Determination Of Primary Sequence Research Articles

Adapter-mediated Substrate Selection for Endoplasmic Reticulum-associated Degradation

During endoplasmic reticulum (ER)-associated degradation (ERAD), a relatively small number of ubiquitin ligases (E3) must be capable of ubiquitinating an assortment of substrates diverse in both structure and location (ER lumen, membrane, and/or cytosol). Therefore, mechanisms that operate independently of primary sequence determinants must exist to ensure specificity during this process. Here we provide direct evidence for adapter-mediated substrate recruitment for a virus-encoded ERAD E3 ligase, mK3. Members of an ER membrane protein complex that normally functions during major histocompatibility complex class I biogenesis in the immune system are required for mK3 substrate selection. We demonstrate that heterologous substrates could be ubiquitinated by mK3 if they were recruited by these ER accessory molecules to the proper position relative to the ligase domain of mK3. This mechanism of substrate recruitment by adapter proteins may explain the ability of some E3 ligases, including cellular ERAD E3 ligases, to specifically target the ubiquitination of multiple substrates that are unrelated in sequence.

Substrate Requirements for SPPL2b-dependent Regulated Intramembrane Proteolysis

Intramembrane proteolysis is now widely recognized as an important physiological pathway required for reverse signaling and membrane protein degradation. Aspartyl intramembrane cleaving proteases of the GXGD-type play an important regulatory role in health and disease. Besides gamma-secretase/presenilin, signal peptide peptidase (SPP) and SPP-like (SPPL) peptidases also belong to the family of GXGD-type aspartyl proteases. Although recently the first SPPL2a/b substrates have been identified, very little is known about substrate requirements, which allow them to be efficiently processed within the membrane. We demonstrate that similar to gamma-secretase substrates, intramembrane proteolysis of Bri2 (Itm2b) is greatly facilitated by an initial shedding event mediated by ADAM-10. Serial deletions revealed that the length of the ectodomain negatively correlates with efficient intramembrane proteolysis. Bri3 (Itm2c), which is highly homologous to Bri2, fails to be shed. Failure of shedding of Bri3 is accompanied by a lack of intramembrane proteolysis by SPPL2b. Surprisingly, a low molecular weight membrane-retained stub of Bri3 also fails to be processed by SPPL2b, indicating that shedding per se is not sufficient for subsequent intramembrane proteolysis. Extensive domain swapping analysis reveals that primary sequence determinants within the intracellular domain and the transmembrane domain together with short luminal juxtamembrane sequences are required for efficient intramembrane proteolysis.

Structures of Human Arylamine N-Acetyltransferases

A large body of biochemical, kinetic and molecular information, accumulated over the course of more than 80 years, has produced valuable insights into the relationship between the structures and the catalytic functions of the human arylamine N-acetyltransferases NAT1 and NAT2. Much of the groundwork for the determination of human NAT structures and functions was provided by seminal biochemical and enzyme kinetic studies in both human and non-human model systems, the cloning and primary amino acid sequence determination of eukaryotic and prokaryotic NATs, the characterization of naturally occurring and artificially mutated forms of human NATs, elucidation of the crystal structures of several prokaryotic NAT orthologues, and information that has been derived from cross-species comparisons. In 2007 the progress of these studies was aided substantially by the successful crystallization and direct structural analysis of human NAT1 and NAT2. The purpose of this review is to give a brief historical perspective, to summarize our current understanding of human NAT structures and functions based on both earlier and more recent work, and to provide some future insights into the potential applications of this information to the prediction of therapeutic and toxic outcomes associated with the acetylation of primary aromatic amine- and hydrazine-containing chemicals.

