Mass spectrometry based protein biomarkers and drug target discovery and clinical diagnosis in Age-Related progressing neurodegenerative diseases.

Neurodegenerative diseases correspond to overly complex health disorders that are driven by intersecting pathophysiology that are often trapped in vicious cycles of degeneration and cognitive decline. The usual diagnostic route of these diseases is based on postmortem examination that involves identifying pathology that is specific to the disease in the brain. However, in such cases, accurate diagnosis of the specific disease is limited because clinical disease presentations are often complex that do not easily allow to discriminate patient's cognitive, behavioral, and functional impairment profiles. Additionally, an early identification and therapeutic intervention of these diseases is pivotal to slow the progression of neurodegeneration and extend healthy life span. Mass spectrometry-based techniques have proven to be hugely promising in biological sample analysis and discovery of biomarkers including protein and peptide biomarkers for potential drug target discovery. Recent studies on these biomarkers have demonstrated their potential for applications in early diagnostics and identifying therapeutic targets to battle against neurodegenerative diseases. In this review, we have presented principles of mass spectrometry (MS) and the associated workflows in analyzing and imaging biological samples for discovery of biomarkers. We have especially focused on age-related progressing neurodegenerative diseases such as Alzheimer's (AD) and Parkinson's disease (PD), Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal dementia (FTD) and the related MS-based biomarker developments for these diseases. Finally, we present a future perspective discussing the potential research directions ahead.

Defective hydrophobic sliding mechanism and active site expansion in HIV-1 protease drug resistant variant Gly48Thr/Leu89Met: mechanisms for the loss of saquinavir binding potency.

HIV drug resistance continues to emerge; consequently, there is an urgent need to develop next generation antiretroviral therapeutics.1 Here we report on the structural and kinetic effects of an HIV protease drug resistant variant with the double mutations Gly48Thr and Leu89Met (PRG48T/L89M), without the stabilizing mutations Gln7Lys, Leu33Ile, and Leu63Ile. Kinetic analyses reveal that PRG48T/L89M and PRWT share nearly identical Michaelis-Menten parameters; however, PRG48T/L89M exhibits weaker binding for IDV (41-fold), SQV (18-fold), APV (15-fold), and NFV (9-fold) relative to PRWT. A 1.9 Å resolution crystal structure was solved for PRG48T/L89M bound with saquinavir (PRG48T/L89M-SQV) and compared to the crystal structure of PRWT bound with saquinavir (PRWT-SQV). PRG48T/L89M-SQV has an enlarged active site resulting in the loss of a hydrogen bond in the S3 subsite from Gly48 to P3 of SQV, as well as less favorable hydrophobic packing interactions between P1 Phe of SQV and the S1 subsite. PRG48T/L89M-SQV assumes a more open conformation relative to PRWT-SQV, as illustrated by the downward displacement of the fulcrum and elbows and weaker interatomic flap interactions. We also show that the Leu89Met mutation disrupts the hydrophobic sliding mechanism by causing a redistribution of van der Waals interactions in the hydrophobic core in PRG48T/L89M-SQV. Our mechanism for PRG48T/L89M-SQV drug resistance proposes that a defective hydrophobic sliding mechanism results in modified conformational dynamics of the protease. As a consequence, the protease is unable to achieve a fully closed conformation that results in an expanded active site and weaker inhibitor binding.

Effect of incorporating a thiophene tail in the scaffold of acetazolamide on the inhibition of human carbonic anhydrase isoforms I, II, IX and XII.

The high resolution crystal structure of 5-(2-thienylacetamido)-1,3,4-thiadiazole-2-sulfonamide complexed to human (h) carbonic anhydrase (CA, EC 4.2.1.1) isoform hCA II is reported. The compound binds in a similar manner with acetazolamide when the sulfamoyl-thiadiazolyl-acetamido fragment of the two compounds is considered, but the thienyl tail was positioned in the subpocket 2, rarely observed by other investigated CA inhibitors. This positioning allows interaction with amino acid residues (such as Asn67, Ile91, Gln92 and Val121 which are variable in other isoforms of medicinal chemistry interest, such as hCA I, IX and XII. Indeed, the investigated sulfonamide was a medium potency hCA I and II inhibitor but was highly effective as a hCA IX and XII inhibitor. This different behavior with respect to acetazolamide (a promiscuous inhibitor of all these isoforms) has been explained by resolving the crystal structure, and may be used to design more isoform-selective compounds.

