Search

Peptide mapping analysis is a regulatory expectation to verify the primary structure of a recombinant product sequence and to monitor post-translational modifications (PTMs). Although proteolytic digestion has been used for decades, it remains a labour-intensive procedure that can be challenging to accurately reproduce. Here, we describe a fast and reproducible protocol for protease digestion that is automated using immobilised trypsin on magnetic beads, which has been incorporated into an optimised peptide mapping workflow to show method transferability across laboratories. The complete workflow has the potential for use within a multi-attribute method (MAM) approach in drug development, production and QC laboratories. The sample preparation workflow is simple, ideally suited to inexperienced operators and has been extensively studied to show global applicability and robustness for mAbs by performing sample digestion and LC-MS analysis at four independent sites in Europe. LC-MS/MS along with database searching was used to characterise the protein and determine relevant product quality attributes (PQAs) for further testing. A list of relevant critical quality attributes (CQAs) was then established by creating a peptide workbook containing the specific mass-to-charge (m/z) ratios of the modified and unmodified peptides of the selected CQAs, to be monitored in a subsequent test using LC-MS analysis. Data is provided that shows robust digestion efficiency and low levels of protocol induced PTMs. Graphical abstract Electronic supplementary material The online version of this article (10.1007/s00216-020-02809-z) contains supplementary material, which is available to authorized users.

Disulfide bond reduction within antibody mass spectrometry workflows is typically carried out using chemical reducing agents to produce antibody subunits for middle-down and middle-up analysis. In this contribution we offer an online electrochemical reduction method for the reduction of antibodies coupled with liquid chromatography (LC) and mass spectrometry (MS), reducing the disulfide bonds present in the antibody without the need for chemical reducing agents. An electrochemical cell placed before the analytical column and mass spectrometer facilitated complete reduction of NISTmAb inter- and intrachain disulfide bonds. Reduction and analysis were carried out under optimal solvent conditions using a trapping column and switching valve to facilitate solvent exchange during analysis. The level of reduction was shown to be affected by electrochemical potential, temperature and solvent organic content, but with optimization, complete disulfide bond cleavage was achieved. The use of an inline electrochemical cell offers a simple, rapid, workflow solution for liquid chromatography mass spectrometry analysis of antibody subunits.

This contribution addresses the experimental proof of the relaxation coupling of the 7Li (I = 3/2) longitudinal magnetization orders in the solid-state electrolyte Li10GeP2S12 (LGPS). This effect was theoretically described by Korb and Petit in 1988 but has not yet been shown experimentally. In a 2D-T1/spin-alignment echo (SAE) experiment, the inverse Laplace transformation of the spectral component over two time dimensions revealed the asymmetric course of the spin-lattice relaxation following from the coupling of all longitudinal orders. These observations were supported by Multi-quantum-filter experiments and by simulations of the 2D-T1/SAE experiment with a lithium spin system. Since the asymmetric relaxation effects are directly dependent on the velocities and degrees of freedom of ion motion they could be used especially in fast Li-ion conductors as a separation tool for environments with different mobility processes.

We studied the habitat selection of pronghorn (Antilocapra americana) during seasonal migration; an important period in an animal’s annual cycle associated with broad-scale movements. We further decompose our understanding of migration habitat itself as the product of both broad- and fine-scale behavioral decisions and take a multi-scale approach to assess pronghorn spring and fall migration across the transboundary Northern Sagebrush Steppe region. We used a hierarchical habitat selection framework to assess a suite of natural and anthropogenic features that have been shown to influence selection patterns of pronghorn at both broad (migratory neighborhood) and fine (migratory pathway) scales. We then combined single-scale predictions into a scale-integrated step selection function (ISSF) map to assess its effectiveness in predicting migration route habitat. During spring, pronghorn selected for native grasslands, areas of high forage productivity (NDVI), and avoided human activity (i.e., roads and oil and natural gas wells). During fall, pronghorn selected for native grasslands, larger streams and rivers, and avoided roads. We detected avoidance of paved roads, unpaved roads, and wells at broad spatial scales, but no response to these features at fine scales. In other words, migratory pronghorn responded more strongly to anthropogenic features when selecting a broad neighborhood through which to migrate than when selecting individual steps along their migratory pathway. Our results demonstrate that scales of migratory route selection are hierarchically nested within each other from broader (second-order) to finer scales (third-order). In addition, we found other variables during particular migratory periods (i.e., native grasslands in spring) were selected for across scales indicating their importance for pronghorn. The mapping of ungulate migration habitat is a topic of high conservation relevance. In some applications, corridors are mapped according to telemetry location data from a sample of animals, with the assumption that the sample adequately represents habitat for the entire population. Our use of multi-scale modelling to predict resource selection during migration shows promise and may offer another relevant alternative for use in future conservation planning and land management decisions where telemetry-based sampling is unavailable or incomplete.

The multi-scale hierarchical structure of tooth enamel enables it to withstand a lifetime of damage without catastrophic failure. While many previous studies have investigated structure-function relationships in enamel, the effects of crystal misorientation on mechanical performance have not been assessed. To address this issue, in the present study, we review previously published polarization-dependent imaging contrast (PIC) maps of mouse and human enamel, and parrotfish enameloid, in which crystal orientations were measured and displayed in every 60-nm-pixel. By combining those previous results with the PIC maps of sheep enamel presented here we discovered that, in all enamel(oid)s, adjacent crystals are slightly misoriented, with misorientation angles in the 0°-30° range, and mean 2°-8°. Within this limited range, misorientation is positively correlated with literature hardness values, demonstrating an important structure-property relation, not previously identified. At greater misorientation angles 8°30°, this correlation is expected to reverse direction, but data from different non-enamel systems, with more diverse crystal misorientations, are required to determine if and where this occurs. STATEMENT OF SIGNIFICANCE: We identify a structure-function relationship in tooth enamels from different species: crystal misorientation correlates with hardness, contributing to the remarkable mechanical properties of enamel in diverse animals.