Crystal Structure of the Hydroxyquinol 1,2-Dioxygenase from Nocardioides simplex 3E, a Key Enzyme Involved in Polychlorinated Aromatics Biodegradation

Hydroxyquinol 1,2-dioxygenase (1,2-HQD) catalyzes the ring cleavage of hydroxyquinol (1,2,4-trihydroxybenzene), a central intermediate in the degradation of aromatic compounds including a variety of particularly recalcitrant polychloro- and nitroaromatic pollutants. We report here the primary sequence determination and the analysis of the crystal structure of the 1,2-HQD from Nocardioides simplex 3E solved at 1.75 A resolution using the multiple wavelength anomalous dispersion of the two catalytic irons (1 Fe/293 amino acids). The catalytic Fe(III) coordination polyhedron composed by the side chains of Tyr164, Tyr197, His221, and His223 resembles that of the other known intradiol-cleaving dioxygenases, but several of the tertiary structure features are notably different. One of the most distinctive characteristics of the present structure is the extensive openings and consequent exposure to solvent of the upper part of the catalytic cavity arranged to favor the binding of hydroxyquinols but not catechols. A co-crystallized benzoate-like molecule is also found bound to the metal center forming a distinctive hydrogen bond network as observed previously also in 4-chlorocatechol 1,2-dioxygenase from Rhodococcus opacus 1CP. This is the first structure of an intradiol dioxygenase specialized in hydroxyquinol ring cleavage to be investigated in detail.

Retraction: A proteomic approach for investigation of photosynthetic apparatus in plants

The proteome of the photosynthetic apparatus of barley (Hordeum vulgare), obtained by analysis of thylakoids without any previous fractionation, was mapped by native electrophoresis followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) as the second dimension two-dimensional-blue native (2-D/BN)/SDS-PAGE). This protocol provided an excellent alternative to the 2-D-isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis for 2-D separation of the most hydrophobic thylakoid proteins. Monocots and dicots showed significant differences in the first dimension while in the second dimension patterns appeared similar. Identification of each spot was performed by internal peptide primary sequence determination using both nano-electrospray ionization tandem mass spectrometry and, to a lesser extent, peptide mass fingerprinting matrix-assisted laser desorption/ionization-time of flight using MALDI-TOF. This is due in particular to the fact that a limited number of peptides was obtained after trypsin digestion of these highly hydrophobic proteins. A larger number of peptides from hydrophilic intermembrane domains of transmembrane proteins were detected. Despite this, about 70% of the expected proteins were identified, including proteins with grand average of hydropathicity scores higher than 0.5. It is therefore reasonable to assert that protein hydrophobicity is not the limiting factor. Small proteins were not well identified with trypsin digestion. Instead some of these could be identified using acid hydrolysis. The method presented here does not require prefractionation of different thylakoid complexes and consequently gives confidence in comparing the proteome of the photosynthetic apparatus before and after treatment. It thus allows us to understand the molecular mechanisms underlying physiological adaptations of higher plants and to perform screening of photosynthetic mutants.

Primary sequence determinants responsible for site-selective dephosphorylation of the PDGF β-receptor by the receptor-like protein tyrosine phosphatase DEP-1

Site-selective dephosphorylation of receptor tyrosine kinases contributes to receptor regulation. The receptor-like protein tyrosine phosphatase DEP-1 site-selectively dephosphorylates the PDGF β-receptor. DEP-1 dephosphorylation of original and chimeric phospho-peptides spanning the preferred pY1021 and the less preferred pY857 and pY562 sites was analyzed. Double substitutions of basic residues at −4 and +3 of pY857 and pY562 peptides improved affinity. Substitutions of single amino acids indicated preference for an acidic residue at position −1 and a preference against a basic residue at position +3. DEP-1 site-selective dephosphorylation of PDGF β-receptor is thus determined by the primary sequence surrounding phosphorylation sites and involves interactions with residues spanning at least between positions −1 and +3.

Intensely punctate meiotic recombination in the class II region of the major histocompatibility complex.

