Cloning of the human α2-macroglobulin gene and detection of mutations in two functional domains: the bait region and the thiolester site

Summary Overlapping genomic clones of the human ₂-macroglobulin (2M) gene were isolated from a cosmid library and were used to map 80 kb of the chromosomal region of this gene. Fragments carrying the two exons encoding the bait region and the exon encoding the thiolester site were partially sequenced and PCR primers were designed for the amplification of both functional domains. By direct genomic sequencing of these domains in 30 healthy individuals and in 30 patients with chronic lung disease three mutations were detected. The first was a sequence polymorphism occurring near the thiolester site of the gene, changing Val¹⁰⁰⁰(GTC) to Ile¹⁰⁰⁰(ATC), with allele frequencies of 0.30 (GTC) and 0.70 (ATC), respectively. No difference of 2M serum levels was observed for these two alleles. The second mutation occured within the thiolester site of one patient, changing Cys⁹⁷²(TGT) to Tyr⁹⁷²(TAT). Since activation of the internal thiolester formed between Cys⁹⁷² and Gln⁹⁷⁵ in each of the subunits of the tetrameric 2M is involved in the covalent cross-linking of the activating proteinase, this mutation is predicted to interfere with 2M function. The 2M serum level was within the normal range in this patient. In one healthy individual we detected an alteration of the bait region sequence, which is usually encoded by two different exons separated by an intron of size 1.6kb. In this individual, PCR amplification of genomic DNA using the bait region primers produced the common fragment of size 1.8 kb and an additional variant fragment of size 0.23kb. This finding, and the genomic sequencing data of this individual, indicate that he carries two different alleles of the 2M gene: one with the regular structure (bait exon I-intron-bait exon II), the other with the two bait exons fused into one. Direct genomic sequencing of the two 2M functional domains is a useful tool for the detection of the genetic, and possibly the functional, heterogeneity of 2M. This, in turn, may provide some insight into the hitherto unknown physiological role(s) of 2M, by studying in vivo effects of naturally ocurring mutations of the gene.     

Structure of APP-C991–99 and implications for role of extra-membrane domains in function and oligomerization

The 99 amino acid C-terminal fragment of Amyloid Precursor Protein APP-C99 (C99) is cleaved by γ-secretase to form Aβ peptide, which plays a critical role in the etiology of Alzheimer's Disease (AD). The structure of C99 consists of a single transmembrane domain flanked by intra and intercellular domains. While the structure of the transmembrane domain has been well characterized, little is known about the structure of the flanking domains and their role in C99 processing by γ-secretase. To gain insight into the structure of full-length C99, REMD simulations were performed for monomeric C99 in model membranes of varying thickness. We find equilibrium ensembles of C99 from simulation agree with experimentally-inferred residue insertion depths and protein backbone chemical shifts. In thin membranes, the transmembrane domain structure is correlated with extra-membrane structural states and the extra-membrane domain structural states become less correlated to each other. Mean and variance of the transmembrane and G₃₇G₃₈ hinge angles are found to increase with thinning membrane. The N-terminus of C99 forms β-strands that may seed aggregation of Aβ on the membrane surface, promoting amyloid formation. In thicker membranes the N-terminus forms α-helices that interact with the nicastrin domain of γ-secretase. The C-terminus of C99 becomes more α-helical as the membrane thickens, forming structures that may be suitable for binding by cytoplasmic proteins, while C-terminal residues essential to cytotoxic function become α-helical as the membrane thins. The heterogeneous but discrete extra-membrane domain states analyzed here open the path to new investigations of the role of C99 structure and membrane in amyloidogenesis. This article is part of a Special Issue entitled: Protein Aggregation and Misfolding at the Cell Membrane Interface edited by Ayyalusamy Ramamoorthy.     