Structural study of the location of the phenyl tail of benzene sulfonamides and the effect on human carbonic anhydrase inhibition.

The crystal structure of 4-phenylacetamidomethyl-benzenesulfonamide (4ITP) bound to human carbonic anhydrase (hCA, EC 4.2.1.1) II is reported. 4ITP is a medium potency hCA I and II inhibitor (KIs of 54-75nM), a strong mitochondrial CA VA/VB inhibitor (KIs of 8.3-8.6nM) and a weak transmembrane CA inhibitor (KIs of 136-212nM against hCA IX and XII). This elongated compound binds in an extended conformation to hCA II, with its tail lying towards the hydrophobic half of the active site whereas the sulfonamide moiety coordinates the zinc ion. The present structure was compared to that of structurally related aromatic sulfonamides, such as 4-phenylacetamido-benzene-sulfonamide (3OYS), 4-(2-mercaptophenylacetamido)-benzene-sulfonamide (2HD6) and 4-(3-nitrophenyl)-ureido-benzenesulfonamide (3N2P). Homology models of the hCA I, VA, VB, IX and XII structures were build which afforded an understanding of the amino acids involved in the binding of these compounds to these isoforms. The main conclusion of the study is that the orientation of the tail moiety and the presence of flexible linkers as well polar groups in it, strongly influence the potency and the selectivity of the sulfonamides for the inhibition of cytosolic, mitochondrial or transmembrane CA isoforms.

The malignant brain tumor (MBT) domain protein SFMBT1 is an integral histone reader subunit of the LSD1 demethylase complex for chromatin association and epithelial-to-mesenchymal transition.

Chromatin readers decipher the functional readouts of histone modifications by recruiting specific effector complexes for subsequent epigenetic reprogramming. The LSD1 (also known as KDM1A) histone demethylase complex modifies chromatin and represses transcription in part by catalyzing demethylation of dimethylated histone H3 lysine 4 (H3K4me2), a mark for active transcription. However, none of its currently known subunits recognizes methylated histones. The Snai1 family transcription factors are central drivers of epithelial-to-mesenchymal transition (EMT) by which epithelial cells acquire enhanced invasiveness. Snai1-mediated transcriptional repression of epithelial genes depends on its recruitment of the LSD1 complex and ensuing demethylation of H3K4me2 at its target genes. Through biochemical purification, we identified the MBT domain-containing protein SFMBT1 as a novel component of the LSD1 complex associated with Snai1. Unlike other mammalian MBT domain proteins characterized to date that selectively recognize mono- and dimethylated lysines, SFMBT1 binds di- and trimethyl H3K4, both of which are enriched at active promoters. We show that SFMBT1 is essential for Snai1-dependent recruitment of LSD1 to chromatin, demethylation of H3K4me2, transcriptional repression of epithelial markers, and induction of EMT by TGFß. Carcinogenic metal nickel is a widespread environmental and occupational pollutant. Nickel alters gene expression and induces EMT. We demonstrate the nickel-initiated effects are dependent on LSD1-SFMBT1-mediated chromatin modification. Furthermore, in human cancer, expression of SFMBT1 is associated with mesenchymal markers and unfavorable prognosis. These results highlight a critical role of SFMBT1 in epigenetic regulation, EMT, and cancer.

Structural effect of phenyl ring compared to thiadiazole based adamantyl-sulfonamides on carbonic anhydrase inhibition.

