however, its physiological roles remain unknown. Here, we demonstrate that Drosophila CAP regulates the organization of two actin-rich structures in Drosophila: muscle attachment sites (MASs), which connect somatic muscles to the body wall; and scolopale cells, which form an integral component of the fly chordotonal organs and mediate mechanosensation. Drosophila CAP mutants exhibit aberrant junctional invaginations and perturbation of the cytoskeletal organization at the MAS. CAP depletion also results in collapse of scolopale cells within chordotonal organs, leading to deficits in larval vibration sensation and adult hearing. We investigate the roles of different CAP protein domains in its recruitment to, and function at, various muscle subcellular compartments. Depletion of the CAP-interacting protein Vinculin results in a marked reduction in CAP levels at MASs, and vinculin mutants partially phenocopy Drosophila CAP mutants. These results show that CAP regulates junctional membrane and cytoskeletal organization at the membrane-cytoskeletal interface of stretch-sensitive structures, and they implicate integrin signaling through a CAP/Vinculin protein complex in stretch-sensitive organ assembly and function.

Cell-type-specific roles of Na+/K+ ATPase subunits in Drosophila auditory mechanosensation.

Ion homeostasis is a fundamental cellular process particularly important in excitable cell activities such as hearing. It relies on the Na(+)/K(+) ATPase (also referred to as the Na pump), which is composed of a catalytic α subunit and a ß subunit required for its transport to the plasma membrane and for regulating its activity. We show that α and ß subunits are expressed in Johnston's organ (JO), the Drosophila auditory organ. We knocked down expression of α subunits (ATPα and α-like) and ß subunits (nrv1, nrv2, and nrv3) individually in JO with UAS/Gal4-mediated RNAi. ATPα shows elevated expression in the ablumenal membrane of scolopale cells, which enwrap JO neuronal dendrites in endolymph-like compartments. Knocking down ATPα, but not α-like, in the entire JO or only in scolopale cells using specific drivers, resulted in complete deafness. Among ß subunits, nrv2 is expressed in scolopale cells and nrv3 in JO neurons. Knocking down nrv2 in scolopale cells blocked Nrv2 expression, reduced ATPα expression in the scolopale cells, and caused almost complete deafness. Furthermore, knockdown of either nrv2 or ATPα specifically in scolopale cells causes abnormal, electron-dense material accumulation in the scolopale space. Similarly, nrv3 functions in JO but not in scolopale cells, suggesting neuron specificity that parallels nrv2 scolopale cell-specific support of the catalytic ATPα. Our studies provide an amenable model to investigate generation of endolymph-like extracellular compartments.

Physiological, anatomical, and behavioral changes after acoustic trauma in Drosophila melanogaster.

Noise-induced hearing loss (NIHL) is a growing health issue, with costly treatment and lost quality of life. Here we establish Drosophila melanogaster as an inexpensive, flexible, and powerful genetic model system for NIHL. We exposed flies to acoustic trauma and quantified physiological and anatomical effects. Trauma significantly reduced sound-evoked potential (SEP) amplitudes and increased SEP latencies in control genotypes. SEP amplitude but not latency effects recovered after 7 d. Although trauma produced no gross morphological changes in the auditory organ (Johnston's organ), mitochondrial cross-sectional area was reduced 7 d after exposure. In nervana 3 heterozygous flies, which slightly compromise ion homeostasis, trauma had exaggerated effects on SEP amplitude and mitochondrial morphology, suggesting a key role for ion homeostasis in resistance to acoustic trauma. Thus, Drosophila exhibit acoustic trauma effects resembling those found in vertebrates, including inducing metabolic stress in sensory cells. This report of noise trauma in Drosophila is a foundation for studying molecular and genetic sequelae of NIHL.

Biosynthesis and roles of phospholipids in mitochondrial fusion, division and mitophagy.

Mitochondria move, fuse and divide in cells. The dynamic behavior of mitochondria is central to the control of their structure and function. Three conserved mitochondrial dynamin-related GTPases (i.e., mitofusin, Opa1 and Drp1 in mammals and Fzo1, Mgm1 and Dnm1 in yeast) mediate mitochondrial fusion and division. In addition to dynamins, recent studies demonstrated that phospholipids in mitochondria also play key roles in mitochondrial dynamics by interacting with dynamin GTPases and by directly changing the biophysical properties of the mitochondrial membranes. Changes in phospholipid composition also promote mitophagy, which is a selective mitochondrial degradation process that is mechanistically coupled to mitochondrial division. In this review, we will discuss the biogenesis and function of mitochondrial phospholipids.

Rapid autoxidation of ferrous heme-Aß complexes relevant to Alzheimer's disease.