The Neurobiology of LRRK2 and its Role in the Pathogenesis of Parkinson’s Disease

Leucine-rich repeat kinase 2 (LRRK2) is a large, widely expressed protein of largely unknown function. Mutations in the gene encoding LRRK2 have been linked to multiple diseases, including a prominent association with familial and sporadic Parkinson’s disease (PD), as well as inflammatory bowel disorders such as Crohn’s disease. The LRRK2 protein possesses both kinase and GTPase signaling domains, as well as multiple protein interaction domains. Experimental studies in both cellular and in vivo models of mutant LRRK2-induced neurodegeneration have given clues to potential function(s) of LRRK2, yet much remains unknown. For example, while it is known that intact kinase and GTPase activity are required for mutant forms of the protein to trigger cell death, the specific targets of these enzymatic activities that mediate the death of neurons are not known. In this review, we discuss the evidence linking LRRK2 to various cellular/neuronal activities such as extrinsic death and inflammatory signaling, lysosomal protein degradation, the cytoskeletal system and neurite outgrowth, vesicle trafficking, mitochondrial dysfunction, as well as multiple points of interaction with several other genes linked to the pathogenesis of PD. In order for more effective therapeutic strategies to be envisioned and implemented, the mechanisms underlying LRRK2-mediated neurodegeneration need to be better characterized. Furthermore, insights into LRRK2-associated PD pathogenesis can potentially advance our understanding of the more common sporadic forms of PD.     

Expression of full-length and truncated trkB in human striatum and substantia nigra neurons: implications for Parkinson’s disease

Brain derived neurotrophic factor (BDNF) is a potent mediator of cell survival and differentiation and can reverse neuronal injury associated with Parkinson’s disease (PD). Tropomyosin receptor kinase B (trkB) is the high affinity receptor for BDNF. There are two major trkB isoforms, the full-length receptor (trkB.tk⁺) and the truncated receptor (trkB.t1), that mediate the diverse, region specific functions of BDNF. Both trkB isoforms are widely distributed throughout the brain, but the isoform specific distribution of trkB.t1 and trkB.tk⁺ to human neurons is not well characterized. Therefore, we report the regional and neuronal distribution of trkB.tk⁺ and trkB.t1 in the striatum and substantia nigra pars compacta (SNpc) of human autopsy tissues from control and PD cases. In both PD and control tissues, we found abundant, punctate distribution of trkB.tk⁺ and trkB.t1 proteins in striatum and SNpc neurons. In PD, trkB.tk⁺ is decreased in striatal neurites, increased in striatal somata, decreased in SNpc somata and dendrites, and increased in SNpc axons. TrkB.t1 is increased in striatal somata, decreased in striatal axons, and increased in SNpc distal dendrites. We believe changes in trkB isoform distribution and expression levels may be markers of pathology and affect the neuronal response to BDNF.     

Connexin 26 (GJB2) mutations in the Turkish population: implications for the origin and high frequency of the 35delG mutation in Caucasians

Mutations in the Connexin 26 (GJB2/Cx26) gene are responsible for more than half of all cases of prelingual non-syndromic recessive deafness in many Caucasian populations. To determine the importance of Cx26 mutations as a cause of deafness in Turks we screened 11 families with prelingual non-syndromic deafness, seven (64%) of which were found to carry the 35delG mutation. We subsequently screened 674 Turkish subjects with no known hearing loss and found twelve 35delG heterozygotes (1.78%; 95% confidence interval: 0.9%-3%) but no examples of the 167delT mutation. To search for possible founder effects, we typed chromosomes carrying the 35delG mutation for closely linked polymorphic markers in samples from Turkey and United States and compared the allele frequencies with those of hearing subjects. The data showed a modest degree of disequilibrium in both populations. Analyses of two pedigrees from Turkey demonstrated both conserved and different haplotypes, suggesting possible founder effects and multiple origins of the 35delG mutation.     

On the role of excitonic interactions in carotenoid–phthalocyanine dyads and implications for photosynthetic regulation

In two recent studies, energy transfer was reported in certain phthalocyanine–carotenoid dyads between the optically forbidden first excited state of carotenoids (Car S₁) and phthalocyanines (Pcs) in the direction Pc → Car S₁ (Kloz et al., J Am Chem Soc 133:7007–7015, 2011) as well as in the direction Car S₁ → Pc (Liao et al., J Phys Chem A 115:4082–4091, 2011). In this article, we show that the extent of this energy transfer in both directions is closely correlated in these dyads. This correlation and the additional observation that Car S₁ is instantaneously populated after Pc excitation provides evidence that in these compounds excitonic interactions can occur. Besides pure energy transfer and electron transfer, this is the third type of tetrapyrrole–carotenoid interaction that has been shown to occur in these model compounds and that has previously been proposed as a photosynthetic regulation mechanism. We discuss the implications of these models for photosynthetic regulation. The findings are also discussed in the context of a model in which both electronic states are disordered and in which the strength of the electronic coupling determines whether energy transfer, excitonic coupling, or electron transfer occurs.