There is considerable interest in understanding patterns of linkage disequilibrium (LD) in the human genome, to aid investigations of human evolution and facilitate association studies in complex disease. The relative influences of meiotic crossover distribution and population history on LD remain unclear, however. In particular, it is uncertain to what extent crossovers are clustered into 'hot spots, that might influence LD patterns. As a first step to investigating the relationship between LD and recombination, we have analyzed a 216-kb segment of the class II region of the major histocompatibility complex (MHC) already characterized for familial crossovers. High-resolution LD analysis shows the existence of extended domains of strong association interrupted by patchwork areas of LD breakdown. Sperm typing shows that these areas correspond precisely to meiotic crossover hot spots. All six hot spots defined share a remarkably similar symmetrical morphology but vary considerably in intensity, and are not obviously associated with any primary DNA sequence determinants of hot-spot activity. These hot spots occur in clusters and together account for almost all crossovers in this region of the MHC. These data show that, within the MHC at least, crossovers are far from randomly distributed at the molecular level and that recombination hot spots can profoundly affect LD patterns.

Sequence determination and resonance assignments of an Azomonas siderophore using 13C natural abundance 13C–1H HNCA experiment

The determination of the amino acid sequence of small peptides as siderophores is commonly performed using long-range HMBC correlation experiments. However, this type of experiment can lead to ambiguities in the assignment resulting in erroneous primary sequence determination. In this paper, we propose to use an HNCA type experiment on a 15 N-labelled sample in order to avoid these ambiguities. This experiment was successfully used to determine the primary sequence of azoverdin, a siderophore produced by Azomonas . La détermination de séquences primaires de petits peptides comme les sidérophores bactériens s’appuie en général sur des spectres RMN de corrélation carbone–proton à longue distance de type HMBC. Cependant, ce type d’expérience ne permet pas de résoudre toutes les ambiguïtés d’attribution et peut conduire à des erreurs de séquence primaire. Dans cet article, nous proposons d’utiliser une expérience de type HNCA sur un échantillon enrichi à l’azote 15 afin de lever ces ambiguïtés. Cette expérience a été appliquée avec succès pour déterminer la séquence primaire de l’azoverdine, un sidérophore d’ Azomonas .

Identification of Sequence Determinants That Direct Different Intracellular Folding Pathways for Aquaporin-1 and Aquaporin-4

Homologous aquaporin water channels utilize different folding pathways to acquire their transmembrane (TM) topology in the endoplasmic reticulum (ER). AQP4 acquires each of its six TM segments via cotranslational translocation events, whereas AQP1 is initially synthesized with four TM segments and subsequently converted into a six membrane-spanning topology. To identify sequence determinants responsible for these pathways, peptide segments from AQP1 and AQP4 were systematically exchanged. Chimeric proteins were then truncated, fused to a C-terminal translocation reporter, and topology was analyzed by protease accessibility. In each chimeric context, TM1 initiated ER targeting and translocation. However, AQP4-TM2 cotranslationally terminated translocation, while AQP1-TM2 failed to terminate translocation and passed into the ER lumen. This difference in stop transfer activity was due to two residues that altered both the length and hydrophobicity of TM2 (Asn(49) and Lys(51) in AQP1 versus Met(48) and Leu(50) in AQP4). A second peptide region was identified within the TM3-4 peptide loop that enabled AQP4-TM3 but not AQP1-TM3 to reinitiate translocation and cotranslationally span the membrane. Based on these findings, it was possible to convert AQP1 into a cotranslational biogenesis mode similar to that of AQP4 by substituting just two peptide regions at the N terminus of TM2 and the C terminus of TM3. Interestingly, each of these substitutions disrupted water channel activity. These data thus establish the structural basis for different AQP folding pathways and provide evidence that variations in cotranslational folding enable polytopic proteins to acquire and/or maintain primary sequence determinants necessary for function.

Ligation-mediated PCR for quantitative in vivo footprinting.

Ligation-mediated polymerase chain reaction (LM-PCR) is a genomic analysis technique for determination of (1) primary DNA nucleotide sequences (2) cytosine methylation patterns (3) DNA lesion formation and repair, and (4) in vivo protein-DNA footprints. However, LM-PCR can be limited by the multiple steps required and the relatively short stretch of sequence (usually <200 bp) that can be analyzed per reaction. We report here a simplified, one-day LM-PCR protocol in which all pipetting steps can be performed by a robotic workstation and which, moreover, provides longer reads (>350 bp) and enhanced signal quality by use of nonradioactive detection and a LI-COR DNA sequencing instrument. Sensitivity comparable to radiolabeling is achieved using oligonucleotide primers that are 5'-end labeled with infrared fluorochromes. We showed that the technique could be used for sensitive and reproducible in vivo photofootprinting of the human phosphoglycerate kinase 1 (PGK1) promoter, as well as providing good Maxam-Gilbert sequence information. The methods described here should allow high-throughput, high-resolution analysis of transcription factor binding and chromatin structure, and also may be useful for sequencing gaps that are refractory to cloning.

