Biography

Dmitri Kudryashov received his MD from the Russian Medical State University (RGMU) in Moscow and PhD from the Russian Academy of Medical Sciences and Cardiology Research Center, where he studied the function of the Myosin Light Chain Kinase (MLCK) family of proteins (Mentors Dr. Vladimir Shirinsky and Dr. Alexander Vorotnikov). Dmitri continued his research on the actin and myosin cytoskeleton as a postdoctoral fellow at UCLA in Prof. Emil Reisler's group. For his postdoctoral work, Dr. Kudryashov received the Paul Boyer Award for Outstanding Postdoctoral Studies in Biochemistry and Herbert Newby McCoy Award. Dr. Kudryashov joined the Department of Biochemistry at OSU in 2011 and was promoted to Associate Professor at the Department of Chemistry and Biochemistry in 2017.

Research Overview

1. Actin cytoskeleton in health and disease.

Actin is one of the most abundant and functionally versatile proteins on our planet. As a major component of the cytoskeleton, actin serves as a track for myosin-based motility and thus is involved in muscle contraction, organelle transport, and closure of the contractile ring. In addition, polymerization of actin itself also serves motor purposes and thus drives exo- and endocytosis, cell division, migration, invasion, and other cellular processes of high physiological and pathological relevance. Invasion of cancer cells underlies metastatic dissemination of human cancers and is considered as a potential therapeutic target. Currently, our interests in this large area are focused in the following directions:

Actin is involved in the pathogenesis of infectious diseases as an element of the innate immune system but also as a target that can be hijacked by many bacterial and viral toxins. We are interested in both.

Actin binding proteins: Plastins. We investigate the role of actin binding protein plastin in functions of normal (e.g., immune, intestinal, and bone cells) and cancer cells. Recently, we showed that the bundling activity of plastins is regulated in a broad range via a robust but tunable auto-inhibitory association of its actin-binding domains (ABD1 and ABD2; Schwebach et al., 2022). The allosteric auto-inhibition is regulated by phosphorylation and likely by interactions with protein partners and allows plastins to organize actin in various types of assemblies from aligned bundles (as found in microvilli and stereocilia) to loosely arranged meshes (as in lamellipodia). The study is published in Nature Structural&Molecular Biology. The discovered domain separation opens opportunities for the involvement of plastins in mechanotransduction, which is currently under investigation.

Plastin-2 in immune cells and cancer. Of the three mammalian isoforms, L-plastin (a.k.a. Plastin-2 or LCP1), is found in immune cells but is also often elevated in tumors and associated with their increased metastatic activity. We compare the properties of L-plastin with two other human plastins (T- and I-plastins) in order to understand what makes the L-isoform particularly suitable for invasion.

Plastins in osteogenesis and beyond. For the ubiquitously expressed T-plastin isoform (a.k.a. Plastin-3), we established that mutations affecting its sensitivity to Ca²⁺ in either direction lead to osteoporosis (Schwebach et al., 2020). Since none of the four explored osteogenesis imperfecta mutations are located in the Ca^2+-binding EF-hands region, their location allowed us to predict the hitherto unknown relationships between the actin-binding core and regulatory domains of plastins. The role of plastins in the pathogenesis of other hereditary and acquired diseases is under investigation.

2. Actin-specific bacterial toxins

Integration of research on bacterial toxins at the biochemical and cellular levels allowed us to discover or update our understanding of several highly detrimental yet very elegant pathogenic mechanisms.

Actin Crosslinking Domain. Thus, while studying ACD (actin crosslinking domain) toxin from V.cholerae (also found in several other Gram-negative bacteria) we predicted that the toxin-produced covalent actin oligomers should possess highly unusual properties that great amplify the toxicity. Indeed, we found that ACD is highly effective even at very low doses, when only a small population of actin in the affected cells is cross-linked. We discovered that the cross-linked oligomers bind tightly and interfere with the activity of several families of vital cytoskeletal proteins (e.g., formins, Ena/VASP, NPFs, Spire, and Cobl). This potent toxicity is due to avidity (i.e., multisite interactions) of the oligomers to G-actin domains, several copies of which are present in all of the targeted proteins. This study is funded by NIH IGMS R01 grant and published in Science, Current Biology, and other journals (Heisler el al. 2015; Kudryashova et al. 2018).