Aedes aegypti is a critical vector for transmitting Zika, dengue, chikungunya, and yellow fever viruses to humans. Genetic strategies to limit mosquito survival based upon sex distortion or disruption of development may be valuable new tools to control Ae. aegypti populations. We identified six genes with expression limited to pupal development ; osi8 and osi11 (Osiris protein family), CPRs and CPF (cuticle protein family), and stretchin (a muscle protein). Heritable CRISPR/Cas9-mediated gene knockout of these genes did not reveal any defects in pupal development. However, stretchin-null mutations (strnΔ35/Δ41) resulted in flightless mosquitoes with an abnormal open wing posture. The inability of adult strnΔ35/Δ41 mosquitoes to fly restricted their escape from aquatic rearing media following eclosion , and substantially reduced adult survival rates. Transgenic strains which contain the EGFP marker gene under the control of strn regulatory regions (0.8 kb, 1.4 kb, and 2.2 kb upstream, respectively), revealed the gene expression pattern of strn in muscle-like tissues in the thorax during late morphogenesis from L 4 larvae to young adults. We demonstrated that Ae. aegypti pupae-specific strn is critical for adult mosquito flight capability and a key late-acting lethal target for mosquito-borne disease control.

The mechanisms by which colicins, protein toxins produced by Escherichia coli, kill other E. coli, have become much better understood in recent years. Most colicins initially bind to an outer membrane protein receptor, and then search for a separate nearby outer membrane protein translocator that serves as a pathway into target cells. Many colicins use the outer membrane porin, OmpF, as that translocator, while using a different primary receptor. Colicin N is unique among known colicins in that only OmpF had been identified as being required for uptake of the colicin and it was presumed to somehow serve as both receptor and translocator. Genetic screens also identified a number of genes required for lipopolysaccharide (LPS) synthesis as uniquely required for killing by colicin N, but not by other colicins. In this issue of Molecular Microbiology, Johnson et al. show that the receptor-binding domain of colicin N binds to LPS, and does not require OmpF for that binding. LPS of a minimal length is required for binding, explaining the requirement for specific elements of the LPS biosynthetic pathway. For colicin N, the receptor-binding domain does not recognize a protein, but rather the most abundant component of the outer membrane itself, LPS.

Of the steps involved in the killing of Escherichia coli by colicins, binding to a specific outer-membrane receptor was the best understood and earliest characterized. Receptor binding was believed to be an indispensable step in colicin intoxication, coming before the less well-understood step of translocation across the outer membrane to present the killing domain to its target. In the process of identifying the translocator for colicin Ia, I created chimaeric colicins, as well as a deletion missing the entire receptor-binding domain of colicin Ia. The normal pathway for colicin Ia killing was shown to require two copies of Cir: one that serves as the primary receptor and a second copy that serves as translocator. The novel Ia colicins retain the ability to kill E. coli, even in the absence of receptor binding, as long as they can translocate via their Cir translocator. Experiments to determine whether colicin M uses a second copy of its receptor, FhuA, as its translocator were hampered by precipitation of colicin M chimaeras in inclusion bodies. Nevertheless, I show that receptor binding can be bypassed for killing, as long as a translocation pathway is maintained for colicin M. These experiments suggest that colicin M, unlike colicin Ia, may normally use a single copy of FhuA as both its receptor and its translocator. Colicin E1 can kill in the absence of receptor binding, using translocation through TolC.

TonB-dependent transporters (TBDTs), which transport iron-chelating siderophores and vitamin B12 across the outer membrane of gram negative bacteria, share a conserved architecture of a 22-stranded beta-barrel with an amino-terminal plug domain occluding the barrel. We previously reported that we could induce TBDTs to reversibly open in planar lipid bilayers via the use of urea and that these channels were responsive to physiological concentrations of ligands. Here we report that in the presence of urea, trypsin can cleave the amino-terminal 67 residues of the plug of the TonB-dependent transporter FhuA, as assessed by gel shift and mass spectrometry assays. On the bilayer, trypsin treatment in the presence of urea resulted in the induced conductance no longer being reversed upon removal of urea, suggesting that urea opens intact FhuA channels by pulling the plug at least partly out of the barrel, and that removal of the urea then allows reinsertion of the plug into the barrel. When expressed separately, the FhuA plug domain was found to be a mostly unfolded structure that was able to occlude isolated FhuA beta-barrels inserted into the membrane. Thus, although folded in the barrel, the plug need not be folded upon exiting the barrel. The rate of insertion of the beta-barrels into the membrane was tremendously increased in the presence of an osmotic gradient provided by either urea or glycerol. Negative staining electron microscopy showed that FhuA in a detergent solution formed vesicles, thus explaining why an osmotic gradient promoted the insertion of FhuA into membranes.

Filamentous inclusions made of α-synuclein are found in nerve cells and glial cells in a number of human neurodegenerative diseases, including Parkinson disease, dementia with Lewy bodies, and multiple system atrophy. The assembly and spreading of these inclusions are likely to play an important role in the etiology of common dementias and movement disorders. Both α-synuclein and the homologous β-synuclein are abundantly expressed in the central nervous system; however, β-synuclein is not present in the pathological inclusions. Previously, we observed a poor correlation between filament formation and the presence of residues 73–83 of α-synuclein, which are absent in β-synuclein. Instead, filament formation correlated with the mean β-sheet propensity, charge, and hydrophilicity of the protein (global physicochemical properties) and β-strand contiguity calculated by a simple algorithm of sliding averages (local physicochemical property). In the present study, we rendered β-synuclein fibrillogenic via one set of point mutations engineered to enhance global properties and a second set engineered to enhance predominantly β-strand contiguity. Our findings show that the intrinsic physicochemical properties of synucleins influence their fibrillogenic propensity via two distinct but overlapping modalities. The implications for filament formation and the pathogenesis of neurodegenerative diseases are discussed.