     

Dissociation between the processivity and total activity of γ-secretase: implications for the mechanism of Alzheimer's disease-causing presenilin mutations

The amyloid β-peptide (Aβ), strongly implicated in the pathogenesis of Alzheimer's disease (AD), is produced from the amyloid β-protein precursor (APP) through consecutive proteolysis by β- and γ-secretases. The latter protease contains presenilin as the catalytic component of a membrane-embedded aspartyl protease complex. Missense mutations in presenilin are associated with early-onset familial AD, and these mutations generally both decrease Aβ production and increase the ratio of the aggregation-prone 42-residue form (Aβ42) to the 40-residue form (Aβ40). The connection between these two effects is not understood. Besides Aβ40 and Aβ42, γ-secretase produces a range of Aβ peptides, the result of initial cutting at the ε site to form Aβ48 or Aβ49 and subsequent trimming every three or four residues. Thus, γ-secretase displays both overall proteolytic activity (ε cutting) and processivity (trimming) toward its substrate APP. Here we tested whether a decrease in total activity correlates with decreased processivity using wild-type and AD-mutant presenilin-containing protease complexes. Changes in pH, temperature, and salt concentration that reduced the overall activity of the wild-type enzyme did not consistently result in increased proportions of longer Aβ peptides. Low salt concentrations and acidic pH were notable exceptions that subtly alter the proportion of individual Aβ peptides, suggesting that the charged state of certain residues may influence processivity. Five different AD mutant complexes, representing a broad range of effects on overall activity, Aβ42:Aβ40 ratios, and ages of disease onset, were also tested, revealing again that changes in total activity and processivity can be dissociated. Factors that control initial proteolysis of APP at the ε site apparently differ significantly from factors affecting subsequent trimming and the distribution of Aβ peptides.     

New mutations in the human p53 gene — a regulator of the cell cycle and carcinogenesis

Mutations in the tumor suppressor gene p53 often lead to disarrangement of the cell cycle and of genetic integrity control of cells that may contribute to tumor development. We studied p53 gene mutations in 26 primary tumors of colorectal cancer patients. Mutations in p53 were found in 17 tumors (65.4%). All point mutations affected the DNA binding domain of p53 and were localized in exons 4-8 of the gene. Mutant p53 isoforms with altered domain structure and/or with alternative C-terminus arising from frameshift mutations or abnormal splicing were found in six tumors. Mutations Leu111Gln and Ser127Phe were shown in colorectal cancer for the first time. Isoforms p53-305 with C(4) insertion in codons 300/301 and p53i9* including an additional 44 nucleotides of the 3 -end of intron 9 were discovered for the first time. Mutations of p53 were associated with lymph node metastases and III/IV stage of tumors that are signs of unfavorable prognosis in colorectal cancer.     

1H, 13C and 15N resonance assignments of the human filamin A tandem immunoglobulin-like domains 16–17 and 18–19

Filamins are large actin-binding and cross-linking proteins which act as linkers between the cytoskeleton and various signaling proteins. Filamin A (FLNa) is the most abundant of the three filamin isoforms found in humans. FLNa contains an N-terminal actin-binding domain and 24 immunoglobulin-like (Ig) domains. The Ig domains are responsible for the FLNa dimerization and most of the interactions that FLNa has with numerous other proteins. There are several crystal and solution structures from isolated single Ig domains of filamins in the PDB database, but only few from longer constructs. Here, we present nearly complete chemical shift assignments of FLNa tandem Ig domains 16–17 and 18–19. Chemical shift mapping between FLNa tandem Ig domain 16–17 and isolated domain 17 suggests a novel domain–domain interaction mode.     