TccC3 toxin of Photorhabdus Luminescens.  P. Luminescens is a glowing killer of insects that produces several toxins targeting the actin cytoskeleton. We added to the understanding of the toxin's pathogenic mechanisms by showing that TccC3 can modify (ADP-mono-ribosylate) only filamentous actin (F-actin) and only its most dynamic subpopulation free of tropomyosin (Dong et al., 2022). Furthermore, we confirmed the previously established inhibition of actin disassembly by ADF/cofilin, but also showed that it is effective only when actin is heavily modified. We discovered that bundling of the modified actin by tandem-CH domain proteins (e.g., plastins and α-actinins) is strongly inhibited, which correlates with potent uncoupling of the cortical actin from the cytoplasmic membrane that reveals itself as profound blebbing.

Other actin-specific toxins and tools. Many more exciting mechanisms of actin-specific are under investigation, some of which will be published soon, while others would have to wait. Currently, we are exploring the effects on the actin cytoskeleton of ADP-ribosylating toxins produced by salmonella, clostridium, aeromonas, and other bacteria, along with the actin-nucleating (and more, check again soon!) vibrio toxins VopF and VopL as well as actin-bundling (and more!) toxins VopM and VopV. Keep tuned, and you will not be bored! With the enriched knowledge of these and other toxins' mechanisms, we will soon be way better equipped with powerful toxin-based tools for studying the role of actin in various cellular events (e.g., inflammation, apoptosis, DNA-repair, etc.).

3. Selective ablation of cancer cells by bacterial toxins

Several bacterial toxins have attracted significant interest as potent antitumor agents (called immunotoxins) due to their incredible toxicity (yes, protein toxins are the deadliest compounds on the planet!), potential selectivity, ability to penetrate the cell membrane, high resistance against host defense systems, and demonstrated capacity to kill cells by mechanisms independent of the drug-resistant phenotypes of most tumors. Yet, the specificity of the currently recognized toxins towards tumor cells is limited due to i) a cross-specificity of the toxin receptors; ii) non-specific acquisition of the immunotoxins by metabolically active tissues. To address the problem, we split toxins into two inactive parts and deliver split variants of potent bacterial toxins via two different pathways uniquely represented only on the surface of cancer cells. The idea is that the fully functional toxin will be assembled only in the cytoplasm of a doubly targeted cancer cell. The first proof-of-principle study was funded by NIH NCI R21 and Pelotonia (OSU CCC) grants, patented, and published in PNAS (Purde et al. 2020).

4. Selective inactivation of bacterial toxins and viral proteins by human defensins.

Defensins are a family of short cationic immune peptides with a broad repertoire of anti-microbial activities. Defensins disorganize bacterial cell membranes and inactivate bacterial toxins and viral proteins while showing little effect on the host's proteins. The amazing selectivity of defensins against various unrelated toxins remained a major puzzle until recently. We proposed and demonstrated that the elusive property targeted by defensins is not a particular sequence but thermodynamics plasticity of the toxins, which is a necessary element for their effective molding from soluble to membrane-embedded molecules or for unfolding required for translocation into the host cell cytosol via narrow pores. The study was published in Immunity, Scientific Reports, Biochemical Journal, Journal of Molecular Biology, and other journals.

To address these and other scientific problems, we employ a highly interdisciplinary combination of biochemical, biophysical, structural, and cell biology approaches, including but not limited to methods of fluorescence spectroscopy, circular dichroism, calorimetry, mass spectrometry, electron microscopy, single-molecule in vitro and cell imaging, X-ray crystallography, and others. We also enjoy many fruitful collaborations with our colleagues, who are generously providing their expertise in SS NMR (Dr. Polenova, U Delaware), X-ray crystallography and MD simulations (Dr. Sotomayor, OSU), cryo-EM (Dr. Egelman, U Virginia), microbiology (Dr. Yount, OSU; Dr. Gunn, NCH), neurosciences (Dr. Zuchero, Stanford), actin biochemistry (Dr. Kovar, UChicago), cell biology (Dr. de Lanerolle; U Illinois), to name a few.

