Publications by authors named "Kamil Khafizov"

29 Publications

  • Page 1 of 1

Association of , and Genes Polymorphisms With the Calcium Urolithiasis Development in Russian Population.

Front Genet 2021 12;12:621049. Epub 2021 May 12.

Department of Medical Genetics, Ministry of Public Health of the Russian Federation, I. M. Sechenov First Moscow State Medical University, Sechenov University, Moscow, Russia.

Kidney stone disease is an urgent medical and social problem. Genetic factors play an important role in the disease development. This study aims to establish an association between polymorphisms in genes coding for proteins involved in calcium metabolism and the development of calcium urolithiasis in Russian population. In this case-control study, we investigated 50 patients with calcium urolithiasis (experimental group) and 50 persons lacking signs of kidney stone disease (control group). For molecular genetic analysis we used a previously developed gene panel consisting of 33 polymorphisms in 15 genes involved in calcium metabolism: , , , , , , , , , , , , , , and High-throughput target sequencing was utilized to study the loci of interest. Odds ratios and 95% confidence intervals were used to estimate the association between each SNP and risk of urolithiasis development. Multifactor dimensionality reduction analysis was also carried out to analyze the gene-gene interaction. We found statistically significant (unadjusted -value < 0.05) associations between calcium urolithiasis and the polymorphisms in the following genes: rs1042636 (OR = 3.18 for allele A), rs1801197 (OR = 6.84 for allele A), and rs6486795 (OR = 2.25 for allele C). The maximum OR was shown for AA genotypes in loci rs1042636 () and rs1801197 () (OR = 4.71, OR = 11.8, respectively). After adjustment by Benjamini-Hochberg FDR we found only (rs1801197) was significantly associated with the risk of calcium urolithiasis development. There was no relationship between recurrent course of the disease and family history of urolithiasis in investigated patients. Thus we found a statistically significant association of polymorphism rs1801197 (gene ) with calcium urolithiasis in Russian population.
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http://dx.doi.org/10.3389/fgene.2021.621049DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8153711PMC
May 2021

Refining pairwise sequence alignments of membrane proteins by the incorporation of anchors.

PLoS One 2021 30;16(4):e0239881. Epub 2021 Apr 30.

Computational Structural Biology Section, National Institutes of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD, United States of America.

The alignment of primary sequences is a fundamental step in the analysis of protein structure, function, and evolution, and in the generation of homology-based models. Integral membrane proteins pose a significant challenge for such sequence alignment approaches, because their evolutionary relationships can be very remote, and because a high content of hydrophobic amino acids reduces their complexity. Frequently, biochemical or biophysical data is available that informs the optimum alignment, for example, indicating specific positions that share common functional or structural roles. Currently, if those positions are not correctly matched by a standard pairwise sequence alignment procedure, the incorporation of such information into the alignment is typically addressed in an ad hoc manner, with manual adjustments. However, such modifications are problematic because they reduce the robustness and reproducibility of the aligned regions either side of the newly matched positions. Previous studies have introduced restraints as a means to impose the matching of positions during sequence alignments, originally in the context of genome assembly. Here we introduce position restraints, or "anchors" as a feature in our alignment tool AlignMe, providing an aid to pairwise global sequence alignment of alpha-helical membrane proteins. Applying this approach to realistic scenarios involving distantly-related and low complexity sequences, we illustrate how the addition of anchors can be used to modify alignments, while still maintaining the reproducibility and rigor of the rest of the alignment. Anchored alignments can be generated using the online version of AlignMe available at www.bioinfo.mpg.de/AlignMe/.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0239881PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8087094PMC
September 2021

Ngari virus (Orthobunyavirus, Peribunyaviridae) in ixodid ticks collected from cattle in Guinea.

Acta Trop 2021 Feb 9;214:105790. Epub 2020 Dec 9.

Central Research Institute of Epidemiology, 111123, Novogireevskaya st. 3A, Moscow, Russia.

Ngari virus is a mosquito-borne virus belonging to the genus Orthobunyavirus (Peribunyaviridae family). This virus is pathogenic to humans and causes severe illness. Ngari virus is present in several African countries, including Madagascar. Here, we report the detection of Ngari virus in ixodid ticks collected from cows in Guinea. A tick survey was conducted in March-November of 2018 in six regions of Guinea. The sample comprised 710 pools, with a total of 2067 ticks belonging to five species collected from 197 cows. At the initial stage, we screened a subsample of tick pools of vector-borne viruses with a multiplex genus-specific primer panel. In the second stage of the study, we narrowed the search and screened all the samples by qPCR for the detection of Ngari virus. All positive samples were sequenced with primers flanking Ngari virus-specific fragments on the S and M segments. We found Ngari virus in 12 pools that were formed from engorged ticks collected from livestock in three villages of the Kindia and Kankan regions. Sequencing of the S and M segments confirmed that the detected viruses belong to Ngari virus, and the viruses were most similar to the strain Adrar, which was isolated in Mauritania. We detected viral RNA in ticks of the following species: Amblyomma variegatum, Rhipicephalus geigyi, and Rh. (Boophilus) spp. There is no evidence that ixodid ticks are competent vectors of the Ngari virus. Most likely, the ticks obtained the virus through blood from an infected host. The study of engorged ticks can be recommended as a simpler approach for the wide screening of the Ngari virus and subsequent testing of cattle and mosquitos in those locations where the PCR-positive ticks were collected.
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http://dx.doi.org/10.1016/j.actatropica.2020.105790DOI Listing
February 2021

Isolation and characterization of Wad Medani virus obtained in the tuva Republic of Russia.