C4 polymorphism in the dog: Molecular heterogeneity of the C4α and C4γ subunit chains

Using an immunoblotting technique and goat antihuman C4, we observed five distinct electrophoretic variants of C4 in a panel of 60 random dogs. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of immunoprecipitated C4 showed that dog C4 is composed of three polypeptide subunit chains (, , and ) and that structural variability occurs within the - and -chain regions. Two distinct molecular weight forms of both the C4- ( _A and _B) and C4-( _A and _B) chain were detected. The variant forms of C4 and C4 were found in association with particular C4 allotypes.     

Conformational States of Trifluoroacetic Acid–Treated Cytochrome c in the Presence of Salts and Alcohols

We have carried out a systematic investigation of salts- and alcohols-induced conformational alterations on the trifluoroacetic acid (TFA)-treated ferricytochrome c by soret absorption spectroscopy, far UV circular dichroism (CD), tryptophan fluorescence, and 1-anilino-8-naphthalene sulfonate (ANS) binding. TFA induces the unfolding of native cytochrome c obtained from horse heart leading to loss of secondary structure. The addition of increasing concentration of salts and alcohols leads to increase in MRE value at 222 and 208 nm indicating an increase in the alpha-helical content leading to formation of compact dimensional structure. Cytochrome c is a heme protein in which the resonance energy of tryptophan is transferred to heme resulting in quenched tryptophan fluorescence. Addition of alcohols leads to increase in tryptophan and ANS fluorescence. The tryptophan and ANS fluorescence in case of salts shows decreased fluorescence intensity. TFA-induced unfolded cytochrome c showed the soret absorption maximum at 394 nm. However, an intermediate state in presence of alcohols and salts showed the absorption maxima at 398 nm and 402 nm, respectively. Among all the salts and alcohols studied, K3Fe(CN)6 and butanol were found to be most effective as examined by the above-mentioned spectroscopic techniques. The order of effectiveness of alcohols was found to be butanol > propanol > ethanol > methanol. The following effective trend in the case of salts was obtained: K3Fe(CN)6 > K2SO4>KClO4 > KCl. These results suggest that alcohols induce an intermediate with molten globule-like conformation on the TFA unfolded state, whereas salts induce a refolded intermediate approaching native-like conformation.     

A review of shark agonistic displays: comparison of display features and implications for shark–human interactions

Agonistic displays in 23 species of sharks of six families are described and illustrated. These displays are reviewed in terms of ethological concepts and shark hydrodynamic models. Shark agonistic displays feature many common elements rendering them readily distinguishable from normal swimming and pseudodisplays caused by sharksucker irritation. Shark agonistic displays are most readily elicited by rapid, direct diver approach when food is absent and potential escape routes restricted. Such displays appear to be motivated by defence of self or the immediately surrounding space rather than defence of territory or resources. Costs and benefits of display versus attack in shark–shark and shark–diver contests are evaluated using payoff matrices and optimal strategies are identified. Shark–human interactions are modelled in terms of a system of nested critical approach distances. For divers faced with a displaying shark, responses which may decrease the likelihood of defensive attack are suggested. Recommendations for future work on shark agonistic behaviour are offered.     

Sequence complementarity between human noncoding RNAs and SARS-CoV-2 genes: What are the implications for human health?

ObjectivesTo investigate in silico the presence of nucleotide sequence complementarity between the RNA genome of Severe Acute Respiratory Syndrome CoronaVirus-2 (SARS-CoV-2) and human non-coding (nc)RNA genes.MethodsThe FASTA sequence (NC_045512.2) of each of the 11 SARS-CoV-2 isolate Wuhan-Hu-1 genes was retrieved from NCBI.nlm.nih.gov/gene and the Ensembl.org library interrogated for any base-pair match with human ncRNA genes. SARS-CoV-2 gene-matched human ncRNAs were screened for functional activity using bioinformatic analysis. Finally, associations between identified ncRNAs and human diseases were searched in GWAS databases.ResultsA total of 252 matches were found between the nucleotide sequence of SARS-CoV-2 genes and human ncRNAs. With the exception of two small nuclear RNAs, all of them were long non-coding (lnc)RNAs expressed mainly in testis and central nervous system under physiological conditions. The percentage of alignment ranged from 91.30% to 100% with a mean nucleotide alignment length of 17.5 ± 2.4. Thirty-three (13.09%) of them contained predicted R-loop forming sequences, but none of these intersected the complementary sequences of SARS-CoV-2. However, in 31 cases matches fell on ncRNA regulatory sites, whose adjacent coding genes are mostly involved in cancer, immunological and neurological pathways. Similarly, several polymorphic variants of detected non-coding genes have been associated with neuropsychiatric and proliferative disorders.ConclusionThis pivotal in silico study shows that SARS-CoV-2 genes have Watson-Crick nucleotide complementarity to human ncRNA sequences, potentially disrupting ncRNA epigenetic control of target genes. It remains to be elucidated whether this could result in the development of human disease in the long term.     