The lab welcomes enthusiastic and energetic biochemists, cell and structural biologists, and biophysicists at all levels: undergraduate, graduate, and postgraduate. To express your interest in joining the group, please email Dr. Dmitri Kudryashov.

Publications

52. Dong S, Zheng W, Pinkerton N, Hansen J, Tikunova SB, Davis JP, Heissler SM, Kudryashova E, Egelman EH, Kudryashov DS. Photorhabdus luminescens TccC3 Toxin Targets the Dynamic Population of F-Actin and Impairs Cell Cortex Integrity. Int J Mol Sci (2022), 23(13), 7026; doi.org/10.3390/ijms23137026.

51. Schwebach CL, Kudryashova E, Agrawal R, Zheng W, Egelman EH, Kudryashov DS. Allosteric regulation controls actin-bundling properties of human plastins. Nat Struct Mol Biol (2022) doi: 10.1038/s41594-022-00771-1. PMID: 35589838

Highlighted in Phys.org,  EurekAlert! AAAS, The Medical News, Technology.org, Newswise, ScienMag, AZO Life Sciences, Earth

50. Kraus J, Russell RW, Kudryashova E, Xu C, Katyal N, Perilla JR, Kudryashov DS, Polenova T. Magic Angle Spinning NMR Atomic-Resolution Structure of Human Cofilin-2 Assembled on Actin Filaments Reveals Isoform-Specific Conformation and Binding Mode. Nat Commun (2022) 13(1):2114. doi: 10.1038/s41467-022-29595-9. PMID: 35440100

49. Kudryashova E, Zani A, Vilmen G, Sharma A, Lu W, Yount JS, Kudryashov DS. Inhibition of SARS-CoV-2 Infection by Human Defensin HNP1 and Retrocyclin RC-101. J Mol Biol (2022) 434(6):167225. doi: 10.1016/j.jmb.2021.167225. PMID: 34487793

48. Shi G, Kenney AD, Kudryashova E, Zani A, Zhang L, Lai KK, Hall-Stoodley L, Robinson RT, Kudryashov DS, Compton AA, Yount JS. Opposing activities of IFITM proteins in SARS-CoV-2 infection. EMBO J (2021) 40(3):e106501. doi: 10.15252/embj.2020106501. PMID: 33270927

47. Smith H, Pinkerton N, Heisler DB, Kudryashova E, Hall AR, Karch KR, Norris A, Wysocki V, Sotomayor M, Reisler E, Vavylonis D, Kudryashov DS. Rounding out the understanding of ACD toxicity with the discovery of cyclic forms of actin oligomers. Int J Mol Sci (2021) 22(2):718. doi: 10.3390/ijms22020718. Int J Mol Sci. 2021. PMID: 33450834

46. Schwebach CL, Kudryashova E, Kudryashov DS. Plastin 3 in X-Linked Osteoporosis: Imbalance of Ca²⁺-Dependent Regulation Is Equivalent to Protein Loss. Front Cell Dev Biol (2021) 8:635783. doi: 10.3389/fcell.2020.635783. Review. PMID: 33553175

45. Purde V, Kudryashova E, Heisler DB, Shakya R, Kudryashov DS. Intein-mediated cytoplasmic reconstitution of a split toxin enables selective cell ablation in mixed populations and tumor xenografts. Proc Natl Acad Sci U S A. (2020) 117(36):22090-22100. PMID: 32839344

 44. Schwebach CL, Kudryashova E, Zheng W, Orchard M, Smith H, Runyan LA, Egelman EH, Kudryashov DS. Osteogenesis imperfecta mutations in plastin 3 lead to impaired calcium regulation of actin bundling. Bone Research, NPG (2020) 8:21. PMID: 32509377

43. Purde V, Busch F, Kudryashova E, Wysocki V, Kudryashov D. Oligomerization Affects the Ability of Human Cyclase-Associated Proteins 1 and 2 to Promote Actin Severing by Cofilins. International Journal of Molecular Sciences. (2019) 20(22):5647. 10.3390/ijms20225647. PMID: 31718088