Ticks Tick Borne Dis 2021 03 25;12(2):101612. Epub 2020 Nov 25.

Chumakov Institute of Poliomyelitis and Viral Encephalitides FSBSI Chumakov FSC R&D IBP RAS, Moscow, Russia; Institute for Translational Medicine and Biotechnology, Sechenov First Moscow State Medical University, Moscow, Russia.

Wad Medani virus (WMV) belongs to the genus Orbivirus and is a poorly studied arbovirus with unclear medical significance. Presently, a limited number of WMV strains are characterized and available in NCBI GenBank, some isolated many years ago. A new WMV strain was isolated in 2012 from Dermacentor nuttalli ticks collected from sheep in the Tuva Republic, Russia, and sequenced using high-throughput methods. Complete coding sequences were obtained revealing signs of multiple intersegment reassortments. These point to a high variability potential in WMV that may lead to the formation of strains with novel properties. These new data on WMV can promote better understanding of: ecological features of its circulation; relationships within the genus Orbivirus; and the medical significance of the virus.
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http://dx.doi.org/10.1016/j.ttbdis.2020.101612DOI Listing
March 2021

Lethal Outcome of Leptospirosis in Southern Russia: Characterization of Isolated from a Deсeased Teenager.

Int J Environ Res Public Health 2020 06 14;17(12). Epub 2020 Jun 14.

Pasteur Institute, Federal Service on Consumers' Rights Protection and Human Well-Being Surveillance, 197101 Saint-Petersburg, Russia.

This article describes a lethal case of leptospirosis that occurred in Southern Russia. The strain was isolated and characterized using a microscopic agglutination test, MALDI-TOF mass spectrometry, targeted PCR, and high-throughput sequencing. We show that molecular and mass-spectrometry methods can be an alternative to conventional methods of leptospirosis diagnostics and study, which require highly qualified staff and can be performed only at specialized laboratories. We also report the first whole genome of . isolated in Russia.
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http://dx.doi.org/10.3390/ijerph17124238DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7344687PMC
June 2020

Current Trends in Diagnostics of Viral Infections of Unknown Etiology.

Viruses 2020 02 14;12(2). Epub 2020 Feb 14.

FSBI "Center of Strategic Planning" of the Ministry of Health, 119435 Moscow, Russia.

Viruses are evolving at an alarming rate, spreading and inconspicuously adapting to cutting-edge therapies. Therefore, the search for rapid, informative and reliable diagnostic methods is becoming urgent as ever. Conventional clinical tests (PCR, serology, etc.) are being continually optimized, yet provide very limited data. Could high throughput sequencing (HTS) become the future gold standard in molecular diagnostics of viral infections? Compared to conventional clinical tests, HTS is universal and more precise at profiling pathogens. Nevertheless, it has not yet been widely accepted as a diagnostic tool, owing primarily to its high cost and the complexity of sample preparation and data analysis. Those obstacles must be tackled to integrate HTS into daily clinical practice. For this, three objectives are to be achieved: (1) designing and assessing universal protocols for library preparation, (2) assembling purpose-specific pipelines, and (3) building computational infrastructure to suit the needs and financial abilities of modern healthcare centers. Data harvested with HTS could not only augment diagnostics and help to choose the correct therapy, but also facilitate research in epidemiology, genetics and virology. This information, in turn, could significantly aid clinicians in battling viral infections.
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http://dx.doi.org/10.3390/v12020211DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7077230PMC
February 2020

Genetic diversity of Kemerovo virus and phylogenetic relationships within the Great Island virus genetic group.

Ticks Tick Borne Dis 2020 03 17;11(2):101333. Epub 2019 Nov 17.

Martsinovsky Institute of Medical Parasitology, Tropical and Vector Borne Diseases, Sechenov First Moscow State Medical University, Moscow, Russia; Saint-Petersburg Pasteur Institute, Federal Service on Consumers' Rights Protection and Human Well-Being Surveillance, Saint-Petersburg, Russia.

Kemerovo virus (KEMV) is a member of the Great Island virus genetic group, belonging to the tick-borne arboviruses of the genus Orbivirus within the family Reoviridae. Nine strains of KEMV, which were isolated from various locations in Russia, were sequenced by high-throughput sequencing to study their intraspecific diversity and the interspecific relationships of viruses within the Great Island genetic group. For the first time, multiple reassortment within KEMV was reliably demonstrated. Different types of independently emerged alternative reading frames in segment 9 and heterogeneity of the viral population in one of the KEMV strains were found. The hypothesis of the role of an alternative open reading frame (ORF) in segment 9 in KEMV cellular tropism was not confirmed in this study.
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http://dx.doi.org/10.1016/j.ttbdis.2019.101333DOI Listing
March 2020

Improved Protocols of ITS1-Based Metabarcoding and Their Application in the Analysis of Plant-Containing Products.

Genes (Basel) 2019 02 7;10(2). Epub 2019 Feb 7.

Skolkovo Institute of Science and Technology, Nobel St. 3, Moscow 143026, Russia.