Nine-haem cytochrome c from Desulfovibrio desulfuricans ATCC 27774:primary sequence determination, crystallographic refinement at 1.8 and modelling studies of its interaction with the tetrahaem cytochrome c3.

A monomeric nine-haem cytochrome c (9Hcc) with 292 amino acid residues was isolated from cells of the sulfate- and nitrate-reducing bacterium Desulfovibrio desulfuricans ATCC 27774 grown under both nitrate- and sulfate-respiring conditions. The nucleotide sequence encoding the 292 residues was determined, allowing the correction of about 10% of the previous primary structure, determined from 1.8 A electron density maps. The refinement at 1.8 A resolution of the structural model was completed, giving an R-value of 16.5%. The nine haem groups are arranged into two tetrahaem clusters, located at both ends of the molecule, with Fe-Fe distances and local protein fold very similar to tetrahaem cytochromes c3, and the extra haem is located asymmetrically between the two regions. The new primary sequence determination confirmed the 39% sequence homology found between this cytochrome and the C-terminal region (residues 229-514) of the high-molecular-weight cytochrome c (Hmc) from D. vulgaris Hildenborough, providing strong evidence of structural similarity between 9Hcc and the C-terminal region of Hmc. The interaction between 9Hcc and the tetrahaem cytochrome c3 from the same organism was studied by modelling methods, and the results suggest that a specific interaction is possible between haem 4 of tetrahaem cytochrome c3 and haem 1 or haem 2 of 9Hcc, in agreement with previous kinetic experiments which showed the catalytic effect of the tetrahaem cytochrome c3 upon the reduction of 9Hcc by the [NiFe] hydrogenase from D. desulfuricans ATCC 27774. These studies suggest a role for 9Hcc as part of the assembly of redox proteins involved in recycling the molecular hydrogen released by the cell as a result of substrate oxidation.

Determination of Primary Amino Acid Sequence and Unique Three-Dimensional Structure of WGH1, a Monoclonal Human IgM Antibody with Anti-PR3 Specificity

Transformed B cells making monoclonal IgM-λ anti-PR3 antibody WGH1 from a patient with Wegener's granulomatosis were used to prepare mRNA and synthesize cDNA. PCR primers for human μ and λ chains were then employed to amplify heavy- and light-chain V-regions followed by cloning into pCR2-1 vector and sequencing. Molecular modeling of VH regions employed knowledge-based homology modeling to obtain minimum energy conformation. The VH sequence was subgroup III with marked overall homology to VH1.9III. The VHCDR3 region of WGH1 was unique, consisting of 21 amino acid residues which included seven tyrosines as well as three negatively charged aspartic acid residues. The VL region was subgroup II with a negatively charged glutamic acid at position 100 in CDR3. Molecular modeling of VH revealed a major conformational difference in the shape of CDR3 compared with other antibodies for which three-dimensional structures have been determined. Monoclonal antibody WGH1 reacting with PR3 (a highly positively charged molecule) shows a unique reactive cassette within VHCDR3 with a number of negatively charged aspartic acid residues. WGH1 VHCDR3 contains a loop which shows a major projection not usually recorded in other previously studied antibody molecules.