42. Targeting and inactivation of bacterial toxins by human defensins.(2017) Kudryashova E, Seveau SM, Kudryashov DS. Biological Chemistry. 26;398(10):1069-1085. Review. PMID: 28593905

41. The Roles of Actin-Binding Domains 1 and 2 in the Calcium-Dependent Regulation of Actin Filament Bundling by Human Plastins. (2017) Schwebach CL, Agrawal R, Lindert S, Kudryashova E, Kudryashov DS. Journal of Molecular Biology. 429(16):2490-2508. PMID: 28694070

40. DeActs: genetically encoded tools for perturbing the actin cytoskeleton in single cells. (2017) Harterink M, da Silva ME, Will L, Turan J, Ibrahim A, Lang AE, van Battum EY, Pasterkamp RJ, Kapitein LC, Kudryashov D, Barres BA, Hoogenraad CC, Zuchero JB. Nature Methods. 14(5):479-482. PMID:28394337

39. Structural Analysis of Human Cofilin 2/Filamentous Actin Assemblies: Atomic-Resolution Insights from Magic Angle Spinning NMR Spectroscopy.(2017) Yehl J., Kudryashova E., Reisler E., Kudryashov D., Polenova T. Scientific Reports. 17;7:44506. PMID:28303963

38. Kudryashova E, Heisler DB, Kudryashov DS, "Pathogenic mechanisms of actin cross-linking toxins – peeling away the layers". In Current Topics in Microbiology and Immunology – Springer Book Series. (2016)

37. Kudryashova E, Koneru, Kvaratskhelia M, Strömstedt A, Lu W, Kudryashov D. "Thermodynamic instability of viral proteins is a pathogen-associated molecular pattern targeted by human defensins" (2016) Scientific Reports. 6: 32499

36. Serebryannyy LA, Parilla M, Annibale P, Cruz CM, Laster K, Gratton E, Kudryashov D, Kosak ST, Gottardi CJ, de Lanerolle P," Persistent nuclear actin filaments inhibit transcription by RNA polymerase II." J Cell Sci. (2016) 129(18):3412-25.

35. Vilitkevich EL, Khapchaev AY, Kudryashov DS, Nikashin AV, Schavocky JP, Lukas TJ, Watterson DM, Shirinsky VP. Phosphorylation Regulates Interaction of 210-kDa Myosin Light Chain Kinase N-terminal Domain with Actin Cytoskeleton. (2015) Biochemistry (Mosc). 80(10):1288-97.

34. Kudryashova E, Lu W, Kudryashov DS "Defensins versus pathogens: an unfolding study." (2015) Oncotarget 6(30):28533-4 - Invited Editorial.

33. Heisler DB, Kudryashova E, Grinevich DO, Suarez C, Winkelman JD, Birukov KG, Kotha SR, Parinandi NL, Vavylonis D, Kovar DR, Kudryashov DS  "ACD toxin–produced actin oligomers poison formin-controlled actin polymerization." (2015) Science 349(6247):535-9.                                                                                   Highlighted in Genetic Engineering & Biotechnology News (GEN), CHEMIE.DE, Bionity.com, Nature Reviews Microbiology, EurekAlert! AAAS, News Medical, Microbe World, Science Daily, Science Newsline Biology, Medical News Today, NewsUnited, NewsWise, sott.net, phys.org.

32. Kudryashova E, Seveau S, Lu W, Kudryashov DS  "Retrocyclins neutralize bacterial toxins by potentiating their unfolding." (2015) Biochemical Journal 367(2): 311-320.

31. Wang N, Wang M, Zhu YH, Grosel, TW, Sun D, Kudryashov DS, Wu JQ  "The Rho-GEF Gef3 interacts with the septin complex and activates the GTPase Rho4 during fission yeast cytokinesis." (2015) Molecular Biology of the Cell 26(2): 238-255.

30. Ge P, Oztug Durer ZA, Kudryashov DS, Zhou HZ, Reisler E  "CryoEM reveals different coronin binding modes for ADP- and ADP-BeFx- actin filaments." (2014) Nature Structural & Molecular Biology 21(12): 1075-1081. F1000Prime recommended.