Plants are widely used for food and beverage preparation, most often in the form of complex mixtures of dried and ground parts, such as teas, spices or herbal medicines. Quality control of such products is important due to the potential health risks from the presence of unlabelled components or absence of claimed ones. A promising approach to analyse such products is DNA metabarcoding due to its high resolution and sensitivity. However, this method's application in food analysis requires several methodology optimizations in DNA extraction, amplification and library preparation. In this study, we present such optimizations. The most important methodological outcomes are the following: 1) the DNA extraction method greatly influences amplification success; 2) the main problem for the application of metabarcoding is DNA purity, not integrity or quantity; and 3) the "non-amplifiable" samples can be amplified with polymerases resistant to inhibitors. Using this optimized workflow, we analysed a broad set of plant products (teas, spices and herbal remedies) using two NGS platforms. The analysis revealed the problem of both the presence of extraneous components and the absence of labelled ones. Notably, for teas, no correlation was found between the price and either the absence of labelled components or presence of unlabelled ones; for spices, a negative correlation was found between the price and presence of unlabelled components.
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http://dx.doi.org/10.3390/genes10020122DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6409534PMC
February 2019

Sequencing and genetic characterization of two strains Paramushir virus obtained from the Tyuleniy Island in the Okhotsk Sea (2015).

Ticks Tick Borne Dis 2019 02 12;10(2):269-279. Epub 2018 Nov 12.

Saint-Petersburg Pasteur Institute, Center, Federal Service on Consumers' Rights Protection and Human Well-Being Surveillance, Saint-Petersburg, Russia; Martsinovsky Institute of Medical Parasitology, Tropical and Vector Borne Diseases, Sechenov First Moscow State Medical University, Moscow, Russia. Electronic address:

Paramushir virus belongs to Sakhalin virus genogroup within Orthonairovirus genus and is one of the poorly studied viruses with unknown pathogenicity. At the moment, only one nearly complete sequence of Paramushir virus genome, isolated in 1972, is available. Two new strains of PARV were isolated in 2015 from a sample collected at the Tyuleniy Island in the Okhotsk Sea and sequenced using a combination of high throughput sequencing and specific multiplex PCR. Both strains are closely related to the early sequenced PARV strain LEIV-1149 K. The signs of intersegment reassortment and probable recombination were revealed, which point to a high variability potential of Paramushir virus and may lead to the formation of strains with novel properties, different from those of the predecessors. The new data regarding Paramushir virus can promote a better understanding of the diversity and relations within Orthonairovirus genus and help define intragenic demarcation criteria, which have not yet been established.
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http://dx.doi.org/10.1016/j.ttbdis.2018.11.004DOI Listing
February 2019

The Study of Viral RNA Diversity in Bird Samples Using De Novo Designed Multiplex Genus-Specific Primer Panels.

Adv Virol 2018 12;2018:3248285. Epub 2018 Aug 12.

Central Research Institute of Epidemiology, Moscow 111123, Russia.

Advances in the next generation sequencing (NGS) technologies have significantly increased our ability to detect new viral pathogens and systematically determine the spectrum of viruses prevalent in various biological samples. In addition, this approach has also helped in establishing the associations of viromes with many diseases. However, unlike the metagenomic studies using rRNA for the detection of bacteria, it is impossible to create universal oligonucleotides to target all known and novel viruses, owing to their genomic diversity and variability. On the other hand, sequencing the entire genome is still expensive and has relatively low sensitivity for such applications. The existing approaches for the design of oligonucleotides for targeted enrichment are usually involved in the development of primers for the PCR-based detection of particular viral species or genera, but not for families or higher taxonomic orders. In this study, we have developed a computational pipeline for designing the oligonucleotides capable of covering a significant number of known viruses within various taxonomic orders, as well as their novel variants. We have subsequently designed a genus-specific oligonucleotide panel for targeted enrichment of viral nucleic acids in biological material and demonstrated the possibility of its application for virus detection in bird samples. We have tested our panel using a number of collected samples and have observed superior efficiency in the detection and identification of viral pathogens. Since a reliable, bioinformatics-based analytical method for the rapid identification of the sequences was crucial, an NGS-based data analysis module was developed in this study, and its functionality in the detection of novel viruses and analysis of virome diversity was demonstrated.
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http://dx.doi.org/10.1155/2018/3248285DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6109506PMC
August 2018

Targeted sequencing reveals complex, phenotype-correlated genotypes in cystic fibrosis.

BMC Med Genomics 2018 02 13;11(Suppl 1):13. Epub 2018 Feb 13.

Moscow Institute of Physics and Technology, Department of Biological and Medical Physics, Dolgoprudny, Moscow Region, Russian Federation, 141700.

Background: Cystic fibrosis (CF) is one of the most common life-threatening genetic disorders. Around 2000 variants in the CFTR gene have been identified, with some proportion known to be pathogenic and 300 disease-causing mutations have been characterized in detail by CFTR2 database, which complicates its analysis with conventional methods.

Methods: We conducted next-generation sequencing (NGS) in a cohort of 89 adult patients negative for p.Phe508del homozygosity. Complete clinical and demographic information were available for 84 patients.

Results: By combining MLPA with NGS, we identified disease-causing alleles in all the CF patients. Importantly, in 10% of cases, standard bioinformatics pipelines were inefficient in identifying causative mutations. Class IV-V mutations were observed in 38 (45%) cases, predominantly ones with pancreatic sufficient CF disease; rest of the patients had Class I-III mutations. Diabetes was seen only in patients homozygous for class I-III mutations. We found that 12% of the patients were heterozygous for more than two pathogenic CFTR mutations. Two patients were observed with p.[Arg1070Gln, Ser466*] complex allele which was associated with milder pulmonary obstructions (FVC 107 and 109% versus 67%, CI 95%: 63-72%; FEV 90 and 111% versus 47%, CI 95%: 37-48%). For the first time p.[Phe508del, Leu467Phe] complex allele was reported, observed in four patients (5%).

Conclusion: NGS can be a more information-gaining technology compared to standard methods. Combined with its equivalent diagnostic performance, it can therefore be implemented in the clinical practice, although careful validation is still required.
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http://dx.doi.org/10.1186/s12920-018-0328-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5836842PMC
February 2018

Comparative analysis of inverted repeats of polypod fern (Polypodiales) plastomes reveals two hypervariable regions.

