Center for Molecular Medicine Cologne

Hoppe, Thorsten - C 08

Role of UNC45B missense mutations in progressive congenital muscle disease


The fundamental importance of chaperones for muscle integrity strongly suggests that correcting chaperone function might be a therapeutic option to tackle hereditary myopathies, however, no proven therapies exist at the moment. 

Monitoring myopathy-related defects in Caenorhabditis elegans and patient cells combined with structure-function analysis of UNC45B mutations will provide novel insights into myofibrillogenesis and regeneration mechanisms of therapeutic relevance for the treatment of muscle diseases.

Skeletal muscle integrity and function is constantly challenged by stress and strain placed upon sarcomeric and structural proteins.

Therefore, pathogenic mutations in chaperones or co-chaperones often trigger inherited neuromuscular disorders collectively termed chaperonopathies. In contrast to the rather broad role of general chaperones in protein homeostasis and myofibrillar integrity, the assembly of myosin requires precise temporal and spatial control, which is governed by the highly specialized co-chaperone UNC-45. The repetitive arrangement of UNC-45 oligomers defines the periodicity of myosin organization in growing sarcomers. However, despite the conserved role in myofibrillogenesis from C. elegans to vertebrates, the relevance of muscle-specific UNC-45 dysfunction for the pathology and pathogenesis of human myopathies remained completely unclear.

The research program is particularly focussed to explore the patho-mechanism associated with patient-derived mutations in the myosin-directed co-chaperone UNC45B. The detailed characterization of UNC45B function in the formation and maintenance of skeletal muscle is important for the molecular understanding of congenital muscle diseases and other forms of muscle weakness.

Our Aims

Muscular dystrophies are disabling and fatal forms of hereditary muscle diseases. The conserved, myosin-directed co-chaperone UNC-45 is essential for muscle formation and integrity, however, little is known about the impact of UNC-45 defects in the context of human myopathies. This proposal will utilize a multidisciplinary approach that synergizes protein biochemistry with C. elegans and cell culture approaches to address the role of UNC45 in a degenerative myopathy.

The goals of this proposal are

  1. to characterize structure-functional properties of disease-associated UNC45B mutations.
  2. to explore the role of UNC45B mutations on myofibrillogenesis in C. elegans.
  3. to define myopathy-related UNC45B dysfunction in patient muscle cells.

Previous Work

Proper muscle development and function require a complex system of structural and motor proteins organized into contractile units called the sarcomeres. The sarcomeric repeat is a supra-molecular structure, in which actin and myosin filaments together with associated proteins are arranged in a precise order that coordinates actin-myosin cross bridges with filament gliding and muscle contraction. The folding, stability, and organization of sarcomeric proteins into muscle filaments is governed by molecular chaperones (Kim et al., 2008). 

The fundamental importance of chaperones for the development and maintenance of skeletal muscle is underscored by recent studies indicating that chaperone dysfunction is responsible for many hereditary myopathies. These so called chaperonopathies are directly caused by pathogenic mutations in genes encoding chaperones and co-chaperones. The first described entity was a myofibrillar myopathy associated with mutations in the heat shock protein (HSP) HSPB5 (CRYAB), although most later identified chaperonopathies were Charcot-Marie-Tooth-types of neuropathies caused by HSPB1, HSPB8, CCTdelta/MKKS, and DNAJB2 gene defects. 

In contrast to the rather broad regulation of myofibrillar integrity by these general chaperones, the precise temporal and spatial control of myosin assembly is governed by the Hsp90 co-chaperone UNC-45 (Kim et al., 2008). Recent work of our lab importantly contributed to the mechanistic understanding of UNC-45 function in muscle formation and maintenance. We found that UNC-45 oligomers serve as multisite-docking platforms, which support precisely defined collaboration with Hsp70 and Hsp90 to form myosin filaments (Gazda et al., 2013). Thus, UNC-45 provides substrate specificity for the partner chaperones during late stages of myofibrillogenesis. Detailed analysis of structure-related mutations indicated that the elongated part of the conserved UCS (UNC-45/CRO1/She4p) domain of UNC-45 acts as myosin-binding site (Figure 1).