Two closely related scaffolds were identified through an uHTS campaign as desirable starting points for the development of Rho-Kinase (ROCK) inhibitors. Here, we describe our hit-to-lead evaluation process which culminated in the rapid discovery of potent leads such as 22 which successfully demonstrated an early in vivo proof of concept for anti-hypertensive activity.

Colicin Ia, a channel-forming bactericidal protein, uses the outer membrane protein, Cir, as its primary receptor. To kill E. coli, it must cross this membrane. The crystal structure of Ia receptor-binding domain bound to Cir, a 22-stranded plugged β-barrel protein, suggests that the plug does not move. Therefore, another pathway is needed for the colicin to cross the outer membrane, but no “second receptor” has ever been identified for TonB-dependent colicins, such as Ia. We show that if the receptor-binding domain of colicin Ia is replaced by that of colicin E3, this chimera effectively kills cells, provided they have the E3 receptor (BtuB), Cir, and TonB. This is consistent with wild-type Ia using one Cir as its primary receptor (BtuB in the chimera) and a second Cir as the translocation pathway for its N-terminal translocation (T) domain and its channel-forming C-terminal domain. Deletion of colicin Ia’s receptor-binding domain results in a protein that kills E. coli, albeit less effectively, provided they have Cir and TonB. We show that purified T-domain competes with Ia and protects E. coli from being killed by it. Thus, in addition to binding to colicin Ia’s receptor-binding domain, Cir also binds weakly to its translocation domain.

Colicin Ia is a 69 kDa protein that kills susceptible Escherichia coli cells by binding to a specific receptor in the outer membrane, colicin I receptor (70 kDa), and subsequently translocating its channel forming domain across the periplasmic space, where it inserts into the inner membrane and forms a voltage-dependent ion channel. We determined crystal structures of colicin I receptor alone and in complex with the receptor binding domain of colicin Ia. The receptor undergoes large and unusual conformational changes upon colicin binding, opening at the cell surface and positioning the receptor binding domain of colicin Ia directly above it. We modelled the interaction with full-length colicin Ia to show that the channel forming domain is initially positioned 150 A above the cell surface. Functional data using full-length colicin Ia show that colicin I receptor is necessary for cell surface binding, and suggest that the receptor participates in translocation of colicin Ia across the outer membrane.

The Pim family of Ser/Thr kinases has been implicated in the process of lymphomagenesis and cell survival. Known substrates of Pim kinases are few and poorly characterized. In this study we set out to identify novel Pim-2 substrates using the Kinase Substrate Tracking and Elucidation (KESTREL) approach. Two potential substrates, eukaryotic initiation factor 4B (eIF4B) and apoptosis inhibitor 5 (API-5), were identified from rat thymus extracts. Sequence comparison of the Pim-2 kinase phosphorylation sites of eIF4B and mouse BAD, the only other known Pim-2 substrate, revealed conserved amino acids preceding the phosphorylated serine residue. Stepwise replacement of the conserved residues produced a consensus sequence for Pim kinase recognition: RXRHXS. Pim-1 and Pim-2 catalyzed the phosphorylation of this recognition sequence 20-fold more efficiently than the original (K/R-K/R-R-K/R-L-S/T-a; a = small chain amino acid) Pim-1 phosphorylation site. The identification of the novel Pim kinase consensus sequence provides a more sensitive and versatile peptide based assay for screening modulators of Pim kinase activity.

A 3300-base segment of Escherichia coli chromosomal DNA, cloned into pBR322, will complement a methionine auxotroph in which the lesion is a defective methionyl-tRNA synthetase with a much reduced affinity for methionine. Crude extracts of these transformants contain elevated levels of a protein which has a subunit molecular weight of 66000, methionyl-tRNA synthetase aminoacylation activity in vitro and which cross-reacts with anti-(methionyl-tRNA synthetase) antibodies. This polypeptide is very slightly larger than the well-characterised and crystallised tryptic fragment of methionyl-tRNA synthetase. A DNA sequence of 1750 residues at one end of the cloned insert codes for a non-terminated open reading frame in which we can locate a large number of methionyl-tRNA synthetase tryptic and chymotryptic peptides. We have also sequenced 300 nucleotides upstream of this coding segment where we find a large invert repeat in the putative methionyl-tRNA synthetase promoter region.

A novel inhibitor of p38 mitogen-activated protein kinase (p38), CMPD1, identified by high-throughput screening, is characterized herein. Unlike the p38 inhibitors described previously, this inhibitor is substrate selective and noncompetitive with ATP. In steady-state kinetics experiments, CMPD1 was observed to prevent the p38alpha-dependent phosphorylation (K(i)(app) = 330 nM) of the splice variant of mitogen-activated protein kinase-activated protein kinase 2 (MK2a) that contains a docking domain for p38alpha and p38beta, but it did not prevent the phosphorylation of ATF-2 (K(i)(app) > 20 microM). In addition to kinetic studies, isothermal titration calorimetry and surface plasmon resonance experiments were performed to elucidate the mechanism of inhibition. While isothermal titration calorimetry analysis indicated that CMPD1 binds to p38alpha, CMPD1 was not observed to compete with ATP for p38alpha, nor was it able to interrupt the binding of p38alpha to MK2a observed by surface plasmon resonance. Therefore, deuterium exchange mass spectrometry (DXMS) was employed to study the p38alpha.CMPD1 inhibitory complex, to provide new insight into the mechanism of substrate selective inhibition. The DXMS data obtained for the p38alpha.CMPD1 complex were compared to the data obtained for the p38alpha.MK2a complex and a p38alpha.active site binding inhibitor complex. Alterations in the DXMS behavior of both p38alpha and MK2a were observed upon complex formation, including but not limited to the interaction between the carboxy-terminal docking domain of MK2a and its binding groove on p38alpha. Alterations in the D(2)O exchange of p38alpha produced by CMPD1 suggest that the substrate selective inhibitor binds in the vicinity of the active site of p38alpha, resulting in perturbations to regions containing nucleotide binding pocket residues, docking groove residues (E160 and D161), and a Mg(2+) ion cofactor binding residue (D168). Although the exact mechanism of substrate selective inhibition by this novel inhibitor has not yet been disclosed, the results suggest that CMPD1 binding in the active site region of p38alpha induces perturbations that may result in the suboptimal positioning of substrates and cofactors in the transition state, resulting in selective inhibition of p38alpha activity.