BMC Plant Biol 2017 12 28;17(Suppl 2):255. Epub 2017 Dec 28.

M.V. Lomonosov Moscow State University, 119991, Moscow, Russia.

Background: Ferns are large and underexplored group of vascular plants (~ 11 thousands species). The genomic data available by now include low coverage nuclear genomes sequences and partial sequences of mitochondrial genomes for six species and several plastid genomes.

Results: We characterized plastid genomes of three species of Dryopteris, which is one of the largest fern genera, using sequencing of chloroplast DNA enriched samples and performed comparative analysis with available plastomes of Polypodiales, the most species-rich group of ferns. We also sequenced the plastome of Adianthum hispidulum (Pteridaceae). Unexpectedly, we found high variability in the IR region, including duplication of rrn16 in D. blanfordii, complete loss of trnI-GAU in D. filix-mas, its pseudogenization due to the loss of an exon in D. blanfordii. Analysis of previously reported plastomes of Polypodiales demonstrated that Woodwardia unigemmata and Lepisorus clathratus have unusual insertions in the IR region. The sequence of these inserted regions has high similarity to several LSC fragments of ferns outside of Polypodiales and to spacer between tRNA-CGA and tRNA-TTT genes of mitochondrial genome of Asplenium nidus. We suggest that this reflects the ancient DNA transfer from mitochondrial to plastid genome occurred in a common ancestor of ferns. We determined the marked conservation of gene content and relative evolution rate of genes and intergenic spacers in the IRs of Polypodiales. Faster evolution of the four intergenic regions had been demonstrated (trnA- orf42, rrn16-rps12, rps7-psbA and ycf2-trnN).

Conclusions: IRs of Polypodiales plastomes are dynamic, driven by such events as gene loss, duplication and putative lateral transfer from mitochondria.
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http://dx.doi.org/10.1186/s12870-017-1195-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5751766PMC
December 2017

Reconstructed Serine 288 in the Left Flipper Region of the Rat P2X7 Receptor Stabilizes Nonsensitized States.

Biochemistry 2017 07 26;56(26):3394-3402. Epub 2017 Jun 26.

A. I. Virtanen Institute, University of Eastern Finland , Kuopio, Finland.

Serine 275, a conserved residue of the left flipper region of ATP-gated P2X3 receptors, plays a key role in both agonist binding and receptor desensitization. It is conserved in most of the P2X receptors except P2X7 and P2X6. By combining experimental patch-clamp and modeling approaches, we explored the role of the corresponding residue in the rat P2X7 receptor (rP2X7) by replacing the phenylalanine at position 288 with serine and characterizing the membrane currents generated by either the wild-type (WT) or the mutated rP2X7 receptor. F288S, an rP2X7 mutation, slowed the deactivation subsequent to 2 and 20 s applications of 1 mM ATP. F288S also prevented sensitization (a progressive current growth) observed with the WT in response to a 20 s application of 1 mM ATP. Increasing the ATP concentration to 5 mM promoted sensitization also in the mutated rP2X7 receptor, accelerating the deactivation rate to typical WT values. YO-PRO1 uptake in cells expressing either the WT or the F288S P2X7 receptor was consistent with recorded membrane current data. Interestingly, in the human P2X7 (hP2X7) receptor, substitution Y288S did not change the deactivation rate, while the Y288F mutant generated a "rat-like" phenotype with a fast deactivation rate. Our combined experimental, kinetic, and molecular modeling data suggest that the rat F288S novel phenotype is due to a slower rate of ATP binding and/or unbinding and stabilization of nonsensitized receptor states.
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http://dx.doi.org/10.1021/acs.biochem.7b00258DOI Listing
July 2017

Computational approaches to study the effects of small genomic variations.

J Mol Model 2015 Oct 8;21(10):251. Epub 2015 Sep 8.

Moscow Institute of Physics and Technology, Dolgoprudny, Moscow Region, Russian Federation,

Advances in DNA sequencing technologies have led to an avalanche-like increase in the number of gene sequences deposited in public databases over the last decade as well as the detection of an enormous number of previously unseen nucleotide variants therein. Given the size and complex nature of the genome-wide sequence variation data, as well as the rate of data generation, experimental characterization of the disease association of each of these variations or their effects on protein structure/function would be costly, laborious, time-consuming, and essentially impossible. Thus, in silico methods to predict the functional effects of sequence variations are constantly being developed. In this review, we summarize the major computational approaches and tools that are aimed at the prediction of the functional effect of mutations, and describe the state-of-the-art databases that can be used to obtain information about mutation significance. We also discuss future directions in this highly competitive field.
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http://dx.doi.org/10.1007/s00894-015-2794-yDOI Listing
October 2015

AlignMe--a membrane protein sequence alignment web server.

Nucleic Acids Res 2014 Jul 21;42(Web Server issue):W246-51. Epub 2014 Apr 21.