Heme bound Aß peptides have been reported to reduce O2 by 2e- to H2O2 which may result in oxidative stress commonly encountered in Alzheimer's disease. In this study we report the first instance of rapid freeze quench trapping and characterizing the heme(III)-O2˙- intermediate involved in the heme-Aß induced formation of partially reduced oxygen species (PROS) in physiologically relevant aqueous medium using absorption and resonance Raman spectroscopy. The kinetics of this process indicates a key role of the Tyr10 residue, unique to human Aß, in the generation of H2O2 from O2.

Heme-Aß in SDS micellar environment: Active site environment and reactivity.

Alzheimer's disease (AD), the most common cause of dementia, is a progressive neurodegenerative disorder that causes brain cell death. Oxidative stress derived from the accumulation of redox cofactors like heme in amyloid plaques originating from amyloid ß (Aß) peptides has been implicated in the pathogenesis of AD. In the past our group has studied the interactions and reactivities of heme with soluble oligomeric and aggregated forms of Aß. In this manuscript we report the interaction of heme with Aß that remains membrane bound using membrane mimetic SDS (sodium dodecyl sulfate) micellar medium. Employing different spectroscopic techniques viz. circular dichroism (CD), absorption (UV-Vis), electron paramagnetic resonance (EPR) and resonance Raman (rR) we find that Aß binds heme using one of its three His (preferentially His13) in SDS micellar medium. We also find that Arg5 is an essential distal residue responsible for higher peroxidase activity of heme bound Aß in this membrane mimetic environment than free heme. This peroxidase activity exerted by even membrane bound heme-Aß can potentially be more detrimental as the active site remains close to membranes and can hence oxidise the lipid bilayer of the neuronal cell, which can induce cell apoptosis. Thus, heme-Aß in solution as well as in membrane-bound form are detrimental.

Insights from Self-Assembled Aggregates of Amyloid ß Peptides on Gold Surfaces.

Amyloid ß (Aß) peptides mutated at different positions using a cysteine moiety assemble on Au electrodes using the thiol functionality of cysteine. Self-assembled monolayers (SAMs) of Aß on Au surfaces can act as abiological platforms that allow the mimicking of fibrils and oligomeric Aß via the formation of controlled large and small peptide aggregates. These Aß constructs bind with heme and Cu and exhibit different reactivities. These abiological platforms can also be used to investigate potential drugs that can interact with heme and Cu-Aß. SAM formation of Aß mutants allows the study of different morphology and structure as well as behavior changes on binding with different metals and cytochrome c (Cyt c). This review provides a detailed insight into the structure and reactivities of various Aß aggregated on Au electrodes mimicking the cell membrane.

Spin state dependent peroxidase activity of heme bound amyloid ß peptides relevant to Alzheimer's disease.

The colocalization of heme rich deposits in the senile plaque of Aß in the cerebral cortex of the Alzheimer's disease (AD) brain along with altered heme homeostasis and heme deficiency symptoms in AD patients has invoked the association of heme in AD pathology. Heme bound Aß complexes, depending on the concentration of the complex or peptide to heme ratio, exhibit an equilibrium between a high-spin mono-His bound peroxidase-type active site and a low-spin bis-His bound cytochrome b type active site. The high-spin heme-Aß complex shows higher peroxidase activity than free heme, where compound I is the reactive oxidant. It is also capable of oxidizing neurotransmitters like serotonin in the presence of peroxide, owing to the formation of compound I. The low-spin bis-His heme-Aß complex on the other hand shows enhanced peroxidase activity relative to high-spin heme-Aß. It reacts with H2O2 to produce two stable intermediates, compound 0 and compound I, which are characterized by absorption, EPR and resonance Raman spectroscopy. The stability of compound I of low-spin heme-Aß is accountable for its enhanced peroxidase activity and oxidation of the neurotransmitter serotonin. The effect of the second sphere Tyr10 residue of Aß on the formation and stability of the intermediates of low-spin heme-Aß has also been investigated. The higher stability of compound I for low-spin heme-Aß is likely due to H-bonding interactions involving Tyr10 in the distal pocket.

In situ transduction of cells in human corneal limbus using adeno-associated viruses: an ex vivo study.

