Stanford Advisors

All Publications

  • Broad-spectrum activity against mosquito-borne flaviviruses achieved by a targeted protein degradation mechanism. Nature communications Liu, H. Y., Li, Z., Reindl, T., He, Z., Qiu, X., Golden, R. P., Donovan, K. A., Bailey, A., Fischer, E. S., Zhang, T., Gray, N. S., Yang, P. L. 2024; 15 (1): 5179


    Viral genetic diversity presents significant challenges in developing antivirals with broad-spectrum activity and high barriers to resistance. Here we report development of proteolysis targeting chimeras (PROTACs) targeting the dengue virus envelope (E) protein through coupling of known E fusion inhibitors to ligands of the CRL4CRBN E3 ubiquitin ligase. The resulting small molecules block viral entry through inhibition of E-mediated membrane fusion and interfere with viral particle production by depleting intracellular E in infected Huh 7.5 cells. This activity is retained in the presence of point mutations previously shown to confer partial resistance to the parental inhibitors due to decreased inhibitor-binding. The E PROTACs also exhibit broadened spectrum of activity compared to the parental E inhibitors against a panel of mosquito-borne flaviviruses. These findings encourage further exploration of targeted protein degradation as a differentiated and potentially advantageous modality for development of broad-spectrum direct-acting antivirals.

    View details for DOI 10.1038/s41467-024-49161-9

    View details for PubMedID 38898037

    View details for PubMedCentralID PMC11187112

  • The non-muscle actinopathy-associated mutation E334Q in cytoskeletal γ-actin perturbs interaction of actin filaments with myosin and ADF/cofilin family proteins ELIFE Greve, J. N., Marquardt, A., Heiringhoff, R., Reindl, T., Thiel, C., Di Donato, N., Taft, M. H., Manstein, D. J. 2024; 12


    Various heterozygous cytoskeletal γ-actin mutations have been shown to cause Baraitser-Winter cerebrofrontofacial syndrome, non-syndromic hearing loss, or isolated eye coloboma. Here, we report the biochemical characterization of human cytoskeletal γ-actin carrying mutation E334Q, a mutation that leads to a hitherto unspecified non-muscle actinopathy. Following expression, purification, and removal of linker and thymosin β4 tag sequences, the p.E334Q monomers show normal integration into linear and branched actin filaments. The mutation does not affect thermal stability, actin filament nucleation, elongation, and turnover. Model building and normal mode analysis predict significant differences in the interaction of p.E334Q filaments with myosin motors and members of the ADF/cofilin family of actin-binding proteins. Assays probing the interactions of p.E334Q filaments with human class 2 and class 5 myosin motor constructs show significant reductions in sliding velocity and actin affinity. E334Q differentially affects cofilin-mediated actin dynamics by increasing the rate of cofilin-mediated de novo nucleation of actin filaments and decreasing the efficiency of cofilin-mediated filament severing. Thus, it is likely that p.E334Q-mediated changes in myosin motor activity, as well as filament turnover, contribute to the observed disease phenotype.

    View details for DOI 10.7554/eLife.93013

    View details for Web of Science ID 001180948900001

    View details for PubMedID 38446501

    View details for PubMedCentralID PMC10942649

  • Distinct actin-tropomyosin cofilament populations drive the functional diversification of cytoskeletal myosin motor complexes ISCIENCE Reindl, T., Giese, S., Greve, J. N., Reinke, P. Y., Chizhov, I., Latham, S. L., Mulvihill, D. P., Taft, M. H., Manstein, D. J. 2022; 25 (7): 104484


    The effects of N-terminal acetylation of the high molecular weight tropomyosin isoforms Tpm1.6 and Tpm2.1 and the low molecular weight isoforms Tpm1.12, Tpm3.1, and Tpm4.2 on the actin affinity and the thermal stability of actin-tropomyosin cofilaments are described. Furthermore, we show how the exchange of cytoskeletal tropomyosin isoforms and their N-terminal acetylation affects the kinetic and chemomechanical properties of cytoskeletal actin-tropomyosin-myosin complexes. Our results reveal the extent to which the different actin-tropomyosin-myosin complexes differ in their kinetic and functional properties. The maximum sliding velocity of the actin filament as well as the optimal motor density for continuous unidirectional movement, parameters that were previously considered to be unique and invariant properties of each myosin isoform, are shown to be influenced by the exchange of the tropomyosin isoform and the N-terminal acetylation of tropomyosin.