Computational Structural Biology Group, Max Planck Institute of Biophysics, Frankfurt am Main 60438, Germany

We present a web server for pair-wise alignment of membrane protein sequences, using the program AlignMe. The server makes available two operational modes of AlignMe: (i) sequence to sequence alignment, taking two sequences in fasta format as input, combining information about each sequence from multiple sources and producing a pair-wise alignment (PW mode); and (ii) alignment of two multiple sequence alignments to create family-averaged hydropathy profile alignments (HP mode). For the PW sequence alignment mode, four different optimized parameter sets are provided, each suited to pairs of sequences with a specific similarity level. These settings utilize different types of inputs: (position-specific) substitution matrices, secondary structure predictions and transmembrane propensities from transmembrane predictions or hydrophobicity scales. In the second (HP) mode, each input multiple sequence alignment is converted into a hydrophobicity profile averaged over the provided set of sequence homologs; the two profiles are then aligned. The HP mode enables qualitative comparison of transmembrane topologies (and therefore potentially of 3D folds) of two membrane proteins, which can be useful if the proteins have low sequence similarity. In summary, the AlignMe web server provides user-friendly access to a set of tools for analysis and comparison of membrane protein sequences. Access is available at http://www.bioinfo.mpg.de/AlignMe.
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http://dx.doi.org/10.1093/nar/gku291DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4086118PMC
July 2014

Gene regulation by PAX6: structural-functional correlations of missense mutants and transcriptional control of Trpm3/miR-204.

Mol Vis 2014 6;20:270-82. Epub 2014 Mar 6.

Department of Ophthalmology and Visual Sciences, Albert Einstein College of Medicine, Bronx, NY ; Department of Genetics, Albert Einstein College of Medicine, Bronx, NY.

Purpose: Pax6 is a key regulatory gene for eye, brain, and pancreas development. It acts as a transcriptional activator and repressor. Loss-of-function of Pax6 results in down- and upregulation of a comparable number of genes, although many are secondary targets. Recently, we found a prototype of a Pax6-binding site that acts as a transcriptional repressor. We also identified the Trpm3 gene as a Pax6-direct target containing the miR-204 gene located in intron 6. Thus, there are multiple Pax6-dependent mechanisms of transcriptional repression in the cell. More than 50 Pax6 missense mutations have been identified in humans and mice. Two of these mutations, N50K (Leca4) and R128C (Leca2), were analyzed in depth resulting in different numbers of regulated genes and different ratios of down- and upregulated targets. Thus, additional studies of these mutants are warranted to better understand the molecular mechanisms of the mutants' action.

Methods: Mutations in PAX6 and PAX6(5a), including G18W, R26G, N50K, G64V, R128C, and R242T, were generated with site-directed mutagenesis. A panel of ten luciferase reporters driven by six copies of Pax6-binding sites representing a spectrum of sites that act as repressors, moderate activators, and strong activators were used. Two additional reporters, including the Pax6-regulated enhancer from mouse Trpm3 and six copies of its individual Pax6-binding site, were also tested in P19 cells.

Results: PAX6 (N50K) acted either as a loss-of-function or neutral mutation. In contrast, PAX6 (R128C) and (R242T) acted as loss-, neutral, and gain-of-function mutations. With three distinct reporters, the PAX6 (N50K) mutation broke the pattern of effects produced by substitutions in the surrounding helices of the N-terminal region of the paired domain. All six mutations tested acted as loss-of-function using the Trpm3 Pax6-binding site.

Conclusions: These studies highlight the complexity of Pax6-dependent transcriptional activation and repression mechanisms, and identify the N50K and R128C substitutions as valuable tools for testing interactions between Pax6, Pax6 (N50K), and Pax6 (R128C) with other regulatory proteins, including chromatin remodelers.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3945805PMC
September 2014

Trends in structural coverage of the protein universe and the impact of the Protein Structure Initiative.

Proc Natl Acad Sci U S A 2014 Mar 24;111(10):3733-8. Epub 2014 Feb 24.

Department of Systems and Computational Biology, Department of Biochemistry, New York Structural Genomics Research Consortium, Immune Function Network, and Department of Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, NY 10461.

The exponential growth of protein sequence data provides an ever-expanding body of unannotated and misannotated proteins. The National Institutes of Health-supported Protein Structure Initiative and related worldwide structural genomics efforts facilitate functional annotation of proteins through structural characterization. Recently there have been profound changes in the taxonomic composition of sequence databases, which are effectively redefining the scope and contribution of these large-scale structure-based efforts. The faster-growing bacterial genomic entries have overtaken the eukaryotic entries over the last 5 y, but also have become more redundant. Despite the enormous increase in the number of sequences, the overall structural coverage of proteins--including proteins for which reliable homology models can be generated--on the residue level has increased from 30% to 40% over the last 10 y. Structural genomics efforts contributed ∼50% of this new structural coverage, despite determining only ∼10% of all new structures. Based on current trends, it is expected that ∼55% structural coverage (the level required for significant functional insight) will be achieved within 15 y, whereas without structural genomics efforts, realizing this goal will take approximately twice as long.
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http://dx.doi.org/10.1073/pnas.1321614111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3956173PMC
March 2014

Crystal structure of human Karyopherin β2 bound to the PY-NLS of Saccharomyces cerevisiae Nab2.

J Struct Funct Genomics 2013 Jun 28;14(2):31-5. Epub 2013 Mar 28.

Department of Pharmacology, University of Texas Southwestern, Dallas, TX 75390, USA.

Import-Karyopherin or Importin proteins bind nuclear localization signals (NLSs) to mediate the import of proteins into the cell nucleus. Karyopherin β2 or Kapβ2, also known as Transportin, is a member of this transporter family responsible for the import of numerous RNA binding proteins. Kapβ2 recognizes a targeting signal termed the PY-NLS that lies within its cargos to target them through the nuclear pore complex. The recognition of PY-NLS by Kapβ2 is conserved throughout eukaryotes. Kap104, the Kapβ2 homolog in Saccharomyces cerevisiae, recognizes PY-NLSs in cargos Nab2, Hrp1, and Tfg2. We have determined the crystal structure of Kapβ2 bound to the PY-NLS of the mRNA processing protein Nab2 at 3.05-Å resolution. A seven-residue segment of the PY-NLS of Nab2 is observed to bind Kapβ2 in an extended conformation and occupies the same PY-NLS binding site observed in other Kapβ2·PY-NLS structures.
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http://dx.doi.org/10.1007/s10969-013-9150-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3681870PMC
June 2013

Alignment of helical membrane protein sequences using AlignMe.