This study aimed to evaluate the efficacy of in situ adeno-associated virus (AAV)-mediated gene delivery into the human corneal limbal region via targeted sub-limbal injection technique. Human cadaveric corneal tissues were fixed on an artificial anterior chamber. Feasibility of sub-limbal injection technique was tested using trypan blue and black India ink. An enhanced green fluorescent protein (eGFP) encoding AAV DJ was injected into sub-limbal region. After AAV injection, corneal tissues were incubated in air-lift culture and prepared for immunohistochemical analysis. Cell survivial and expression of eGFP, stem cell markers (p63α and cytokeratin 19 (KRT19)), and differentiation marker cytokeratin 3 (KRT3) were evaluated using confocal microscopy. Both trypan blue and black India ink stained and were retained sub-limbally establishing specificity of the injection technique. Immunohistochemical analysis of corneas injected with AAV DJ-eGFP indicated that AAV-transduced cells in the limbal region co-express eGFP, p63α, and KRT19 and that these transduced cells were capable of differentiating to KRT3 postitive corneal epithelial cells. Our sub-limbal injection technique can target cells in the human limbus in a reproducible and efficient manner. Thus, we demonstrate that in situ injection of corneal limbus may provide a feasible mode of genetic therapy for corneal disorders with an epithelial etiology.

Interaction of ApoMyoglobin with Heme-hIAPP complex.

Human Islet Amyloid Polypeptide (hIAPP) or amylin, can bind heme and the resultant complexes are prone to generate partially reduced oxygen species (PROS). The formation of PROS and the related oxidative stress highlight the importance of Heme-hIAPP in the onset and development of Type 2 Diabetes mellitus (T2Dm) in humans. In this study, the interaction of Heme-hIAPP with apomyoglobin (ApoMb) has been investigated using a combination of spectroscopic and electrophoresis techniques. Absorption, resonance Raman data and gel electrophoresis results confirm that ApoMb can uptake heme from Heme-hIAPP and constitute a six-coordinate high-spin ferric heme active site identical to that of myoglobin (Mb). The heme transfer reaction has two distinct kinetic steps. A possible mechanism of this reaction involves heme transfer to the apoprotein in the first step followed by a reorganisation of the protein chain to form the active site of native Mb. Increase in the pH of the reaction medium enhances the rate of the second step of heme transfer. This possibly corresponds to the deprotonation of a propionate side chain of the heme moiety at high pH which facilitates secondary interactions with the conserved distal Lys45 residue of horse heart Mb. Additionally, ApoMb sequesters ligand bound heme from Heme-hIAPP. After the heme transfer reaction, the amount of PROS formed by Heme-hIAPP complex diminishes significantly. This not only potentially diminishes heme-induced toxicity in the pancreatic ß-cells but also produces Mb which has well-documented functions throughout the respiratory system and can thereby likely reduce the risks associated with T2Dm.

A Small Molecule that Binds an RNA Repeat Expansion Stimulates Its Decay via the Exosome Complex.

Many diseases are caused by toxic RNA repeats. Herein, we designed a lead small molecule that binds the structure of the r(CUG) repeat expansion [r(CUG)exp] that causes myotonic dystrophy type 1 (DM1) and Fuchs endothelial corneal dystrophy (FECD) and rescues disease biology in patient-derived cells and in vivo. Interestingly, the compound's downstream effects are different in the two diseases, owing to the location of the repeat expansion. In DM1, r(CUG)exp is harbored in the 3' untranslated region, and the compound has no effect on the mRNA's abundance. In FECD, however, r(CUG)exp is located in an intron, and the small molecule facilitates excision of the intron, which is then degraded by the RNA exosome complex. Thus, structure-specific, RNA-targeting small molecules can act disease specifically to affect biology, either by disabling the gain-of-function mechanism (DM1) or by stimulating quality control pathways to rid a disease-affected cell of a toxic RNA (FECD).

Peroxidase activity of heme bound amyloid ß peptides associated with Alzheimer's disease.

The amyloid cascade hypothesis attributes the neurodegeneration observed in Alzheimer's disease (AD) to the deposition of the amyloid ß (Aß) peptide into plaques and fibrils in the AD brain. The metal ion hypothesis which implicates several metal ions, viz. Zn2+, Cu2+ and Fe3+, in the AD pathology on account of their abnormal accumulation in the Aß plaques along with an overall dyshomeostasis of these metals in the AD brain was proposed a while back. Metal ion chelators and ionophores, put forward as possible drug candidates for AD, are yet to succeed in clinical trials. Heme, which is widely distributed in the mammalian body as the prosthetic group of several important proteins and enzymes, has been thought to be associated with AD by virtue of its colocalization in the Aß plaques along with the similarity of several heme deficiency symptoms with those of AD and most importantly, due to its ability to bind Aß. This feature article illustrates the active site environment of heme-Aß which resembles those of peroxidases. It also discusses the peroxidase activity of heme-Aß, its ability to effect oxidative degradation of neurotransmitters like serotonin and also the identification of the highly reactive high-valent intermediate, compound I. The effect of second sphere residues on the formation and peroxidase activity of heme-Aß along with the generation and decay of compound I is highlighted throughout the article. The reactivities of heme bound Aß peptides give an alternative theory to understand the possible cause of this disease.