29. Kudryashova E, Quintyn RS, Lu W, Seveau S, Wysocki V, Kudryashov DS  "Human defensins facilitate local unfolding of thermodynamically unstable regions of bacterial protein toxins." (2014) Immunity 41(5): 709-721. Highlighted in Immunity, NSF Science360 News, Nature Immunology. F1000Prime recommended.

28. Kudryashova E, Heisler D, Zywiec A, Kudryashov DS  "Thermodynamic properties of the effector domains of MARTX toxins suggest their unfolding for translocation across the host membrane." (2014) Molecular Microbiology 92(5): 1056-1071.

27. Lyon AN, Pineda RH, Kudryashova E, Kudryashov DS, Beattie CE  "Calcium binding is essential for plastin-3 function in Smn-deficient motor neurons." (2014) Human Molecular Genetics 23(8): 1990-2004.

26. Kudryashov DS, Reisler E "ATP and ADP actin states." (2013) Biopolymers 99(4): 245–256.

25. Durer ZA, Kudryashov DS, Sawaya MR, Altenbach C, Hubbell W, Reisler E  "Structural States and Dynamics of the D-Loop in Actin." (2012) Biophysical Journal 103(5): 930-939.

24. Kudryashova E, Kalda C, Kudryashov DS "Glutamyl Phosphate Is an Activated Intermediate in Actin Crosslinking by Actin Crosslinking Domain (ACD) Toxin." (2012) PLoS One 7(9): e45721.

23. Galkin VE, Orlova A, Kudryashov DS, Solodukhin A, Reisler E Schröder GF, Egelman EH  "Remodeling of actin filaments by ADF/cofilin proteins." (2011) Proceedings of the National Academy of Sciences USA 108(51): 20568-20572.

22. Kudryashov DS, Grintsevich EE, Rubenstein PA, Reisler E  "A Nucleotide State-sensing Region on Actin." (2010) Journal of Biological Chemistry 285(33): 25591-25601.

21. Grintsevich EE, Galkin VE, Orlova A, Ytterberg AJ, Mikati MM, Kudryashov DS, Loo JA, Egelman EH, Reisler E  "Mapping of Drebrin Binding Site on F-Actin." (2010) Journal of Molecular Biology 398(4): 542-554.

20. Oztug Durer ZA, Diraviyam K, Sept D, Kudryashov DS, Reisler E  "F-Actin Structure Destabilization and DNase I Binding Loop Fluctuations Mutational Cross-Linking and Electron Microscopy Analysis of Loop States and Effects on F-Actin." (2010) Journal of Molecular Biology 395(3): 544-557.

19. Kudryashov DS, Durer ZA, Ytterberg AJ, Sawaya MR, Pashkov I, Yeates TO, Ogorzalek Loo R, Loo J, Satchell KJ, Reisler E  "Connecting actin monomers by iso-peptide bond is a toxicity mechanism of the Vibrio cholerae MARTX toxin."  (2008) Proceedings of the National Academy of Sciences USA 105(47): 18537-42.

18. Sawaya* MR, Kudryashov* DS, Pashkov* I, Reisler E, Yeates TO  "Multiple crystal structures of actin dimers and their implications for interactions in the actin filament." (2008) Acta Crystallographica Section D, Biological crystallography 64: 454-65. * Co-first authors.

17. Kudryashov* DS, Cordero* CL, Reisler E, Satchell KJ  "Characterization of the enzymatic activity of the actin cross-linking domain from the Vibrio cholerae MARTX(Vc) toxin." (2008) Journal of Biological Chemistry 283(1): 445-452. * Co-first authors.

16. Cordero* CL, Kudryashov* DS, Reisler E, Satchell KJ  "The actin cross-linking domain of the Vibrio cholerae RTX toxin directly catalyzes the covalent cross-linking of actin." (2006) Journal of Biological Chemistry 281(43): 32366-32374. * Co-first authors.

15. Kudryashov^# DS, Galkin VE, Orlova A, Phan M, Egelman EH, Reisler E  "Cofilin cross-bridges adjacent actin protomers and replaces part of the longitudinal F-actin interface." (2006) Journal of Molecular Biology 358(3): 785-97. ^# Corresponding author.