We investigated the inhibitory activity of sulfonamides incorporating adamantyl moieties against the physiologically relevant human (h) CA (EC 4.2.1.1) isoforms hCA I, II III (cytosolic), IX and XII (transmembrane, tumor-associated). The presence of a benzenesulfonamide instead of an 1,3,4-thiadiazole-sulfonamide fragment in the molecule of CA inhibitors (CAIs) drastically affects both inhibition efficacy and binding within the enzyme active site, as rationalized by means of X-ray crystallography of the adduct of hCA II with 4-(1-adamantylcarboxamidomethyl)benzenesulfonamide. Comparing the present X-ray structure with that of the corresponding 1,3,4-thiadiazole-sulfonamide compound possessing the 1-adamantylcarboxamide moiety, important differences of binding emerged, which explain the highly different inhibition profile of the two compounds against the investigated CA isoforms, most of which (CA I, II, IX and XII) are important drug targets.

Preliminary X-ray crystallographic analysis of α-carbonic anhydrase from Thiomicrospira crunogena XCL-2.

Thiomicrospira crunogena XCL-2 is a novel sulfur-oxidizing chemolithoautotroph that plays a significant role in the sustainability of deep-sea hydrothermal vent communities. This recently discovered gammaproteobacterium encodes and expresses four carbonic anhydrases (CAs) from three evolutionarily and structurally distinct CA families: an α-CA, two ß-CAs and a γ-CA. In order to characterize and elucidate the physiological roles of these CAs, X-ray crystallographic structural studies have been initiated on the α-CA. The α-CA crystallized in space group C2. The crystals diffracted to a maximum resolution of 2.6 Å, with unit-cell parameters a = 127.1, b = 102.2, c = 105.0 Å, ß = 127.3°, and a calculated Matthews coefficient of 2.04 Å(3) Da(-1) with four identical protein molecules in the crystallographic asymmetric unit. A preliminary solution was determined by molecular replacement with the PHENIX AutoMR wizard, which had an initial TFZ score of 17.9. Refinement of the structure is currently in progress.

Structurally conserved Nop56/58 N-terminal domain facilitates archaeal box C/D ribonucleoprotein-guided methyltransferase activity.

Box C/D RNA-protein complexes (RNPs) guide the 2'-O-methylation of nucleotides in both archaeal and eukaryotic ribosomal RNAs. The archaeal box C/D and C'/D' RNP subcomplexes are each assembled with three sRNP core proteins. The archaeal Nop56/58 core protein mediates crucial protein-protein interactions required for both sRNP assembly and the methyltransferase reaction by bridging the L7Ae and fibrillarin core proteins. The interaction of Methanocaldococcus jannaschii (Mj) Nop56/58 with the methyltransferase fibrillarin has been investigated using site-directed mutagenesis of specific amino acids in the N-terminal domain of Nop56/58 that interacts with fibrillarin. Extensive mutagenesis revealed an unusually strong Nop56/58-fibrillarin interaction. Only deletion of the NTD itself prevented dimerization with fibrillarin. The extreme stability of the Nop56/58-fibrillarin heterodimer was confirmed in both chemical and thermal denaturation analyses. However, mutations that did not affect Nop56/58 binding to fibrillarin or sRNP assembly nevertheless disrupted sRNP-guided nucleotide modification, revealing a role for Nop56/58 in methyltransferase activity. This conclusion was supported with the cross-linking of Nop56/58 to the target RNA substrate. The Mj Nop56/58 NTD was further characterized by solving its three-dimensional crystal structure to a resolution of 1.7 Å. Despite low primary sequence conservation among the archaeal Nop56/58 homologs, the overall structure of the archaeal NTD domain is very well conserved. In conclusion, the archaeal Nop56/58 NTD exhibits a conserved domain structure whose exceptionally stable interaction with fibrillarin plays a role in both RNP assembly and methyltransferase activity.

Conformational variability of different sulfonamide inhibitors with thienyl-acetamido moieties attributes to differential binding in the active site of cytosolic human carbonic anhydrase isoforms.