The discovery of novel 5,7-disubstituted[1,6]naphthyridines as potent inhibitors of Spleen Tyrosine Kinase (SYK) is discussed. The SAR reveals the necessity for a 7-aryl group with preference towards para substitution and that this in combination with 5-aminoalkylamino substituents further improved the potency of the compounds. The initial SAR as well as a survey of the other positions is discussed in detail.

The bacterial toxin colicin Ia forms voltage-gated channels in planar lipid bilayers. The toxin consists of three domains, with the carboxy-terminal domain (C-domain) responsible for channel formation. The C-domain contributes four membrane-spanning segments and a 68-residue translocated segment to the open channel, whereas the upstream domains and the amino-terminal end of the C-domain stay on the cis side of the membrane. The isolated C-domain, lacking the two upstream domains, also forms channels; however, the amino terminus and one of the normally membrane-spanning segments can move across the membrane. (This can be observed as a drop in single-channel conductance.) In longer carboxy-terminal fragments of colicin Ia that include ≤169 residues upstream from the C-domain, the entire upstream region is translocated. Presumably, a portion of the C-domain creates a pathway for the polar upstream region to move through the membrane. To determine the size of this translocation pathway, we have attached “molecular stoppers,” small disulfide-bonded polypeptides, to the amino terminus of the C-domain, and determined whether they could be translocated. We have found that the translocation rate is strongly voltage dependent, and that at voltages ≥90 mV, even a 26-Å stopper is translocated. Upon reduction of their disulfide bonds, all of the stoppers are easily translocated, indicating that it is the folded structure, rather than some aspect of the primary sequence, that slows translocation of the stoppers. Thus, the pathway for translocation is ≥26 Å in diameter, or can stretch to this value. This is large enough for an α-helical hairpin to fit through.

The paired helical filament (PHF), which makes up the major fibrous component of the neurofibrillary lesions of Alzheimer's disease, is composed of hyperphosphorylated and abnormally phosphorylated microtubule-associated protein tau. Previous studies have identified serine and threonine residues phosphorylated in PHF-tau and have shown that tau can be phosphorylated at several of these sites by proline-directed protein kinases and cyclic AMP-dependent protein kinase. Here we have investigated which protein phosphatase activities can dephosphorylate recombinant tau phosphorylated with mitogen-activated protein kinase, glycogen synthase kinase-3 beta, neuronal cdc2-like kinase, or cyclic AMP-dependent protein kinase. We show that protein phosphatase 2A is by far the major protein phosphatase activity in brain that dephosphorylates tau phosphorylated in this manner.

The pathological hallmark of Parkinson's disease is the presence of intracellular inclusions, Lewy bodies, and Lewy neurites, in the dopaminergic neurons of the substantia nigra and several other brain regions. Filamentous α-synuclein is the major component of these deposits and its aggregation is believed to play an important role in Parkinson's disease and several other neurodegenerative diseases. Two homologous proteins, β- and γ-synucleins, are also abundant in the brain. The synucleins are natively unfolded proteins. β-Synuclein, which lacks 11 central hydrophobic residues compared with its homologs, exhibited the properties of a random coil, whereas α- and γ-synucleins were slightly more compact and structured. γ-Synuclein, unlike its homologs, formed a soluble oligomer at relatively low concentrations, which appears to be an off-fibrillation pathway species. Here we show that, although they have similar biophysical properties to α-synuclein, β- And γ-synucleins inhibit α-synuclein fibril formation. Complete inhibition of α-synuclein fibrillation was observed at 4:1 molar excess of β- and γ-synucleins. No significant incorporation of β-synuclein into the fibrils was detected. The lack of fibrils formed by β-synuclein is most readily explained by the absence of a stretch of hydrophobic residues from the middle region of the protein. A model for the inhibition is proposed.

Channel-forming colicins are bactericidal proteins that spontaneously insert into hydrophobic lipid bilayers. We have used magic-angle spinning solid-state nuclear magnetic resonance spectroscopy to examine the conformational differences between the water-soluble and the membrane-bound states of colicin Ia channel domain, and to study the effect of bound colicin on lipid bilayer structure and dynamics. We detected 13 C and 15 N isotropic chemical shift differences between the two forms of the protein, which indicate structural changes of the protein due to membrane binding. The Val Cα signal, unambiguously assigned by double-quantum experiments, gave a 0.6 ppm downfield shift in the isotropic position and a 4 ppm reduction in the anisotropic chemical shift span after membrane binding. These suggest that the α-helices in the membrane-bound colicin adopt more ideal helical torsion angles as they spread onto the membrane. Colicin binding significantly reduced the lipid chain order, as manifested by 2 H quadrupolar couplings. These results are consistent with the model that colicin Ia channel domain forms an extended helical array at the membrane–water interface upon membrane binding.

tau gene mutations cause frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17). Here we have used Xenopus oocyte maturation as an indicator of microtubule function. We show that wild-type four-repeat Tau protein inhibits maturation in a concentration-dependent manner, whereas three-repeat Tau has no effect. Of the seven four-repeat Tau proteins with FTDP-17 mutations tested, five (G272V, DeltaK280, P301L, P301S, and V337M) failed to interfere significantly with oocyte maturation, demonstrating a greatly reduced ability to interact with microtubules. One mutant protein (R406W) almost behaved like wild-type Tau, and one (S305N) inhibited maturation more strongly than wild-type Tau. With the exception of R406W, wild-type Tau and all the mutants studied were similarly phosphorylated during the Xenopus oocyte maturation, and this was independent of their effects on this process. Data obtained with R406W and S305N may be related to charge changes (phosphorylation and basic amino acids). Our results demonstrate variable effects of FTDP-17 mutations on microtubules in an intact cell situation. Those findings establish Xenopus oocyte maturation as a system allowing the study of the functional effects of tau gene mutations in a quantitative manner.