Primary Structure and Function of an A Kinase Anchoring Protein Associated with Calcium Channels

Rapid, voltage-dependent potentiation of skeletal muscle L-type calcium channels requires phosphorylation by cAMP-dependent protein kinase (PKA) anchored via an A kinase anchoring protein (AKAP). Here we report the isolation, primary sequence determination, and functional characterization of AKAP15, a lipid-anchored protein of 81 amino acid residues with a single amphipathic helix that binds PKA. AKAP15 colocalizes with L-type calcium channels in transverse tubules and is associated with L-type calcium channels in transfected cells. A peptide fragment of AKAP15 encompassing the RII-binding domain blocks voltage-dependent potentiation. These results indicate that AKAP15 targets PKA to the calcium channel and plays a critical role in voltage-dependent potentiation and regulation of skeletal muscle contraction. The expression of AKAP15 in the brain and heart suggests that it may mediate rapid PKA regulation of L-type calcium channels in neurons and cardiac myocytes.

Capillary electrophoresis in biochemical and clinical laboratories: Selected attractive examples

As demonstrated by selected examples from our laboratories, CE is a unique methodology for purity control of synthetic as well as natural tissue-isolated biopolymers, a prerequisite before reliable biotestings should be performed. A combination of rapid matrix-assisted laser desorption ionization mass and CE electrophoretic mobility determinations facilitates primary sequence determinations of enzymatic peptide digest mixtures often making costly Edman degradations unnecessary. The enormous separation efficiency and large variety of different possible separation modes in CE, allow detection of single components in complex mixtures which is demonstrated by the apolipoprotein A-I determination in human blood serum in this communication.

Isolation, Primary Sequence Determination, and Disulfide Bond Structure of Cyanovirin-N, an Anti-HIV(Human Immunodeficiency Virus) Protein from the CyanobacteriumNostoc ellipsosporum

A novel anti-HIV protein, cyanovirin-N (CV-N), was isolated from an aqueous cellular extract of the cultured cyanobacterium (blue-green alga)Nostoc ellipsosporum,purified by reverse-phase HPLC, and sequenced by N-terminal Edman degradation of the intact protein and peptide fragments produced by endoproteinase digestions. CV-N consists of a single 101 amino acid chain which exhibits significant internal sequence duplication, but no significant homology to previously described proteins or to the transcription products of known nucleotide sequences. Alignment of residues 1-50 with residues 51-101 reveals 13 conservative amino acid changes as well as direct homology between 16 amino acid residues. CV-N contains four cysteines which form two intrachain disulfide bonds. The positions of the disulfide linkages were established by fast atom bombardment mass spectral studies of peptide fragments generated by a tryptic digestion of the native protein. Reductive cleavage of these crosslinks resulted in loss of anti-HIV activity.

A Predictive Scale for Evaluating Cyclin-dependent Kinase Substrates: A COMPARISON OF p34cdc2 AND p33cdk2

Protein phosphorylation by members of the Cdk (cyclin-dependent kinase) family of protein kinases is necessary for progression through the cell cycle. However, the primary sequence determinants of Cdk substrate specificity have yet to be examined quantitatively. We have used a panel of glutathione S-transferase peptide fusions to investigate the fine-structure specificity of p33(cdk2) and p34(cdc2). Our data indicate that the generally held consensus sequences for p34(cdc2) represent a significant oversimplification of its true specificity and that this specificity is conserved between species. p33(cdk2) and p34(cdc2) have similar but distinct substrate specificities that are affected modestly by the associated cyclin subunit. We derive specific values of phosphorylation efficiencies by these enzymes that can be used to estimate the phosphorylation potential of proposed Cdk substrates.

Structure-infectivity analysis of the human rhinovirus genomic RNA 3' non-coding region.

The specific recognition of genomic positive strand RNAS as templates for the synthesis of intermediate negative strands by the picornavirus replication machinery is presumably mediated by cis-acting sequences within the genomic RNA 3' non-coding region (NCR). A structure-infectivity analysis was conducted on the 44 nt human rhinovirus 14 (HRV14) 3' NCR to identify the primary sequence and/or secondary structure determinants required for viral replication. Using biochemical RNA secondary structure probing techniques, we have demonstrated the existence of a single stem-loop structure contained entirely within the 3' NCR, which appears to be phylogenetically conserved within the rhinovirus genus. We also report the in vivo analysis of a number of 3' NCR deletion mutations engineered into infectious cDNA clones which were designed to disrupt the stem-loop secondary structure to varying degrees. Large deletions (up to 37 nt) resulted in defective growth phenotypes, although they were not lethal. We propose that the absolute requirements for initiation of negative strand synthesis are less stringent than previously postulated, even though defined RNA secondary structure determinants may have evolved to facilitate and/or regulate the process of viral RNA replication.