The X-ray crystal structures of the adducts of human carbonic anhydrase (hCA, EC 4.2.1.1) II complexed with two aromatic sulfonamides incorporating 2-thienylacetamido moieties are reported here. Although, the two inhibitors only differ by the presence of an additional 3-fluoro substituent on the 4-amino-benzenesulfonamide scaffold, their inhibition profiles against the cytosolic isoforms hCA I, II, III, VII and XIII are quite different. These differences were rationalized based on the obtained X-ray crystal structures, and their comparison with other sulfonamide CA inhibitors with clinical applications, such as acetazolamide, methazolamide and dichlorophenamide. The conformations of the 2-thienylacetamido tails in the hCA II adducts of the two sulfonamides were highly different, although the benzenesulfonamide parts were superimposable. Specific interactions between structurally different inhibitors and amino acid residues present only in some considered isoforms have thus been evidenced. These findings can explain the high affinity of the 2-thienylacetamido benzenesulfonamides for some pharmacologically relevant CAs (i.e., isoforms II and VII) being also useful to design high affinity, more selective sulfonamide inhibitors of various CAs.

Comparative analysis of the 15.5kD box C/D snoRNP core protein in the primitive eukaryote Giardia lamblia reveals unique structural and functional features.

Box C/D ribonucleoproteins (RNP) guide the 2'-O-methylation of targeted nucleotides in archaeal and eukaryotic rRNAs. The archaeal L7Ae and eukaryotic 15.5kD box C/D RNP core protein homologues initiate RNP assembly by recognizing kink-turn (K-turn) motifs. The crystal structure of the 15.5kD core protein from the primitive eukaryote Giardia lamblia is described here to a resolution of 1.8 Å. The Giardia 15.5kD protein exhibits the typical α-ß-α sandwich fold exhibited by both archaeal L7Ae and eukaryotic 15.5kD proteins. Characteristic of eukaryotic homologues, the Giardia 15.5kD protein binds the K-turn motif but not the variant K-loop motif. The highly conserved residues of loop 9, critical for RNA binding, also exhibit conformations similar to those of the human 15.5kD protein when bound to the K-turn motif. However, comparative sequence analysis indicated a distinct evolutionary position between Archaea and Eukarya. Indeed, assessment of the Giardia 15.5kD protein in denaturing experiments demonstrated an intermediate stability in protein structure when compared with that of the eukaryotic mouse 15.5kD and archaeal Methanocaldococcus jannaschii L7Ae proteins. Most notable was the ability of the Giardia 15.5kD protein to assemble in vitro a catalytically active chimeric box C/D RNP utilizing the archaeal M. jannaschii Nop56/58 and fibrillarin core proteins. In contrast, a catalytically competent chimeric RNP could not be assembled using the mouse 15.5kD protein. Collectively, these analyses suggest that the G. lamblia 15.5kD protein occupies a unique position in the evolution of this box C/D RNP core protein retaining structural and functional features characteristic of both archaeal L7Ae and higher eukaryotic 15.5kD homologues.

Signature amino acids enable the archaeal L7Ae box C/D RNP core protein to recognize and bind the K-loop RNA motif.

The archaeal L7Ae and eukaryotic 15.5kD protein homologs are members of the L7Ae/15.5kD protein family that characteristically recognize K-turn motifs found in both archaeal and eukaryotic RNAs. In Archaea, the L7Ae protein uniquely binds the K-loop motif found in box C/D and H/ACA sRNAs, whereas the eukaryotic 15.5kD homolog is unable to recognize this variant K-turn RNA. Comparative sequence and structural analyses, coupled with amino acid replacement experiments, have demonstrated that five amino acids enable the archaeal L7Ae core protein to recognize and bind the K-loop motif. These signature residues are highly conserved in the archaeal L7Ae and eukaryotic 15.5kD homologs, but differ between the two domains of life. Interestingly, loss of K-loop binding by archaeal L7Ae does not disrupt C'/D' RNP formation or RNA-guided nucleotide modification. L7Ae is still incorporated into the C'/D' RNP despite its inability to bind the K-loop, thus indicating the importance of protein-protein interactions for RNP assembly and function. Finally, these five signature amino acids are distinct for each of the L7Ae/L30 family members, suggesting an evolutionary continuum of these RNA-binding proteins for recognition of the various K-turn motifs contained in their cognate RNAs.