14. Kudryashova E, Kudryashov D, Kramerova I, Spencer MJ  "Trim32 is a ubiquitin ligase mutated in limb girdle muscular dystrophy type 2H that binds to skeletal muscle myosin and ubiquitinates actin." (2005) Journal of Molecular Biology 354(2): 413-424.

13. Kudryashov DS, Sawaya MR, Adisetiyo H, Norcross T, Hegyi G, Reisler E, Yeates TO  "The crystal structure of a cross-linked actin dimer suggests a detailed molecular interface in F-actin." (2005) Proceedings of the National Academy of Sciences USA 102(37): 13105-13110.

12. Orlova A, Shvetsov A, Galkin VE, Kudryashov DS, Rubenstein PA, Egelman EH, Reisler E "Actin-destabilizing factors disrupt filaments by means of a time reversal of polymerization." (2004) Proceedings of the National Academy of Sciences USA 101(51): 17664-17668.

11. Muhlrad A, Kudryashov D, Michael Peyser Y, Bobkov AA, Almo SC, Reisler E  "Cofilin induced conformational changes in F-actin expose subdomain 2 to proteolysis." (2004) Journal of Molecular Biology 342(5): 1559-1567.

10. Kudryashov DS, Stepanova OV, Vilitkevich EL, Nikonenko TA, Nadezhdina ES, Shanina NA, Lukas TJ, Van Eldik LJ, Watterson DM, Shirinsky VP. "Myosin light chain kinase (210 kDa) is a potential cytoskeleton integrator through its unique N-terminal domain." (2004) Experimental Cell Research 298(2): 407-417.

9. Kudryashov DS, Phillips M, Reisler E "Formation and destabilization of actin filaments with tetramethylrhodamine-modified actin." (2004) Biophysical Journal 87(2): 1136-1145. Editor's Choice Article.

8. Vilitkevich EL, Kudriashev DS, Stepanova OV, Shirinsky VP  "A new actinbinding area of the myosin light chains' high-molecular kinase." (2004) Ross Fiziol Zh Im I.M. Sechenova (Russian journal of physiology, Rus) 90(5): 577-585.

7. Kudryashov DS, Reisler E  "Solution properties of tetramethylrhodamine-modified G-actin." (2003) Biophysical Journal 85(4): 2466-2475.

6. Kudryashov DS, Vorotnikov AV, Dudnakova TV, Stepanova OV, Lukas TJ, Sellers JR, Watterson DM, Shirinsky VP  "Smooth muscle myosin filament assembly under control of a kinase-related protein (KRP) and caldesmon." (2002) Journal of Muscle Research and Cell Motility 23(4): 341-351.

5. Krymsky MA, Kudryashov DS, Shirinsky VP, Lukas TJ, Watterson DM, Vorotnikov AV   "Phosphorylation of kinase-related protein (telokin) in tonic and phasic smooth muscles." (2001) Journal of Muscle Research and Cell Motility 22(5):425-437.

4. Chibalina MV, Kudriashov DS, Shekhonin BV, Shirinskiĭ VP "Functional properties and intracellular localization of high molecular weight isoform of myosin light chain kinase." (2000) Tsitologia (Cytology, Rus) 42(3): 248-255.

3. Vorotnikov AV, Krymsky MA, Chibalina MV, Kudriashov DS, Shirinsky VP  "Differences in contraction and regulatory protein phosphorylation of phasic and tonic smooth muscles." (2000) Tsitologia (Cytology, Rus) 42(4): 378-391.

2. Kudryashov DS, Chibalina MV, Birukov KG, Lukas TJ, Sellers JR, Van Eldik LJ, Watterson DM, Shirinsky VP  "Unique sequence of a high molecular weight myosin light chain kinase is involved in interaction with actin cytoskeleton." (1999) FEBS Letters 463(1-2): 67-71.

1. Bushueva TL, Teplova MV, Bushuev VN, Kudriashov DS, Vorotnikov AV, Shirinskiĭ VP "Stability of the structure of KRP (kinase-related protein)." (1999) Molecular Biology (Rus) 33(2): 227-236.