Coding region and intronic mutations in the tau gene cause frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17). We have previously reported that ABalphaC, a major form of protein phosphatase 2A (PP2A) in brain, binds tightly to tau protein in vitro and is a major tau phosphatase in vivo. Using in vitro assays, we show here that the FTDP-17 mutations G272V, DeltaK280, P301L, P301S, S305N, V337M, G389R, and R406W inhibit by approximately 20-95% the binding of recombinant three-repeat and four-repeat tau isoforms to the ABalphaC holoenzyme and the AC core enzyme of PP2A. Reduction in binding was maximal for tau proteins with the G272V, DeltaK280, and V337M mutations. We also show that tau protein can be specifically coimmunoprecipitated with endogenous PP2A from both rat brain and transfected cell extracts. It is significant that, by using similar coimmunoprecipitation assays, we show that all FTDP-17 mutations tested, including the N279K mutation, alter the ability of tau to associate with cellular PP2A. Taken together, these results indicate that FTDP-17 mutations induce a significant decrease in the binding affinity of tau for PP2A in vivo. We propose that altered protein-protein interactions between PP2A and tau may contribute to FTDP-17 pathogenesis.

The casein milk proteins and the brain proteins alpha-synuclein and tau have been described as natively unfolded with random coil structures, which, in the case of alpha-synuclein and tau, have a propensity to form the fibrils found in a number of neurodegenerative diseases. New insight into the structures of these proteins has been provided by a Raman optical activity study, supplemented with differential scanning calorimetry, of bovine beta- and kappa-casein, recombinant human alpha-, beta- and gamma-synuclein, together with the A30P and A53T mutants of alpha-synuclein associated with familial cases of Parkinson's disease, and recombinant human tau 46 together with the tau 46 P301L mutant associated with inherited frontotemporal dementia. The Raman optical activity spectra of all these proteins are very similar, being dominated by a strong positive band centred at approximately 1318 cm(-1) that may be due to the poly(l-proline) II (PPII) helical conformation. There are no Raman optical activity bands characteristic of extended secondary structure, although some unassociated beta strand may be present. Differential scanning calorimetry revealed no thermal transitions for these proteins in the range 15-110 degrees C, suggesting that the structures are loose and noncooperative. As it is extended, flexible, lacks intrachain hydrogen bonds and is hydrated in aqueous solution, PPII helix may impart a rheomorphic (flowing shape) character to the structure of these proteins that could be essential for their native function but which may, in the case of alpha-synuclein and tau, result in a propensity for pathological fibril formation due to particular residue properties.

The selective and extensive 13C labeling of mostly hydrophobic amino acid residues in a 25 kDa membrane protein, the colicin Ia channel domain, is reported. The novel 13C labeling approach takes advantage of the amino acid biosynthetic pathways in bacteria and suppresses the synthesis of the amino acid products of the citric acid cycle. The selectivity and extensiveness of labeling significantly simplify the solid-state NMR spectra, reduce line broadening, and should permit the simultaneous measurement of multiple structural constraints. We show the assignment of most 13C resonances to specific amino acid types based on the characteristic chemical shifts, the 13C labeling pattern, and the amino acid composition of the protein. The assignment is partly confirmed by a 2D homonuclear double-quantum-filter experiment under magic-angle spinning. The high sensitivity and spectral resolution attained with this 13C-labeling protocol, which is termed TEASE for ten-amino acid selective and extensive labeling, are demonstrated.

Alpha-synuclein (alpha-syn) protein and a fragment of it, called NAC, have been found in association with the pathological lesions of a number of neurodegenerative diseases. Recently, mutations in the alpha-syn gene have been reported in families susceptible to an inherited form of Parkinson's disease. We have shown that human wild-type alpha-syn, mutant alpha-syn(Ala30Pro) and mutant alpha-syn(Ala53Thr) proteins can self-aggregate and form amyloid-like filaments. Here we report that aggregates of NAC and alpha-syn proteins induced apoptotic cell death in human neuroblastoma SH-SY5Y cells. These findings indicate that accumulation of alpha-syn and its degradation products may play a major role in the development of the pathogenesis of these neurodegenerative diseases.

Activation of the transcription factor NF-κB is regulated by the phosphorylation and subsequent degradation of its inhibitory subunit, IκB. A large multiprotein complex, the IκB kinase (IKK), catalyzes the phosphorylation of IκB. The two kinase components of the IKK complex, IKKα and IKKβ, were overexpressed in insect cells and purified to homogeneity. Both purified IKKα and IKKβ specifically catalyzed the phosphorylation of the regulatory serine residues of IκBα. Hence, IKKα and IKKβ were functional catalytic subunits of the IKK complex. Purified IKKα and IKKβ also preferentially phosphorylated serine as opposed to threonine residues of IκBα, consistent with the substrate preference of the IKK complex. Kinetic analysis of purified IKKα and IKKβ revealed that the kinase activity of IKKβ on IκBα is 50–60-fold higher than that of IKKα. The primary difference between the two activities is the K m for IκBα. The kinetics of both IKKα and IKKβ followed a sequential Bi Bi mechanism. No synergistic effects on IκBα phosphorylation were detected between IKKα and IKKβ. Thus, in vitro, IKKα and IKKβ are two independent kinases of IκBα.