Crystal structure of the outer membrane protein OpdK from Pseudomonas aeruginosa.

In Gram-negative bacteria that do not have porins, most water-soluble and small molecules are taken up by substrate-specific channels belonging to the OprD family. We report here the X-ray crystal structure of OpdK, an OprD family member implicated in the uptake of vanillate and related small aromatic acids. The OpdK structure reveals a monomeric, 18-stranded beta barrel with a kidney-shaped central pore. The OpdK pore constriction is relatively wide for a substrate-specific channel (approximately 8 A diameter), and it is lined by a positively charged patch of arginine residues on one side and an electronegative pocket on the opposite side-features likely to be important for substrate selection. Single-channel electrical recordings of OpdK show binding of vanillate to the channel, and they suggest that OpdK forms labile trimers in the outer membrane. Comparison of the OpdK structure with that of Pseudomonas aeruginosa OprD provides the first qualitative insights into the different substrate specificities of these closely related channels.

Structural insight into OprD substrate specificity.

OprD proteins form a large family of substrate-specific outer-membrane channels in Gram-negative bacteria. We report here the X-ray crystal structure of OprD from Pseudomonas aeruginosa, which reveals a monomeric 18-stranded beta-barrel characterized by a very narrow pore constriction, with a positively charged basic ladder on one side and an electronegative pocket on the other side. The location of highly conserved residues in OprD suggests that the structure represents the general architecture of OprD channels.

Unfolding and refolding of leucoagglutinin (PHA-L), an oligomeric lectin from kidney beans (Phaseolus vulgaris).

The unfolding and refolding of Phaseolus vulgaris Leucoagglutinin, a homotetrameric legume lectin, was studied at pH 2.5 and 7.2 using fluorescence, far- and near-UV circular dichroism (CD) spectroscopy, 8-anilino-1-naphthalene sulfonate (ANS) binding and FPLC techniques. This protein was found to refold even at pH 2.5 and also exhibited high refolding yield around 60% at pH 2.5 and 85% at pH 7.2. The refolding at pH 2.5 takes place with the formation of a dimeric intermediate. Although the hydrodynamic radius of the completely renatured protein and the dimer at pH 2.5 was found to be same, the ANS binding as well as far-UV CD spectra of the two were different. The denaturation kinetics at pH 2.5 followed single exponential pattern with the rate of denaturation being independent of protein concentration. The renaturation kinetics on the other hand was dependent on the protein concentration providing further evidence of an intermediate state during refolding. From these experiments the folding pathway of the protein at pH 2.5 was proposed.

The N-terminus of yeast peptide: N-glycanase interacts with the DNA repair protein Rad23.

Yeast peptide: N-glycanase (PNGase) is involved in the proteasomal degradation of misfolded glycoproteins where it interacts with the DNA repair protein Rad23 as first detected in a yeast two-hybrid assay and subsequently confirmed by biochemical in vivo analyses. Limited proteolysis of PNGase with trypsin led to the removal of both an N-terminal and a C-terminal stretch. Based on these truncations the N-terminal region of yeast PNGase was identified as being responsible for binding to Rad23. Secondary structure predictions of this region suggest that it is composed of a single, solvent-exposed alpha-helix. The interaction between PNGase and Rad23 was studied using surface plasmon resonance revealing an equilibrium binding constant of approximately 2.5 microM. The oligomeric nature of Rad23 was also investigated using sedimentation equilibrium analysis. Although Rad23 exists as a dimer in solution, the monomeric form of Rad23 associates with a PNGase monomer in a 1:1 stoichiometric ratio.

Spectroscopic characterization of Phaseolus vulgaris leucoagglutinin.

