Michael Sierks - Academia.edu (original) (raw)

Papers by Michael Sierks

Fibrillar amyloid is the hallmark feature of many protein aggregation diseases, such as Alzheimer... more Fibrillar amyloid is the hallmark feature of many protein aggregation diseases, such as Alzheimer's and Parkinson's diseases. A monoclonal single-chain variable fragment (scFv) targeting insulin fibrils was isolated using phage display technology and an atomic force microscopy (AFM) mica substrate. Specific targeting of the scFv to insulin fibrils but not monomers or other small oligomeric forms, under similar conditions, was demonstrated both by enzyme-linked immunosorbent assays and AFM recognition imaging. The scFv also recognizes β-amyloid fibrils, a hallmark feature of Alzheimer's disease. The results suggest that the isolated scFv possibly targets a shared fibrillar motif—probably the cross-β-sheet characteristic of amyloid fibrils. The techniques outlined here provide additional tools to further study the process of fibril formation. The scFvs isolated can have potential use as diagnostic or therapeutic reagents for protein aggregation diseases. © 2008 Elsevier In...

Scientific reports, Jan 20, 2016

Non-fibrillar soluble oligomeric forms of amyloid-β peptide (oAβ) and tau proteins are likely to ... more Non-fibrillar soluble oligomeric forms of amyloid-β peptide (oAβ) and tau proteins are likely to play a major role in Alzheimer's disease (AD). The prevailing hypothesis on the disease etiopathogenesis is that oAβ initiates tau pathology that slowly spreads throughout the medial temporal cortex and neocortices independently of Aβ, eventually leading to memory loss. Here we show that a brief exposure to extracellular recombinant human tau oligomers (oTau), but not monomers, produces an impairment of long-term potentiation (LTP) and memory, independent of the presence of high oAβ levels. The impairment is immediate as it raises as soon as 20 min after exposure to the oligomers. These effects are reproduced either by oTau extracted from AD human specimens, or naturally produced in mice overexpressing human tau. Finally, we found that oTau could also act in combination with oAβ to produce these effects, as sub-toxic doses of the two peptides combined lead to LTP and memory impairmen...

Neurology, Jan 9, 2018

To utilize a panel of 11 single chain variable fragments (scFvs) that selectively bind disease-re... more To utilize a panel of 11 single chain variable fragments (scFvs) that selectively bind disease-related variants of TAR DNA-binding protein (TDP)-43, β-amyloid, tau, and α-synuclein to assess damage following traumatic brain injury (TBI), and determine if the presence of protein variants could account for the increased risk of various neurodegenerative diseases following TBI. We utilized the panel of 11 scFvs in a sensitive ELISA format to analyze sera from 43 older veterans, 25 who had experienced at least 1 TBI incident during their lifetime (∼29.4 years after TBI), and 18 controls who did not incur TBI, in a cross-sectional study. Each of the 11 scFvs individually could significantly distinguish between TBI and control samples, though they did not detect each TBI sample. Comparing the levels of all 11 variants, all 25 TBI cases displayed higher reactivity compared to the controls and receiver operating characteristic analysis revealed 100% sensitivity and specificity. Higher total...

Bmc Neurosci, 2010

Background: Overexpression and abnormal accumulation of aggregated α-synuclein (αS) have been lin... more Background: Overexpression and abnormal accumulation of aggregated α-synuclein (αS) have been linked to Parkinson's disease (PD) and other synucleinopathies. αS can misfold and adopt a variety of morphologies but recent studies implicate oligomeric forms as the most cytotoxic species. Both genetic mutations and chronic exposure to neurotoxins increase αS aggregation and intracellular reactive oxygen species (ROS), leading to mitochondrial dysfunction and oxidative damage in PD cell models. Results: Here we show that curcumin can alleviate αS-induced toxicity, reduce ROS levels and protect cells against apoptosis. We also show that both intracellular overexpression of αS and extracellular addition of oligomeric αS increase ROS which induces apoptosis, suggesting that aggregated αS may induce similar toxic effects whether it is generated intra-or extracellulary. Conclusions: Since curcumin is a natural food pigment that can cross the blood brain barrier and has widespread medicinal uses, it has potential therapeutic value for treating PD and other neurodegenerative disorders.

Nanomedicine and the Nervous System, 2012

Journal of Alzheimer's disease : JAD, 2012

Misfolding and aggregation of amyloid-β (Aβ) is an important early event in the pathogenesis of A... more Misfolding and aggregation of amyloid-β (Aβ) is an important early event in the pathogenesis of Alzheimer's disease. Aβ is produced by sequential proteolysis of the amyloid-β protein precursor (AβPP) by β- and γ-secretases. A third protease, α-secretase, cleaves AβPP in the middle of the Aβ sequence precluding formation of Aβ. The levels of Aβ generated from AβPP can therefore be controlled by tailoring activity of these proteases toward AβPP. We previously showed that β-secretase proteolysis of AβPP could be selectively inhibited using the single chain antibody fragment (scFv) iBSEC1, which blocks the cleavage site on AβPP, and α-secretase proteolysis of AβPP could be selectively enhanced using a proteolytic scFv (Asec1A) engineered to have α-secretase-like activity. Here we show that DIA10D, a novel tandem bispecific scFv combining iBSEC1 with the ASec1A can control amyloidogenic processing of AβPP by simultaneously inhibiting β-secretase and increasing α-secretase processing ...