The T domain of diphtheria toxin, which extends from residue 202 to 378, causes the translocation of the catalytic A fragment (residues 1–201) across endosomal membranes and also forms ion-conducting channels in planar phospholipid bilayers. The carboxy terminal 57-amino acid segment (322–378) in the T domain is all that is required to form these channels, but its ability to do so is greatly augmented by the portion of the T domain upstream from this. In this work, we show that in association with channel formation by the T domain, its NH2 terminus, as well as some or all of the adjacent hydrophilic 63 amino acid segment, cross the lipid bilayer. The phenomenon that enabled us to demonstrate that the NH2-terminal region of the T domain was translocated across the membrane was the rapid closure of channels at cis negative voltages when the T domain contained a histidine tag at its NH2 terminus. The inhibition of this effect by trans nickel, and by trans streptavidin when the histidine tag sequence was biotinylated, clearly established that the histidine tag was present on the trans side of the membrane. Furthermore, the inhibition of rapid channel closure by trans trypsin, combined with mutagenesis to localize the trypsin site, indicated that some portion of the 63 amino acid NH2-terminal segment of the T domain was also translocated to the trans side of the membrane. If the NH2 terminus was forced to remain on the cis side, by streptavidin binding to the biotinylated histidine tag sequence, channel formation was severely disrupted. Thus, normal channel formation by the T domain requires that its NH2 terminus be translocated across the membrane from the cis to the trans side, even though the NH2 terminus is >100 residues removed from the channel-forming part of the molecule.

The crystal structure of human p56lck SH2 domain in complex with an inhibitor containing the singly chargedp-(carboxymethyl)phenylalanine residue (cmF) as a phosphotyrosine (Tyr(P) or pY) replacement has been determined at 1.8 A resolution. The binding mode of the acetyl-cmF-Glu-Glu-Ile (cmFEEI) inhibitor is very similar to that of the pYEEI inhibitor, confirming that the cmFEEI inhibitor has a similar mechanism of SH2 domain inhibition despite its significantly reduced potency. Observed conformational differences in the side chain of the cmF residue can be interpreted in terms of maintaining similar interactions with the SH2 domain as the Tyr(P) residue. The crystal structure of the free p56lck SH2 domain has been determined at 1.9 A resolution and shows an open conformation for the BC loop and an open phosphotyrosine binding pocket, in contrast to earlier studies on the srcSH2 domain that showed mostly closed conformation. The structural information presented here suggests that the carboxymethyl-phenylalanine residue may be a viable Tyr(P) replacement and represents an attractive starting point for the design and development of SH2 domain inhibitors with better pharmaceutical profiles.

Hyperphosphorylated microtubule-associated protein tau is the major proteinaceous component of the paired helical and straight filaments which constitute a defining neuropathological characteristic of Alzheimer's disease and a number of other neurodegenerative disorders. We have recently shown that full-length recombinant tau assembles into Alzheimer-like filaments upon incubation with heparin. Heparin also promotes phosphorylation of tau by a number of protein kinases, prevents tau from binding to taxol-stabilized microtubules, and produces rapid disassembly of microtubules assembled from tau and tubulin. Here, we have used the above parameters to study the interactions between tau protein and a number of naturally occurring and synthetic glycosaminoglycans. We show that the magnitude of the glycosaminoglycan effects is proportional to their degree of sulfation. Thus, the strongly sulfated glycosaminoglycans dextran sulfate, pentosan polysulfate, and heparin were the most potent, whereas the non-sulfated dextran and hyaluronic acid were without effect. The moderately sulfated glycosaminoglycans heparan sulfate, chondroitin sulfate, and dermatan sulfate had intermediate effects, whereas keratan sulfate had little or no effect. These in vitro interactions between tau protein and sulfated glycosaminoglycans reproduced the known characteristics of paired helical filament-tau from Alzheimer's disease brain. Sulfated glycosaminoglycans are present in nerve cells in Alzheimer's disease brain in the early stages of neurofibrillary degeneration, suggesting that their interactions with tau may constitute a central event in the development of the neuronal pathology of Alzheimer's disease.

One of the key steps involved in T-cell activation is binding of the tyrosine kinase ZAP-70 via its two SH2 domains to peptide segments termed tyrosine-based activation motifs (ITAM) which are present in three of the T-cell receptor (TCR) subunits. The crystal structure of the ZAP-70 SH2 domains complexed to phosphopeptide revealed that the amino-terminal phosphotyrosine-binding pocket is formed at the interface between the two SH2 domains. This study was designed to further characterize the binding between TCR ζ ITAM1 and the ZAP-70 SH2 domains as well as to assess the change in conformation of SH2 domain structure upon ζ ITAM1 binding. BIAcore analysis of wild type and nonfunctional single-point mutants of ZAP-70 SH2 domains demonstrated that the amino-terminal SH2 domain can bind phosphopeptide in the absence of a functional carboxyl-terminal SH2 domain. In addition, the amino-terminal SH2 domain prefers the RREEpYDVLDK sequence of ζ chain ITAM1 over the GQNQLpYNELNL sequence. To assess changes in protein conformation upon ITAM binding to ZAP-70 SH2 domains, fluorescence spectroscopy and analytical ultracentrifugation experiments were performed. A significant blue shift in the tryptophan emission spectrum of the SH2 domains was observed in the presence of saturating amounts of phosphopeptide, indicating a loss in solvent exposure for the tryptophan residues in the protein–phosphopeptide complex. This was accompanied by changes in the frictional coefficient consistent with a compacting of the protein structure. Finally, thermal denaturation experiments showed an increase in stability and cooperativity in unfolding for the protein–phosphopeptide complex relative to the protein alone.