Phaseolus vulgaris leucoagglutinin is a homotetrameric legume lectin possessing the canonical dimeric structure common to other legume lectins. In order to gain insight into the stability of the protein in an acidic environment, it was characterized by CD and fluorescence studies at pH 2.5. This was then compared with the native protein at physiological pH (7.2). The extinction coefficient of the native protein was calculated to be 3.58x10(4) from its UV absorption spectra. The far- and near-UV CD spectra of the protein at pH 2.5 showed very little difference even though the protein was found to exist as a dimer at pH 2.5. The fluorescence emission maxima of the protein upon excitation at 280 nm were found to shift only from 331 nm at pH 7.2 to 333 nm at pH 2.5. Based on the above observation it was concluded that the protein exhibits extreme pH stability especially in the acidic range. The secondary and tertiary structure of the protein is lost only when it is incubated for two days in 6 M GdnHCl at pH 2.5. At pH 7.2 it could be denatured in 6 M GdnHCl after one week of incubation.

Purification and characterization of a thermostable beta-galactosidase from kidney beans (Phaseolus vulgaris L.) cv. PDR14.

Using five different steps, beta-Galactosidase has been purified from kidney beans to apparent electrophoretic homogeneity with approximately 90-fold purification with a specific activity of 281 units mg-1 protein. A single band was observed in native PAGE. Activity staining of the native gel with 5-bromo 4-chloro 3-indoxyl beta-D-galactopyranoside (X-Gal) at pH 4.0 also produced a single band. Analytical gel filtration in Superdex G-75 revealed the molecular mass of the native protein to be approximately 75 kD. 10% SDS-PAGE under reducing conditions showed two subunits of molecular masses, 45 and 30 kD, respectively. Hence, beta-galactosidase from kidney beans is a heterodimer. A typical protein profile with lambda max at 280 nm was observed and A280/A260 ratio was 1.52. The N-terminal sequence of the 45 kD band showed 86% sequence homology with an Arabidopsis thaliana and 85% with Lycopersicon esculentum putative beta-galactosidase sequences. The Electrospray Mass Spectrometric analysis of this band also revealed a peptide fragment that had 90% sequence homology with an Arabidopsis thaliana putative beta-galactosidase sequence. The N-terminal sequencing of the 30 kD band as well as mass spectrometric analysis both by MALDI-TOF and ES MS revealed certain sequences that matched with phytohemagglutinin of kidney beans. The optimum pH of the enzyme was 4.0 and it hydrolysed o- and p-nitrophenyl beta-D galactopyranoside with a Km value of 0.63 mmol/L and 0.74 mmol/L, respectively. The energy of activation calculated from the Arrhenius equation was 14.8 kcal/mol enzyme site. The enzyme was found to be comparatively thermostable showing maximum activity at 67 degrees C. Thermal denaturation of the enzyme at 65 degrees C obeys single exponential decay with first order-rate constant 0.105 min-1. Galactose, a hydrolytic product of this enzyme was a competitive inhibitor with a Ki of 2.7 mmol/L.

Thermal stability of Phaseolus vulgaris leucoagglutinin: a differential scanning calorimetry study.

Phaseolus vulgaris phytohemagglutinin L is a homotetrameric-leucoagglutinating seed lectin. Its three-dimensional structure shows similarity with other members of the legume lectin family. The tetrameric form of this lectin is pH dependent. Gel filtration results showed that the protein exists in its dimeric state at pH 2.5 and as a tetramer at pH 7.2. Contrary to earlier reports on legume lectins that possess canonical dimers, thermal denaturation studies show that the refolding of phytohemagglutinin L at neutral pH is irreversible. Differential scanning calorimetry (DSC) was used to study the denaturation of this lectin as a function of pH that ranged from 2.0 to 3.0. The lectin was found to be extremely thermostable with a transition temperature around 82 degrees C and above 100 degrees C at pH 2.5 and 7.2, respectively. The ratio of calorimetric to vant Hoff enthalpy could not be calculated because of its irreversible-folding behavior. However, from the DSC data, it was discovered that the protein remains in its compact-folded state, even at pH 2.3, with the onset of denaturation occurring at 60 degrees C.