PLoS One 2013 4;8(3):e57731. Epub 2013 Mar 4.

Computational Structural Biology Group, Max Planck Institute of Biophysics, Frankfurt am Main, Germany.

Few sequence alignment methods have been designed specifically for integral membrane proteins, even though these important proteins have distinct evolutionary and structural properties that might affect their alignments. Existing approaches typically consider membrane-related information either by using membrane-specific substitution matrices or by assigning distinct penalties for gap creation in transmembrane and non-transmembrane regions. Here, we ask whether favoring matching of predicted transmembrane segments within a standard dynamic programming algorithm can improve the accuracy of pairwise membrane protein sequence alignments. We tested various strategies using a specifically designed program called AlignMe. An updated set of homologous membrane protein structures, called HOMEP2, was used as a reference for optimizing the gap penalties. The best of the membrane-protein optimized approaches were then tested on an independent reference set of membrane protein sequence alignments from the BAliBASE collection. When secondary structure (S) matching was combined with evolutionary information (using a position-specific substitution matrix (P)), in an approach we called AlignMePS, the resultant pairwise alignments were typically among the most accurate over a broad range of sequence similarities when compared to available methods. Matching transmembrane predictions (T), in addition to evolutionary information, and secondary-structure predictions, in an approach called AlignMePST, generally reduces the accuracy of the alignments of closely-related proteins in the BAliBASE set relative to AlignMePS, but may be useful in cases of extremely distantly related proteins for which sequence information is less informative. The open source AlignMe code is available at https://sourceforge.net/projects/alignme/, and at http://www.forrestlab.org, along with an online server and the HOMEP2 data set.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0057731PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3587630PMC
August 2013

Investigation of the sodium-binding sites in the sodium-coupled betaine transporter BetP.

Proc Natl Acad Sci U S A 2012 Oct 9;109(44):E3035-44. Epub 2012 Oct 9.

Computational Structural Biology Group, Max Planck Institute of Biophysics, 60438 Frankfurt am Main, Germany.

Sodium-coupled substrate transport plays a central role in many biological processes. However, despite knowledge of the structures of several sodium-coupled transporters, the location of the sodium-binding site(s) often remains unclear. Several of these structures have the five transmembrane-helix inverted-topology repeat, LeuT-like (FIRL) fold, whose pseudosymmetry has been proposed to facilitate the alternating-access mechanism required for transport. Here, we provide biophysical, biochemical, and computational evidence for the location of the two cation-binding sites in the sodium-coupled betaine symporter BetP. A recent X-ray structure of BetP in a sodium-bound closed state revealed that one of these sites, equivalent to the Na2 site in related transporters, is located between transmembrane helices 1 and 8 of the FIRL-fold; here, we confirm the location of this site by other means. Based on the pseudosymmetry of this fold, we hypothesized that the second site is located between the equivalent helices 6 and 3. Molecular dynamics simulations of the closed-state structure suggest this second sodium site involves two threonine sidechains and a backbone carbonyl from helix 3, a phenylalanine from helix 6, and a water molecule. Mutating the residues proposed to form the two binding sites increased the apparent K(m) and K(d) for sodium, as measured by betaine uptake, tryptophan fluorescence, and (22)Na(+) binding, and also diminished the transient currents measured in proteoliposomes using solid supported membrane-based electrophysiology. Taken together, these results provide strong evidence for the identity of the residues forming the sodium-binding sites in BetP.
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http://dx.doi.org/10.1073/pnas.1209039109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3497817PMC
October 2012

Metabolomic analysis of patient plasma yields evidence of plant-like α-linolenic acid metabolism in Plasmodium falciparum.

J Infect Dis 2012 Jul 7;206(2):238-48. Epub 2012 May 7.

Division of Infectious Diseases, Department of Medicine, Albert Einstein College of Medicine, Yeshiva University, Bronx, New York, USA.

Metabolomics offers a powerful means to investigate human malaria parasite biology and host-parasite interactions at the biochemical level, and to discover novel therapeutic targets and biomarkers of infection. Here, we used an approach based on liquid chromatography and mass spectrometry to perform an untargeted metabolomic analysis of metabolite extracts from Plasmodium falciparum-infected and uninfected patient plasma samples, and from an enriched population of in vitro cultured P. falciparum-infected and uninfected erythrocytes. Statistical modeling robustly segregated infected and uninfected samples based on metabolite species with significantly different abundances. Metabolites of the α-linolenic acid (ALA) pathway, known to exist in plants but not known to exist in P. falciparum until now, were enriched in infected plasma and erythrocyte samples. In vitro labeling with (13)C-ALA showed evidence of plant-like ALA pathway intermediates in P. falciparum. Ortholog searches using ALA pathway enzyme sequences from 8 available plant genomes identified several genes in the P. falciparum genome that were predicted to potentially encode the corresponding enzymes in the hitherto unannotated P. falciparum pathway. These data suggest that our approach can be used to discover novel facets of host/malaria parasite biology in a high-throughput manner.
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http://dx.doi.org/10.1093/infdis/jis339DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3490690PMC
July 2012

Highly conserved tyrosine 37 stabilizes desensitized states and restricts calcium permeability of ATP-gated P2X3 receptor.