NAC, a 35-residue peptide derived from the neuronal protein alpha-synuclein/NAC precursor, is tightly associated with Abeta fibrils in Alzheimer's disease amyloid, and alpha-synuclein has recently been shown to bind Abeta in vitro. We have studied the interaction between Abeta and synucleins, aiming at determining segments in alpha-synuclein that can account for the binding, as well as identifying a possible interaction between Abeta and the beta-type synuclein. We report that Abeta binds to native and recombinant alpha-synuclein, and to beta-synuclein in an SDS-sensitive interaction (IC50 approx. 20 microM), as determined by chemical cross-linking and solid-phase binding assays. alpha-Synuclein and beta-synuclein were found to stimulate Abeta-aggregation in vitro to the same extent. The synucleins also displayed Abeta-inhibitable binding of NAC and they were capable of forming dimers. Using proteolytic fragmentation of alpha-synuclein and cross-linking to 125I-Abeta, we identified two consecutive binding domains (residues 1-56 and 57-97) by Edman degradation and mass spectrometric analysis, and a synthetic peptide comprising residues 32-57 possessed Abeta-binding activity. To test further the possible significance in pathology, alpha-synuclein was biotinylated and shown to bind specifically to amyloid plaques in a brain with Alzheimer's disease. It is proposed that the multiple Abeta-binding sites in alpha-synuclein are involved in the development of amyloid plaques.

Phosphorylation of tau protein at Ser-262 has been shown to diminish its ability to bind to taxol-stabilized microtubules. The paired helical filaments (PHFs) found in Alzheimer's disease brain are composed of PHF-tau, which is hyperphosphorylated at multiple sites including Ser-262. However, protein kinase(s) able to phosphorylate this site are still under investigation. In this study, the ability of cyclic AMP-dependent protein kinase (cAMP-PK) and calcium/calmodulin-dependent protein kinase II (CaMKII) to phosphorylate tau at Ser-262, as well as Ser-356, is demonstrated by use of a monoclonal antibody (12E8) which has been shown to recognize tau when these sites are phosphorylated. Cleavage of cAMP-PK-phosphorylated tau at cysteine residues by 2-nitro-5-thiocyanobenzoic acid, which cuts the protein into essentially two fragments and separates Ser-262 from Ser-356, revealed that cAMP-PK phosphorylates both Ser-262 and Ser-356. In addition, phosphorylation with cAMP-PK or CaMKII of recombinant tau in which Ser-262, Ser-356 or both had been mutated to alanines, clearly demonstrated that cAMP-PK and CaMKII were able to phosphorylate both sites. Mitogen-activated protein kinase or protein kinase C did not phosphorylate tau at Ser-262 and/or Ser-356. Finally, evidence is presented that phosphorylation of both these sites occurs in cultured nerve cells under certain conditions, indicating their potential physiological relevance.

The chains of the T cell receptor complex play a critical role in the initiation of proximal signaling events upon T cell activation. Three pairs of potential tyrosine phosphorylation sites are located within the cytoplasmic domains of the chains. Subsequent to engagement of the T cell receptor, one or more of these tyrosine residues is phosphorylated. The phosphotyrosine residues, along with flanking amino acids, form an activation motif (and are shared by signaling subunits in the TCR, B cell receptor, and FcγRI) termed tyrosine-based activation motifs (ITAMs). ITAMs serve as binding sites for SH2 domain-containing proteins. Recent evidence suggests that the chains provide docking space for several key signal transduction molecules such as ZAP-70, p56 lck, and Shc. To determine if ZAP-70, p56 lck, and Shc bind to particular chain ITAM sequences, quantitative free-solution measurements of binding affinities (Kd) were obtained by use of surface plasmon resonance technology. The results indicate that binding affinities of distinct SH2 domains to individual and paired phosphorylation sites greatly differ, and may dictate the sequence of signal transduction events.

Colicins are protein toxins produced by E. coli to kill closely related competitor E. coli. Colicins initially attach to target bacteria by binding to outer membrane receptors, most of which are TonB-dependent nutrient transporters—22-stranded -barrels plugged by their amino-terminal domains. One group of colicins, which use the Tol proteins, TolA,B,Q,R, for uptake, have been shown to use OmpF or TolC as their translocators after the initial step when the colicin binds to its receptor. The N-terminal translocation domain of the colicin actually threads into the translocator (Housden et al. (2010) PNAS: 107: 20412).Until recently, no translocator or “second receptor” had been identified for the other family of colicins, those that use TonB and ExbB and D for uptake. Recently, colicin Ia was shown to use a second copy of its primary receptor, Cir, as its translocator (Jakes, K.S. and Finkelstein, A. (2010) Mol. Micro.: 75: 567). In an attempt to determine whether other TonB-dependent colicins also use a second copy of their primary receptor as a translocator, I have made chimera constructs with colicin M, substituting the receptor-binding domain of colicin E3 for that of colicin M, and also deleting the entire receptor-binding domain. These constructs were insoluble and went into inclusion bodies. Dissolving the inclusion bodies in 8M urea, renaturing by dilution and dialysis, and purifying by nickel chelation yielded a small amount of pure protein that had no in vivo killing activity. However, the chimera blocks killing by colicin E3, demonstrating that it binds the E3 receptor, BtuB. Osmotic shock to bypass receptor binding and translocation showed that the enzymatic moiety of the chimera is also active.These results suggest that colicin M translocates differently than colicin Ia and may not normally use a translocator remote from its primary binding site.