Therefore, the repetitive arrangement of UNC-45 oligomers serves as template that defines the periodicity of myosin organization in growing sarcomers (Figure 1 and 2). This idea is strongly supported by point mutations in UNC-45, which trigger myosin disorganization and severe motility defects in C. elegans, Drosophila, Zebrafish, and Xenopus (Figure 3) (Kim et al., 2008). UNC-45 homologs also exist in humans, which indicates a conserved requirement for myosin-directed co-chaperones. Our work has shown that UNC-45 levels are tightly controlled by ubiquitin-dependent proteolysis, which coordinates myosin folding and assembly both in C. elegans and human myoblasts (Hoppe et al., 2004; Janiesch et al., 2007).

Despite its role in myofibrillogenesis and muscle formation, UNC45B mutations have not been linked to human myopathies so far. In search of human patients with mutations in the UNC45B gene we teamed up with the group of Carsten Bönnemann at the National Institute of Neurological Disorders and Stroke/NIH (Bethesda, USA) who is an expert in neuromuscular disease of childhood. In a teenage patient with a progressive, congenital muscle disease we identified a homozygous missense mutation in UNC45B. The UNC45B mutation has a very low allele frequency and homozygosity has never been observed in normal databases. The UNC-45 protein in the patient's muscle is reduced and its localization in muscle appears abnormal. Moreover, the myofibrillar apparatus is focally disrupted.

Meanwhile, more patient families have been identified who exhibit very similar pathologies likely resulting from exactly the same homozygous UNC45B missense mutation as identified in the first patient. Another patient presents with excentric cores on muscle biopsy and a compound heterozygous mutation in UNC45B. According to both the familiar inheritance of the disease symptoms and cell biological data the detected mutations in UNC45B are very likely causative of the progressive muscle defects. In contrast to other chaperonopathies the detected myofibrillar degeneration is not associated with toxic accumulation of protein aggregates. Intriguingly, both amino acids mutated in human UNC45B are conserved and located in the UCS domain of in C. elegans UNC-45, which is required for myosin binding. These residues are in close proximity to established temperature-sensitive unc-45 mutations that cause sarcomeric disorganization and severe movement defects of worms grown at the restrictive temperature of 25°C (Figure 3) (mutant alleles: m94(Glu781Lys) and e286(Leu822Phe) (Barral et al., 1998).

Taken together, we hypothesize that the identified, myopathy-related UNC45B mutations affect myosin binding and/or sarcomere formation rather than causing proteotoxic conditions and protein aggregation.