Distinct cis-acting elements mediate clock, light, and developmental regulation of the Neurospora crassa eas (ccg-2) gene.

The Neurospora crassa eas (ccg-2) gene, which encodes a fungal hydrophobin, is transcriptionally regulated by the circadian clock. In addition, eas (ccg-2) is positively regulated by light and transcripts accumulate during asexual development. To sort out the basis of this complex regulation, deletion analyses of the eas (ccg-2) promoter were carried out to localize the cis-acting elements mediating clock, light, and developmental control. The primary sequence determinants of a positive activating clock element (ACE) were found to reside in a 45-bp region, just upstream from the TATA box. Using a novel unregulated promoter/reporter system developed for this study, we show that a 68-bp sequence encompassing the ACE is sufficient to confer clock regulation on the eas (ccg-2) gene. Electrophoretic mobility shift assays using the ACE reveal factors present in N. crassa protein extracts that recognize and bind specifically to DNA containing this element. Separate regions of the eas (ccg-2) promoter involved in light induction and developmental control are identified and shown not to be required for clock-regulated expression of eas (ccg-2). The distinct nature of the ACE validates its use as a tool for the identification of upstream regulatory factors involved in clock control of gene expression.

Primary sequence determination and molecular modelling of the variable region of an antiMUC1 mucin monoclonal antibody.

Polymerase chain reaction (PCR) products representative of the DNA sequence coding for the variable heavy (VH) and the variable light (VL) chains of an antiMUC1 mucin monoclonal antibody, C595, have been produced. These products were cloned, sequenced, and the primary amino acid sequences of the VH and VL regions deduced. The hypervariable complementarity determining regions (CDRs) and framework regions in the heavy and light chains were located, and homologies with canonical forms for the CDR loops L1, L2, L3, H1 and H2 were identified by database searching. The structure for the H3 loop was calculated directly. Computational molecular modelling was accomplished using the fully automated AbM package (Oxford Molecular, Oxford, U.K.). Energy minimisation was performed using the program InsightII (Biosym, San Diego, California, U.S.A.). The investigation provides a basis for the molecular analysis of the antigen binding site of the C595 antibody with the aim to identify key residues and interactions involved in the immune recognition of the C595 antibody defined epitope, which is expressed in the majority of breast and ovarian carcinomas.

Primary sequence determination of the most basic myonecrotic phospholipase A 2 from the venom of Vipera russelli

The most basic phospholipase A 2 (PLA 2), VRV-PL-VIIIa, was purified from (Sri Lankan) Vipera russelli venom. It is a major component of the venom, contributing over 40% to the whole venom PLA 2 activity. The purity of VRV-PL-VIIIa was ascertained by electrophoresis and by reverse phase high-pressure liquid-chromatography (RP-HPLC). VRV-PL-VIIIa had an apparent mol. wt of 13,000 and was a single polypeptide. The protein was reduced, pyridylethylated and subjected to sequence analysis. The N-terminal amino acid sequence was established up to the 39th residue. Pyridylethylated VRV-PL-VIIIa was digested with endoprotease Glu-C, and several peptides were purified by RP-HPLC; six purified peptides were sequenced. The sequence of the C-terminal was established by sequencing a CNBr-produced peptide purified by RP-HPLC. Several peptides were also generated by digestion with endoprotease Asp-N. Two peptides were sequenced to obtain overlapping regions. The complete structure was deduced from sequences of overlapping peptides and through homology with other group II PLA 2 sequences. Sequence homology was greatest with ammodytoxin A: 99 amino acid residues out of 121 occurred in identical positions. Myotoxin III of Bothrops asper showed 73% homology, 89 out of 121 residues. In agreement with the sequence data, polyclonal antiserum against VRV-PL-VIIIa cross-reacted in ELISA with ammodytoxin A and, to a lesser extent, with caudoxin.