    View details for DOI 10.1016/j.isci.2022.104484

    View details for Web of Science ID 000834811200007

    View details for PubMedID 35720262

    View details for PubMedCentralID PMC9204724

  • Mechanochemical properties of human myosin-1C are modulated by isoform-specific differences in the N-terminal extension JOURNAL OF BIOLOGICAL CHEMISTRY Giese, S., Reindl, T., Reinke, P. A., Zattelman, L., Fedorov, R., Henn, A., Taft, M. H., Manstein, D. J. 2021; 296: 100128


    Myosin-1C is a single-headed, short-tailed member of the myosin class I subfamily that supports a variety of actin-based functions in the cytosol and nucleus. In vertebrates, alternative splicing of the MYO1C gene leads to the production of three isoforms, myosin-1C0, myosin-1C16, and myosin-1C35, that carry N-terminal extensions of different lengths. However, it is not clear how these extensions affect the chemomechanical coupling of human myosin-1C isoforms. Here, we report on the motor activity of the different myosin-1C isoforms measuring the unloaded velocities of constructs lacking the C-terminal lipid-binding domain on nitrocellulose-coated glass surfaces and full-length constructs on reconstituted, supported lipid bilayers. The higher yields of purified proteins obtained with constructs lacking the lipid-binding domain allowed a detailed characterization of the individual kinetic steps of human myosin-1C isoforms in their productive interaction with nucleotides and filamentous actin. Isoform-specific differences include 18-fold changes in the maximum power output per myosin-1C motor and 4-fold changes in the velocity and the resistive force at which maximum power output occurs. Our results support a model in which the isoform-specific N-terminal extensions affect chemomechanical coupling by combined steric and allosteric effects, thereby reducing both the length of the working stroke and the rate of ADP release in the absence of external loads by a factor of 2 for myosin-1C35. As the large change in maximum power output shows, the functional differences between the isoforms are further amplified by the presence of external loads.

    View details for DOI 10.1074/jbc.RA120.015187

    View details for Web of Science ID 000672866400107

    View details for PubMedID 33257319

    View details for PubMedCentralID PMC7948490

  • Variants in exons 5 and 6 of ACTB cause syndromic thrombocytopenia NATURE COMMUNICATIONS Latham, S. L., Ehmke, N., Reinke, P. A., Taft, M. H., Eicke, D., Reindl, T., Stenzel, W., Lyons, M. J., Friez, M. J., Lee, J. A., Hecker, R., Fruehwald, M. C., Becker, K., Neuhann, T. M., Horn, D., Schrock, E., Niehaus, I., Sarnow, K., Gruetzmann, K., Gawehn, L., Klink, B., Rump, A., Chaponnier, C., Figueiredo, C., Knoefler, R., Manstein, D. J., Di Donato, N. 2018; 9: 4250


    Germline mutations in the ubiquitously expressed ACTB, which encodes β-cytoplasmic actin (CYA), are almost exclusively associated with Baraitser-Winter Cerebrofrontofacial syndrome (BWCFF). Here, we report six patients with previously undescribed heterozygous variants clustered in the 3'-coding region of ACTB. Patients present with clinical features distinct from BWCFF, including mild developmental disability, microcephaly, and thrombocytopenia with platelet anisotropy. Using patient-derived fibroblasts, we demonstrate cohort specific changes to β-CYA filament populations, which include the enhanced recruitment of thrombocytopenia-associated actin binding proteins (ABPs). These perturbed interactions are supported by in silico modeling and are validated in disease-relevant thrombocytes. Co-examination of actin and microtubule cytoskeleton constituents in patient-derived megakaryocytes and thrombocytes indicates that these β-CYA mutations inhibit the final stages of platelet maturation by compromising microtubule organization. Our results define an ACTB-associated clinical syndrome with a distinct genotype-phenotype correlation and delineate molecular mechanisms underlying thrombocytopenia in this patient cohort.