J Neurochem 2011 Nov 10;119(4):676-85. Epub 2011 Oct 10.

Department of Neurobiology, AI Virtanen Institute, University of Eastern Finland, Finland.

Tyrosine 37 in the first transmembrane (TM1) domain is highly conserved in ATP-gated P2X receptors suggesting its fundamental role. We tested whether Y37 contributes to the desensitization of P2X3 receptors, which is currently not well understood. By combining electrophysiological, imaging and modeling approaches, we studied desensitization of various Y37 P2X3 mutants and potential partners of Y37. Unlike the membrane current of the WT receptor, which desensitized in seconds, Y37A mutant current did not fully desensitize even after minutes-long applications of β,γ-meATP, α,β-meATP, ATP or 2MeS-ATP. The fractional calcium current was enhanced in the Y37A mutant. Y37F did not rescue the native P2X3 phenotype indicating a role for the hydroxyl group of Y37 for the WT receptor. Homology modeling indicated I318 or I319 in TM2 as potential partners for Y37 in the receptor closed state. We tested this hypothesis by creating a permanent interaction between the two residues via disulfide bond. Whereas single Y37C, I318C and I319C mutants were functional, the double mutants Y37C-I318C and Y37C-I319C were non-functional. Using a cyclic model of receptor operation, we suggest that the conserved tyrosine 37 links TM1 to TM2 of adjacent subunit to stabilize desensitized states and restricts calcium permeability through the ion channel.
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http://dx.doi.org/10.1111/j.1471-4159.2011.07463.xDOI Listing
November 2011

Role of the ectodomain serine 275 in shaping the binding pocket of the ATP-gated P2X3 receptor.

Biochemistry 2011 Oct 9;50(39):8427-36. Epub 2011 Sep 9.

Department of Neurobiology, AI Virtanen Institute, University of Eastern Finland, Kuopio, Finland.

ATP-activated P2X3 receptors expressed in nociceptive sensory neurons play an important role in pain signaling. Basic properties of this receptor subtype, including very strong desensitization, depend on the rate of dissociation of the agonist from the binding site. Even though the rough structure of the ATP binding site has been proposed on the basis of the X-ray structure of the zebrafish P2X4 receptor and mutagenesis studies, the fine subunit-specific structural properties predisposing the receptor to tight capture of the agonist inside the binding pocket have not been elucidated. In this work, by exploring in silico the functional role for the left flipper located in the ectodomain region, we identified within this loop a candidate residue S275, which could contribute to the closure of the agonist-binding pocket. Testing of the S275 mutants using the patch-clamp technique revealed a crucial role for S275 in agonist binding and receptor desensitization. The S275A mutant showed a reduced rate of onset of desensitization and accelerated resensitization and was weakly inhibited by nanomolar agonist. Extracellular calcium application produced inhibition instead of facilitation of membrane currents. Moreover, some full agonists became only partial agonists when applied to the S275A receptor. These effects were stronger with the more hydrophobic mutants S275C and S275V. Taken together, our data suggest that S275 contributes to the closure of the agonist-binding pocket and that effective capture of the agonist provided by the left flipper in calcium-dependent manner determines the high rate of desensitization, slow recovery, and sensitivity to nanomolar agonist of the P2X3 receptor.
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http://dx.doi.org/10.1021/bi200812uDOI Listing
October 2011

The role of trimerization in the osmoregulated betaine transporter BetP.

EMBO Rep 2011 Jun 17;12(8):804-10. Epub 2011 Jun 17.

Department of Structural Biology, Max-Planck Institute of Biophysics, Frankfurt am Main 60438, Germany.

The osmoregulated betaine transporter BetP is a stable trimer. Structural studies have shown that individual protomers can adopt distinct transport conformations, implying a functional role for the trimeric state in transport, although the role of trimerization in regulation is not yet understood. We designed putative monomeric mutants by molecular-dynamics simulations and in silico alanine-scanning mutagenesis. Several mutants including BetP-W101A/T351A were monomeric in detergent as well as in the membrane, as shown by blue native gel electrophoresis, crosslinking and electron microscopy. This monomeric form retains the ability to accumulate betaine, but is no longer regulated by hyperosmotic shock.
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http://dx.doi.org/10.1038/embor.2011.102DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3147255PMC
June 2011

Structural asymmetry in a trimeric Na+/betaine symporter, BetP, from Corynebacterium glutamicum.

J Mol Biol 2011 Apr 31;407(3):368-81. Epub 2011 Jan 31.

Department of Structural Biology, Max Planck Institute of Biophysics, 60438 Frankfurt am Main, Germany.

The Na(+)-coupled symporter BetP catalyzes the uptake of the compatible solute betaine in the soil bacterium Corynebacterium glutamicum. BetP also senses hyperosmotic stress and regulates its own activity in response to stress level. We determined a three-dimensional (3D) map (at 8 Å in-plane resolution) of a constitutively active mutant of BetP in a C. glutamicum membrane environment by electron cryomicroscopy of two-dimensional crystals. The map shows that the constitutively active mutant, which lacks the C-terminal domain involved in osmosensing, is trimeric like wild-type BetP. Recently, we reported the X-ray crystal structure of BetP at 3.35 Å, in which all three protomers displayed a substrate-occluded state. Rigid-body fitting of this trimeric structure to the 3D map identified the periplasmic and cytoplasmic sides of the membrane. Fitting of an X-ray monomer to the individual protomer maps allowed assignment of transmembrane helices and of the substrate pathway, and revealed differences in trimer architecture from the X-ray structure in the tilt angle of each protomer with respect to the membrane. The three protomer maps showed pronounced differences around the substrate pathway, suggesting three different conformations within the same trimer. Two of those protomer maps closely match those of the atomic structures of the outward-facing and inward-facing states of the hydantoin transporter Mhp1, suggesting that the BetP protomer conformations reflect key states of the transport cycle. Thus, the asymmetry in the two-dimensional maps may reflect cooperativity of conformational changes within the BetP trimer, which potentially increases the rate of glycine betaine uptake.
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http://dx.doi.org/10.1016/j.jmb.2011.01.028DOI Listing
April 2011

A study of the evolution of inverted-topology repeats from LeuT-fold transporters using AlignMe.