Carbohydrate Research, 1992

The roles of the aromatic side chains of the glucoamylase from Aspergillus niger in the binding o... more The roles of the aromatic side chains of the glucoamylase from Aspergillus niger in the binding of ligands, as determined by difference spectroscopy using four types of inhibitors (a) valienamine-derived, (b) 1-deoxynojirimycins, (c) D-glucono-1,5-lactone, and (d) maltitol, two types of disaccharide substrates (a) a-(1 +4)-linked and (b) a-(1+6)-linked, and three cyclomalto-oligosaccharides (cyclodextrins, CDs) are discussed. An unusual change in absorbance from 300 to 310-320 mn, obtained only with the valienaminederived inhibitors or when n-glucono-l,%lactone and maltose are combined, is concluded to arise when subsite 2 is occupied in a transition-state-type of complex. The single mutations of two residues thought to be involved in binding, namely, Tyrl16+Ala and Trpl20+Phe, alter, but do not abolish this perturbation. The perturbations in the spectra also suggest that maltose and isomaltose have different modes of binding. The following K,, values (M) were determined: acarbose, ~6 x lo-"; methyl acarviosinide, 1.6 x 10m6; and the D-&co and L-id0 forms of hydrogenated acarbose, 1.4 x lo-* and 5.2 x lo-(', respectively. Therefore, both the valienamine moiety and the chain length of acarbose are important for tight binding. In contrast to the valienamine-derived inhibitors, none of the 1-deoxynojirimycin type protected glucoamylase against inactivating oxidation of tryptophanyl residues, although each had a Kd value of-4 x 10-k. There are two distinct carbohydrate-binding areas in glucoamylase, namely, the active site in the catalytic domain and a starch-granule-binding site in the C-terminal domain. The 01-, p-, and y-CDs have high afhnity for the starch-binding domain and low affinity for the active site, whereas the reverse was found for acarbose.

Biotechnology Progress, 1997

Release of product from the active site is the rate-limiting step in a number of enzymatic reacti... more Release of product from the active site is the rate-limiting step in a number of enzymatic reactions, including maltose hydrolysis by glucoamylase (GA). With GA, an enzymatic conformational change has been associated with the product release step. Solvent characteristics such as viscosity can strongly influence protein conformational changes. Here we show that the rate-limiting step of GA has a rather complex dependence on solvent characteristics. Seven different cosolvents were added to the GA/maltose reaction solution. Five of the cosolvents, all having an ethylene glycol base, resulted in an increase in activity at low concentration of cosolvent and variable decreases in activity at higher concentrations. The increase in enzyme activity was dependent on polymer length of the cosolvent; the longer the polymer, the lower the concentration needed. The maximum increase in catalytic activity at 45°C (40-45%) was obtained with the three longest polymers (degree of polymerization from 200 to 8000). A further increase in activity to 60-65% was obtained at 60°C. The linear relationship between ln(k cat) and (viscosity) 2 obtained with all the cosolvents provides further evidence that product release is the rate-limiting step in the GA catalytic mechanism. A substantial increase in the turnover rate of GA by addition of relatively small amounts of a cosolvent has potential applications for the food industry where high-fructose corn syrup (HFCS) is one of the primary products produced with GA. Since maltodextrin hydrolysis by GA is by far the slowest step in the production of HFCS, increasing the catalytic rate of GA can substantially reduce the process time.

Biochemistry

The catalytic mechanism of glucoamylase (GA) is investigated by comparing kinetic results obtaine... more The catalytic mechanism of glucoamylase (GA) is investigated by comparing kinetic results obtained using R-D-glucosyl fluoride (GF) and maltooligosaccharides as substrates for wild-type and four active site mutant GAs, Tyr116fAla, Trp120fPhe, Asp176fAsn, and Glu400fGln. These replacements decreased the activity (k cat /K M) toward maltose by 6-320-fold. Toward GF, however, Tyr116fAla and Trp120fPhe GAs, showed wild-type and twice wild-type level activity, while Asp176fAsn and Glu400fGln GAs had 22-and 665-fold lower activity, respectively. Glu400, the catalytic base, is suggested to strengthen ground-state binding in subsite 1, and Asp176 does so at subsites 1 and 2. Tyr116 and Trp120 belong to an aromatic cluster that is slightly removed from the catalytic site and not critical for GF hydrolysis, but which is probably involved in maltooligosaccharide transition-state stabilization. Since the mutation of groups near the catalytic site decreased activity for both GF and maltose, but substitution of Tyr116 and Trp120 decreased activity only for maltose, interaction with the substrate aglycon part may be implicated in the rate-limiting step. Rate-limiting aglycon product release was suggested previously for GA-catalyzed hydrolysis [