Nitrite reductase activity of heme and copper bound Aß peptides.

A significant abundance of copper (Cu) and iron in amyloid ß (Aß) plaques, and several heme related metabolic disorders are directly correlated with Alzheimer's disease (AD), and these together with co-localization of Aß plaques with heme rich deposits in the brains of AD sufferers indicates a possible association of the said metals with the disease. Recently, the Aß peptides have been found to bind heme and Cu individually as well as simultaneously. Another significant finding relevant to this is the lower levels of nitrite and nitrate found in the brains of patients suffering from AD. In this study, a combination of absorption and electron paramagnetic resonance spectroscopy and kinetic assays have been used to study the interaction of nitrite with the metal bound Aß complexes. The data indicate that heme(III)-Cu(i)-Aß, heme(II)-Cu(i)-Aß, heme(II)-Aß and Cu(i)-Aß can reduce nitrite to nitric oxide (NO), an important biological messenger also related to AD, and thus behave as nitrite reductases. However these complexes reduce nitrite at different rates with heme(III)-Cu(i)-Aß being the fastest following an inner sphere electron transfer mechanism. The rest of the metal-Aß adducts follow an outer sphere electron transfer mechanism during nitrite reduction. Protonation from the Arg5 residue triggering the N-O bond heterolysis in heme(III) bound nitrite with a simultaneous electron transfer from the Cu(i) center to produce NO is the rate determining step, indicating a proton transfer followed by electron transfer (PTET) mechanism.

Formation of compound I in heme bound Aß-peptides relevant to Alzheimer's disease.

Proteolysis of Amyloid Precursor Protein, APP, results in the formation of amyloid ß (Aß) peptides, which have been associated with Alzheimer's disease (AD). Recently the failure of therapeutic agents that prohibit Aß aggregation and sequester Cu/Zn in providing symptomatic relief to AD patients has questioned the amyloid and metal ion hypothesis. Alternatively, abnormal heme homeostasis and reduced levels of neurotransmitters in the brain are hallmark features of AD. Heme can bind Aß peptides forming a peroxidase type active site which can oxidatively degrade neurotransmitters like serotonin. To date the reactive species responsible for this activity has not been identified. Using rapid kinetics and freeze quenching, we show that heme bound Aß forms a highly reactive intermediate, compound I. Thus, compound I provides a basis for elucidating the oxidative degradation of neurotransmitters like serotonin, resulting in abnormal neurotransmission, a key pathological feature of AD. Site directed mutants indicate that the Arg5 and Tyr10 residues, unique to human Aß, affect the rates of formation and decay of compound I providing insight into their roles in the oxidative degradation of neurotransmitters. Tyr10 can potentially play a natural protective role against the highly reactive oxidant, compound I, in AD.

CHCHD10 mutations promote loss of mitochondrial cristae junctions with impaired mitochondrial genome maintenance and inhibition of apoptosis.

CHCHD10-related diseases include mitochondrial DNA instability disorder, frontotemporal dementia-amyotrophic lateral sclerosis (FTD-ALS) clinical spectrum, late-onset spinal motor neuropathy (SMAJ), and Charcot-Marie-Tooth disease type 2 (CMT2). Here, we show that CHCHD10 resides with mitofilin, CHCHD3 and CHCHD6 within the "mitochondrial contact site and cristae organizing system" (MICOS) complex. CHCHD10 mutations lead to MICOS complex disassembly and loss of mitochondrial cristae with a decrease in nucleoid number and nucleoid disorganization. Repair of the mitochondrial genome after oxidative stress is impaired in CHCHD10 mutant fibroblasts and this likely explains the accumulation of deleted mtDNA molecules in patient muscle. CHCHD10 mutant fibroblasts are not defective in the delivery of mitochondria to lysosomes suggesting that impaired mitophagy does not contribute to mtDNA instability. Interestingly, the expression of CHCHD10 mutant alleles inhibits apoptosis by preventing cytochrome c release.

PARK2/Parkin becomes critical when DNM1L/Drp1 is absent.

Maintaining mitochondrial dynamics and proper execution of mitophagy is crucial for sustaining cellular health. Defects in these processes have been linked to cardiovascular diseases and neurodegeneration. In a recent publication, we reported that the mitochondrial division dynamin protein DNM1L/Drp1 and the E3 ubiquitin ligase PARK2/Parkin work in a synergistic manner to maintain mitochondrial function and structural integrity in the mouse heart and brain.