Isolation of the bovine and human genes for Müllerian inhibiting substance and expression of the human gene in animal cells

We have isolated the bovine and human genes for Müllerian inhibiting substance (MIS), a testicular glycoprotein that causes regression of the Müllerian duct during development of the male embryo. The mRNA sequence of bovine MIS, determined from an analysis of cDNA and genomic clones, codes for a protein of 575 amino acids containing a 24 amino acid leader peptide. The human gene has five exons that code for a protein of 560 amino acids. A comparison of the bovine and human MIS proteins reveals a highly conserved C-terminal domain that shows marked homology with human transforming growth factor-beta and the beta chain of porcine inhibin. Animal cells transfected with the human gene secrete biologically active MIS, which causes regression of the rat Müllerian duct in vitro.     

Flexibility and specificity of the cohesin–dockerin interaction: implications for cellulosome assembly and functionality

Cellulosomes are highly elaborate multi-enzyme complexes of Carbohydrate Active enZYmes (CAZYmes) secreted by cellulolytic microorganisms, which very effectively degrade the most abundant polymers on Earth, cellulose and hemicelluloses. Cellulosome assembly requires that a non-catalytic dockerin module found in cellulosomal enzymes binds to one of the various cohesin domains located in a large molecular scaffold called Scaffoldin. A diversity of cohesin–dockerin binding specificities have been described, the combination of which may result in complex plant cell wall degrading systems, maximising the synergy between enzymes in order to improve catalytic efficiency. Structural studies have allowed the spatial flexibility inherent to the cellulosomal system to be determined. Recent progress achieved from the study of the fundamental cohesin and dockerin units involved in cellulosome assembly will be reviewed.     

Functional interactions between the oxytocin receptor and the β2-adrenergic receptor: implications for ERK1/2 activation in human myometrial cells

The Gq-coupled oxytocin receptor (OTR) and the Gs-coupled β₂-adrenergic receptor (β₂AR) are both expressed in myometrial cells and mediate uterine contraction and relaxation, respectively. The two receptors represent important pharmacological targets as OTR antagonists and β₂AR agonists are used to control pre-term uterine contractions. Despite their physiologically antagonistic effects, both receptors activate the MAP kinases ERK1/2, which has been implicated in uterine contraction and the onset of labor. To determine the signalling pathways involved in mediating the ERK1/2 response, we assessed the effect of blockers of specific G protein-associated pathways. In human myometrial hTERT-C3 cells, inhibition of Gαi as well as inhibition of the Gαq/PKC pathway led to a reduction of both OTR- and β₂AR-mediated ERK1/2 activation. The involvement of Gαq/PKC in β₂AR-mediated ERK1/2 induction was unexpected. To test whether the emergence of this novel signalling mechanism was dependent on OTR expression in the same cell, we conducted experiments in HEK 293 cells that were transfected with the β₂AR alone or co-transfected with the OTR. Using this approach, we found that β₂AR-mediated ERK1/2 responses became sensitive to PKC inhibition only in cells co-transfected with the OTR. Inhibitor studies indicated the involvement of an atypical PKC isoform in this process. We confirmed the specific involvement of PKCζ in this pathway by assessing PKCζ translocation to the cell membrane. Consistent with our inhibitor studies, we found that β₂AR-mediated PKCζ translocation was dependent on co-expression of OTR. The present demonstration of a novel β₂AR-coupled signalling pathway that is dependent on OTR co-expression is suggestive of a molecular interaction between the two receptors.