Biochemistry 2010 Dec 23;49(50):10702-13. Epub 2010 Nov 23.

Computational Structural Biology Group, Max Planck Institute of Biophysics, 60438 Frankfurt am Main, Germany.

X-ray crystal structures have revealed that numerous secondary transporter proteins originally categorized into different sequence families share similar structures, namely, the LeuT fold. The core of this fold consists of two units of five transmembrane helices, whose conformations have been proposed to exchange to form the two alternate states required for transport. That these two units are related implies that LeuT-like transporters evolved from gene-duplication and fusion events. Thus, the origins of this structural repeat may be relevant to the evolution of transport function. However, the lack of significant sequence similarity requires sensitive sequence search methods for analyzing their evolution. To this end, we developed a software application called AlignMe, which can use various types of input information, such as residue hydrophobicity, to perform pairwise alignments of sequences and/or of hydropathy profiles of (membrane) proteins. We used AlignMe to analyze the evolutionary relationships between repeats of the LeuT fold. In addition, we identified proteins from the so-called DedA family that potentially share a common ancestor with these repeats. DedA domains have been implicated in, e.g., selenite uptake; they are found widely distributed across all kingdoms of life; two or more DedA domains are typically found per genome, and some may adopt dual topologies. These results suggest that DedA proteins existed in ancient organisms and may function as dimers, as required for a would-be ancestor of the LeuT fold. In conclusion, we provide novel insights into the evolution of this important structural motif and thus potentially into the alternating-access mechanism of transport itself.
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http://dx.doi.org/10.1021/bi101256xDOI Listing
December 2010

GoLoco motif proteins binding to Galpha(i1): insights from molecular simulations.

Authors:
Kamil Khafizov

J Mol Model 2009 Dec 14;15(12):1491-9. Epub 2009 May 14.

Max Planck Institute of Biophysics, Frankfurt am Main, Germany.

Molecular dynamics simulations, computational alanine scanning and sequence analysis were used to investigate the structural properties of the Galpha(i1)/GoLoco peptide complex. Using these methodologies, binding of the GoLoco motif peptide to the Galpha(i1) subunit was found to restrict the relative movement of the helical and catalytic domains in the Galpha(i1) subunit, which is in agreement with a proposed mechanism of GDP dissociation inhibition by GoLoco motif proteins. In addition, the results provide further insights into the role of the "Switch IV" region located within the helical domain of Galpha, the conformation of which might be important for interactions with various Galpha partners.
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http://dx.doi.org/10.1007/s00894-009-0516-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2847169PMC
December 2009

G protein inactive and active forms investigated by simulation methods.

Proteins 2009 Jun;75(4):919-30

International School for Advanced Studies and INFM-DEMOCRITOS Modeling Center for Research in Atomistic Simulation, via Beirut 4, I-34014 Trieste, Italy.

Molecular dynamics and computational alanine scanning techniques have been used to investigate G proteins in their inactive state (the Galpha(i1)beta(1)gamma(2) heterotrimer) as well as in their empty and monomeric active states (Galpha(i1) subunit). We find that: (i) the residue Q204 of Galpha(i1) plays a key role for binding Gbeta(1)gamma(2) and is classified among the most relevant in the interaction with a key cellular partner, the so-called regulator of G protein signaling protein. The mutation of this residue to L, which is observed in a variety of diseases, provides still fair stability to the inactive state because of the formation of van der Waals interactions. (ii) The empty state turns out to adopt some structural features of the active one, including a previously unrecognized rearrangement of a key residue (K46). (iii) The so-called Switch IV region increases its mobility on passing from the empty to the active state, and, even more, to the inactive state. Such change in mobility could be important for its several structural and functional roles. (iv) A large scale motion of the helical domain in the inactive state might be important for GDP release upon activation by GPCR, consistently with experimental data.
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http://dx.doi.org/10.1002/prot.22303DOI Listing
June 2009

Ligand specificity of odorant receptors.

J Mol Model 2007 Mar 21;13(3):401-9. Epub 2006 Nov 21.

International School for Advanced Studies, via Beirut 4, I-34014, Trieste, Italy.

Odorant receptors belong to class A of the G protein-coupled receptors (GPCRs) and detect a large number of structurally diverse odorant molecules. A recent structural bioinformatic analysis suggests that structural features are conserved across class A of GPCRs in spite of their low sequence identity. Based on this work, we have aligned the sequences of 29 ORs for which ligand binding data are available. Recent site-directed mutagenesis experiments on one such receptor (MOR174-9) provide information that helped to identify nine amino-acid residues involved in ligand binding. Our modeling provides a rationale for amino acids in equivalent positions in most of the odorant receptors considered and helps to identify other amino acids that could be important for ligand binding. Our findings are consistent with most of the previous models and allow predictions for site-directed mutagenesis experiments, which could also validate our model.
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http://dx.doi.org/10.1007/s00894-006-0160-9DOI Listing
March 2007
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