    View details for DOI 10.1038/s41467-018-06713-0

    View details for Web of Science ID 000447123000026

    View details for PubMedID 30315159

    View details for PubMedCentralID PMC6185941

  • Three mammalian tropomyosin isoforms have different regulatory effects on nonmuscle myosin-2B and filamentous beta-actin in vitro JOURNAL OF BIOLOGICAL CHEMISTRY Pathan-Chhatbar, S., Taft, M. H., Reindl, T., Hundt, N., Latham, S. L., Manstein, D. J. 2018; 293 (3): 863-875


    The metazoan actin cytoskeleton supports a wide range of contractile and transport processes. Recent studies have shown how the dynamic association with specific tropomyosin isoforms generates actin filament populations with distinct functional properties. However, critical details of the associated molecular interactions remain unclear. Here, we report the properties of actomyosin-tropomyosin complexes containing filamentous β-actin, nonmuscle myosin-2B (NM-2B) constructs, and either tropomyosin isoform Tpm1.8cy (b.-.b.d), Tpm1.12br (b.-.b.c), or Tpm3.1cy (b.-.a.d). Our results show the extent to which the association of filamentous β-actin with these different tropomyosin cofilaments affects the actin-mediated activation of NM-2B and the release of the ATP hydrolysis products ADP and phosphate from the active site. Phosphate release gates a transition from weak to strong F-actin-binding states. The release of ADP has the opposite effect. These changes in dominant rate-limiting steps have a direct effect on the duty ratio, the fraction of time that NM-2B spends in strongly F-actin-bound states during ATP turnover. The duty ratio is increased ∼3-fold in the presence of Tpm1.12 and 5-fold for both Tpm1.8 and Tpm3.1. The presence of Tpm1.12 extends the time required per ATP hydrolysis cycle 3.7-fold, whereas it is shortened by 27 and 63% in the presence of Tpm1.8 and Tpm3.1, respectively. The resulting Tpm isoform-specific changes in the frequency, duration, and efficiency of actomyosin interactions establish a molecular basis for the ability of these complexes to support cellular processes with widely divergent demands in regard to force production, capacity to move processively, and speed of movement.

    View details for DOI 10.1074/jbc.M117.806521

    View details for Web of Science ID 000422864500009

    View details for PubMedID 29191834

    View details for PubMedCentralID PMC5777259

  • Tropomyosin Isoforms Specify Functionally Distinct Actin Filament Populations In Vitro CURRENT BIOLOGY Gateva, G., Kremneva, E., Reindl, T., Kotila, T., Kogan, K., Gressin, L., Gunning, P. W., Manstein, D. J., Michelot, A., Lappalainen, P. 2017; 27 (5): 705-713


    Actin filaments assemble into a variety of networks to provide force for diverse cellular processes [1]. Tropomyosins are coiled-coil dimers that form head-to-tail polymers along actin filaments and regulate interactions of other proteins, including actin-depolymerizing factor (ADF)/cofilins and myosins, with actin [2-5]. In mammals, >40 tropomyosin isoforms can be generated through alternative splicing from four tropomyosin genes. Different isoforms display non-redundant functions and partially non-overlapping localization patterns, for example within the stress fiber network [6, 7]. Based on cell biological studies, it was thus proposed that tropomyosin isoforms may specify the functional properties of different actin filament populations [2]. To test this hypothesis, we analyzed the properties of actin filaments decorated by stress-fiber-associated tropomyosins (Tpm1.6, Tpm1.7, Tpm2.1, Tpm3.1, Tpm3.2, and Tpm4.2). These proteins bound F-actin with high affinity and competed with α-actinin for actin filament binding. Importantly, total internal reflection fluorescence (TIRF) microscopy of fluorescently tagged proteins revealed that most tropomyosin isoforms cannot co-polymerize with each other on actin filaments. These isoforms also bind actin with different dynamics, which correlate with their effects on actin-binding proteins. The long isoforms Tpm1.6 and Tpm1.7 displayed stable interactions with actin filaments and protected filaments from ADF/cofilin-mediated disassembly, but did not activate non-muscle myosin IIa (NMIIa). In contrast, the short isoforms Tpm3.1, Tpm3.2, and Tpm4.2 displayed rapid dynamics on actin filaments and stimulated the ATPase activity of NMIIa, but did not efficiently protect filaments from ADF/cofilin. Together, these data provide experimental evidence that tropomyosin isoforms segregate to different actin filaments and specify functional properties of distinct actin filament populations.

    View details for DOI 10.1016/j.cub.2017.01.018

    View details for Web of Science ID 000396297100023

    View details for PubMedID 28216317

    View details for PubMedCentralID PMC5344678