Colicins are protein toxins produced by Escherichia coli to kill related bacteria. They must cross the target cell outer membrane (OM), and some must also cross the inner membrane (IM). To accomplish cellular import, colicins have parasitized E. coli nutrient transporters as well as IM and periplasmic proteins normally used to maintain cell wall integrity or provide energy for nutrient uptake through transporters. Colicins have evolved to use both transporters and other membrane proteins through mechanisms different from those employed in physiological substrate uptake. Extended receptor-binding domains allow some colicins to search by lateral diffusion for binding sites on their OM translocators while bound to their primary OM receptor. Transport across the OM is initiated by entry of the unstructured N-terminal translocation domain into the translocator. Periplasmic and IM networks subsequently accomplish insertion of the colicin cytotoxic domain into or across the IM.

VOLTAGE-GATED channels undergo a conformational change in response to changes in transmembrane voltage. Here we use site-directed biotinylation to create conformation-sensitive sites on col-icin la, a bacteriocidal protein that forms a voltage-sensitive mem-brane channel, which can be monitored by electrophysiological methods1,2. We investigated a model of gating developed for the partly homologous colicin El that is based on the insertion of regions of the protein into the membrane in response to cis-positive voltages3–6. Site-directed cysteine mutagenesis, followed by chemi-cal modification, was used to attach a biotin molecule covalently to a series of unique sites on colicin la. The modified protein was incorporated into planar lipid membranes, where the introduced biotin moiety served as a site to bind the water-soluble protein streptavidin, added to one side of the membrane or the other. Our results show that colicin gating is associated with the translocation across the membrane of a segment of the protein of at least 31 amino acids.

Two abundant proteins of 140 and 134 amino acids were purified and sequenced from human brain. They were identified through their reactivity on immunoblots with a partially characterised monoclonal antibody that recognises tau protein in a phosphorylation-dependent manner. The 140 amino acid protein is identical with the precursor of the non-A beta component of Alzheimer's disease amyloid which in turn is highly homologous to synuclein from Torpedo electroplaques and rat brain. The 134 amino acid protein is the human homologue of bovine phosphoneuroprotein 14; it is 61% identical in sequence to the 140 amino acid protein. The previously unrecognised homology between these two proteins defines a family of human brain synucleins. We refer to the 140 and 134 amino acid proteins as alpha-synuclein and beta-synuclein, respectively. Both synucleins are expressed predominantly in brain, where they are concentrated in presynaptic nerve terminals.

Channel-forming colicins are Escherichia coli proteins that form voltage-dependent channels in lipid bilayer membranes and are lethal to sensitive strains of E. coli. Experiments with colicin E1 have led to a model of voltage dependence based on the insertion of alpha-helical segments of the protein into the membrane in response to cis-positive voltages. This model was tested on the partly homologous colicin Ia protein, which offers certain advantages over colicin E1 as a model channel, it is active at neutral pH and exhibits comparatively well-defined single channel conductance. We describe here the creation of a specific probe for locating a particular amino acid residue on one side or the other of a planar lipid bilayer membrane, by using the biotin-streptavidin system. Site-directed mutagenesis was used to change lysine 544 of colicin Ia to cysteine. This placed a unique cysteine at a site expected, by homology to colicin E1, to cross the membrane from the cis to the trans side in association with the opening of the channel. This unique cysteine was biotinylated chemically, so that it could serve as a target for streptavidin. Incubation of the biotinylated mutant colicin with streptavidin blocked its killing activity, in vivo; incubation of wild-type colicin, which lacks cysteine, with streptavidin, did not affect its activity. Channels formed by the biotinylated mutant protein in planar lipid bilayers were abolished by streptavidin added to the cis side of the membrane, if the channels were closed, but not if they were open. Trans streptavidin had no effect on either open or closed channels. Thus, when the channel is closed, residue 544 of colicin Ia is accessible to cis streptavidin in the closed state, but the opening of the channel eliminates this accessibility.

The principal fibrous component of neurofibrillary pathology in Alzheimer's disease, the paired helical filament, is formed from hyperphosphorylated microtubule-associated protein tau. Here we show that recombinant tau protein either in a non-phosphorylated state or following phosphorylation with brain extract can be assembled in vitro into filaments resembling those seen in Alzheimer's disease.

The mechanisms for substrate recognition by two cytoplasmic protein tyrosine phosphatases, PTP-5 and rrbPTP-1, were investigated. Phosphorylation sites on tyrosine-phosphorylated casein, a model PTP substrate, were characterized. Two peptides based on casein phosphorylation sites and one peptide based on the tyrosine phosphorylation site of reduced, carboxamidomethylated and maleylated (RCM) lysozyme were tested as PTP substrates. The three peptides were dephosphorylated by PTP-5 and rrbPTP-1 at rates comparable to those of the corresponding sites on the intact proteins. This indicates that peptides based on the two model PTP substrates, casein and RCM-lysozyme, contained all or most of the structural information necessary for PTP-5 and rrbPTP-1 substrate recognition. Structural elements required for substrate recognition by PTP-5 and rrbPTP-1 were also investigated. Km values for dephosphorylation of three simple aromatic phosphate esters (phosphotyrosine, p-nitrophenyl phosphate, and phenyl phosphate) by rrbPTP-1 were about 5000-fold higher than those obtained for the peptide and protein substrates. This indicates that recognition of protein and peptide substrates involves structural elements in addition to the phosphate group and the aromatic tyrosine ring of phosphotyrosine. Analysis of the effects of truncations and Ala for polar substitutions on the reactivity with PTP-5 and rrbPTP-1 of peptides based on casein, RCM-lysozyme, and angiotensin II indicated that Asp or Glu within the first five residues on the N-terminal side of phosphotyrosine increased peptide reactivity with both PTP's. Asn residues were unable or only weakly able to substitute for Asp residues.(ABSTRACT TRUNCATED AT 250 WORDS)

In this issue, de Zamaroczy et al. show that cleavage of the bacterial toxin colicin D is required for its ability to kill cells. Surprisingly, the cleavage requires the inner membrane peptidase LepB that normally functions in protein secretion.