  1. Frumkin, A., Pokrzywa, W., Karady, I., Hoppe T., and Ben-Zvi A. (2014) Front. Mol. Bio. 1: 21.
  2. Gazda, L., Pokrzywa, W., Hellerschmied, D., Lowe, T., Forne, I., Mueller-Planitz, F., Hoppe, T#., and Clausen, T. (2013) Cell 152: 183-195. (#co-corresponding senior author)
  3. Kim, J., Löwe, T., and Hoppe, T. (2008) Trends Cell Biol. 18: 264-272.
  4. Janiesch, P.C., Kim, J., Mouysset, J., Krause, S., and Hoppe, T. (2007) Nat. Cell. Biol. 9: 379-390.
  5. Hoppe, T., Barral, J.M., Springer, W., Epstein, H.F., and Baumeister, R. (2004) Cell 118: 337-349. 
  • Finger F, Ottens F, and Hoppe T (2021). The Argonaute Proteins ALG-1 and ALG-2 Are Linked to Stress Resistance and Proteostasis. MicroPubl Biol2021. doi:10.17912/micropub.biology.000457.
    • Franz A, Valledor P, Ubieto-Capella P, Pilger D, Galarreta A, Lafarga V, Fernandez-Llorente A, de la Vega-Barranco G, den Brave F, Hoppe T, Fernandez-Capetillo O, and Lecona E (2021). USP7 and VCP(FAF1) define the SUMO/Ubiquitin landscape at the DNA replication fork. Cell Rep37, 109819. doi:10.1016/j.celrep.2021.109819.
    • Muller L, Kutzner CE, Balaji V, and Hoppe T (2021). In Vitro Analysis of E3 Ubiquitin Ligase Function. J Vis Exp. doi:10.3791/62393.
    • Klionsky D.J. et al., (2021). Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition). Autophagy 1, 1-382.
    • Hohfeld J, Benzing T, Bloch W, Furst DO, Gehlert S, Hesse M, Hoffmann B, Hoppe T, Huesgen PF, Kohn M, Kolanus W, Merkel R, Niessen CM, Pokrzywa W, Rinschen MM, Wachten D, and Warscheid B (2021). Maintaining proteostasis under mechanical stress. EMBO Rep22, e52507. doi:10.15252/embr.202152507.
                        • Ottens F, Franz A, and Hoppe T (2021). Build-UPS and break-downs: metabolism impacts on proteostasis and aging. Cell Death Differ28, 505-521. doi:10.1038/s41418-020-00682-y.
                        • Albert MC, Brinkmann K, Pokrzywa W, Gunther SD, Kronke M, Hoppe T, and Kashkar H (2020). CHIP ubiquitylates NOXA and induces its lysosomal degradation in response to DNA damage. Cell Death Dis 11, 740.
                        • Balaji V, and Hoppe T (2020). Regulation of E3 ubiquitin ligases by homotypic and heterotypic assembly. F1000Res 9.
                        • Bayer EA, Stecky RC, Neal L, Katsamba PS, Ahlsen G, Balaji V, Hoppe T, Shapiro L, Oren-Suissa M, and Hobert O (2020). Ubiquitin-dependent regulation of a conserved DMRT protein controls sexually dimorphic synaptic connectivity and behavior. eLife 9.
                        • Donkervoort S., Kutzner C.E., Hu Y., Lornage X., Rendu J., Stojkovic T., Baets J., Neuhaus S.B., Tanboon J., Maroofian R., Bolduc V., Mroczek M., Conijn S., Kuntz N.L., Töpf A., Monges S., Lubieniecki F., McCarty R.M., Chao K.R., Governali S., Böhm J., Boonyapisit K., Malfatti E., Sangruchi T., Horkayne-Szakaly I., Hedberg-Oldfors C., Efthymiou S., Noguchi S., Djeddi S., Iida A., di Rosa G., Fiorillo C., Salpietro V., Darin N., Fauré J., Houlden H., Oldfors A., Nishino I., de Ridder W., Straub V., Pokrzywa W., Laporte J., Foley A.R., Romero N.B., Ottenheijm C., Hoppe T.*, Bönnemann C.G.* (2020). Pathogenic Variants in the Myosin Chaperone UNC-45B Cause Progressive Myopathy with Eccentric Cores. Am. J. Hum. Genet. 107: 1078-1095.
                        • Fatima A, Irmak D, Noormohammadi A, Rinschen MM, Das A, Leidecker O, Schindler C, Sanchez-Gaya V, Wagle P, Pokrzywa W, Hoppe T, Rada-Iglesias A, and Vilchez D (2020). The ubiquitin-conjugating enzyme UBE2K determines neurogenic potential through histone H3 in human embryonic stem cells. Commun Biol 3, 262.
                        • Hoppe T, and Cohen E (2020). Organismal Protein Homeostasis Mechanisms. Genetics 215, 889-901.
                        • Ravanelli S, den Brave F, and Hoppe T (2020). Mitochondrial Quality Control Governed by Ubiquitin. Front Cell Dev Biol 8, 270.
                        Prof. Dr. Thorsten Hoppe CMMC Cologne
                        Prof. Dr. Thorsten Hoppe

                        Institute for Genetics - Lab. for Proteostasis in Development and Aging - CECAD Research Center

                        CMMC - PI - C 08

                        +49 221 478 84218

                        +49 221 478 7789

                        Institute for Genetics - Lab. for Proteostasis in Development and Aging - CECAD Research Center

                        Joseph-Stelzmann-Str. 26

                        50931 Cologne

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                        Publications - Thorsten Hoppe

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                        Group Members

                        Dr. Alexandra Serge
                        Dr. Andre Franz
                        Dr. Ricardo Marchante
                        Dr. Franziska Ottens
                        Dr. Lena Schütter
                        Vishnu Balaji
                        Lylan Bramasole
                        Sotirios Efstathiou
                        Carl Elias Kutzner
                        Leonie Müller
                        Andriana Ntogka
                        Sonia Ravanelli
                        Kavya Vakkayil
                        Agata Lisowski
                        Gabriele Stellbrink
                        Gaby Hopper
                        Karoline Bendig
                        Kerstin Walfort