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. By monitoring myopathy-related defects in Caenorhabditis elegans and patient muscle combined with structure-function analysis of UNC45B mutations, we 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 had remained completely unclear until now.

The CMMC-funded project took advantage of the unique opportunity to explore the patho-mechanism associated with four novel 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.

In the current funding period, we

  1. characterized structure-functional properties of disease-associated UNC45B mutations;
  2. explored the role of UNC45B mutations on myofibrillogenesis in C. elegans; and
  3. defined myopathy-related UNC45B dysfunction in patient muscle.

We identified a common UNC45B variant that acts as a complex hypomorph splice variant. Purified UNC-45B mutants showed changes in folding and solubility. In situ localization studies further demonstrated reduced expression of mutant UNC-45B in muscle combined with abnormal localization away from the A-band towards the Z-disk of the sarcomere. The physiological relevance of these observations was investigated in C. elegans by transgenic expression of conserved UNC-45 missense variants, which showed impaired myosin binding for one and defective muscle function for three. Together, our results demonstrate that UNC-45B impairment manifests as a chaperonopathy with progressive muscle pathology, which discovers the previously unknown conserved role of UNC-45B in myofibrillar organization.

The major findings of this CMMC funded project were published in the American Journal of Human Genetics (Donkervoort, Kutzner et al., 2020).

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 1and 4).

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).

Figure 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).

Figure 3

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). Given the constant use and mechanical stress exposure of the muscle sarcomere, maintenance of muscle function involves a network of protein folding and degradation pathways (Hoppe et al., 2004; Kim et al., 2008). Due to its central role in myosin assembly, UNC-45 protein localization and amount are tightly coordinated with muscle development and stress conditions. In zebrafish it has been shown that UNC-45B shuttles between the Z-disc and the myosin containing A-band of the muscle sarcomere upon eccentric exercise or induced damage to the myofiber (Figure 4B) (Etard et al., 2008). 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). Thus, the growth and maintenance of myofilaments depend on the interplay of protein folding and degradation.

Figure 4

Our findings in the current Funding Period

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. Together, we identified a total of ten patients from eight independent families, who clinically manifest with childhood-onset, progressive proximal and axial muscle weakness and various degrees of respiratory insufficiency. To elucidate the possible origin of the muscle disease, whole exome sequencing was pursued, identifying a recurring homozygous c.2261G>A; p.Arg754Gln missense variant in UNC45B (NM_173167.3) in seven affected individuals from five independent families of various ethnic backgrounds impacting a conserved Arginine in the myosin-binding UCS domain of UNC45B (Figure 4A). Three more patients were found to be compound heterozygous or homozygous for variants in the myosin-binding UCS domain (P2, P9) and the oligomer-forming neck domain of UNC45B (P9, P10) (Figure 4A).

To characterize the structure-functional properties of disease-associated UNC45B mutations, we first used PyMOL modelling of van der Waals overlaps and detected potential steric interferences in the protein structures surrounding the mutation sites (Figure 5A). We next recombinantly expressed the UNC45B mutant proteins in E. coli, subjected them to time-dependent partial trypsin proteolysis experiments and found that the mutant proteins are less susceptible to proteolysis (Figure 5B). Thermal shift assays using the protein stain SYPRO Orange, which intercalates with gradually exposed hydrophobic residues on the protein's surface, revealed more exposed hydrophobic residues for all mutant proteins (Figure 5C). We finally tested for aggregation propensity using a filter trap assay and confirmed the notion, that the UNC45B mutant proteins are aggregation prone and display structural changes (Figure 5D).

Figure 5

To explore the role of UNC45B mutations on myofibrillogenesis in C. elegans, we used a genetic in vivo rescue approach to test whether the muscle-specific expression of UNC45B variantscompensates UNC-45 function and restores myofilament organization in an unc-45 mutant background. Temperature-sensitive unc-45(m94) mutant worms exhibit a severe movement defect and disarrangement of the otherwise highly conserved sarcomere organization when grown at the non-permissive temperature of 25°C. The transgenic expression of the corresponding C. elegans UNC-45 variants in these worms still resulted in defective muscle function for three variants in motility assays in liquid and on a solid agar surface using the ARENA WMicrotracker (NemaMetrix), and in phalloidin staining of muscle filaments (Figure 6A-C). For one variant, immunoprecipitation experiments confirmed that myosin binding was impaired suggesting conformational changes of the myosin-binding canyon in the UCS domain for this variant (Figure 6D).

Figure 6

To define the myopathy-related UNC45B dysfunction in patient muscle, we collaborated with the laboratories of Carsten Bönnemann, Coen Ottenheijm, Jocelyn Laporte, and many clinical pathologists all over the world. Histological analyses of patient muscle biopsies showed internalized nuclei, cytoplasmic bodies, oxidative defects, and reduced oxidative activity with core-like regions, consistent with eccentric cores, among others. Electron microscopy unveiled large areas of myofibrillar disorganization and unstructured cores (Figure 7A-C). Single muscle fiber mechanics revealed a type I fiber dominance but otherwise no impairment of fiber contractile function. Immunofluorescence location studies suggested a reduction and mislocalization of UNC45B variant proteins compared to wild type (Figure 7D-E). In muscle RNA sequencing, a recurring UNC45B variant was identified to act as a complex hypomorph splice variant causing the inclusion of an in-frame STOP codon and subsequent degradation of the nonsense transcript via nonsense-mediated decay (Figure 7F).

Figure 7

Taken together, we report that UNC45B impairment manifests as a chaperonopathy with progressive muscle pathology, which discovers the previously unknown, conserved role of UNC45B in myofibrillar organization. The disease mechanism involves alterations in UNC45B mRNA splicing and UNC45B protein levels, as well as disruptions of myosin binding, folding, and localization.

  • Frumkin, A., Pokrzywa, W., Karady, I., Hoppe T., and Ben-Zvi A. (2014) Front. Mol. Bio. 1: 21.
  • 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)
  • Kim, J., Löwe, T., and Hoppe, T. (2008) Trends Cell Biol. 18: 264-272.
  • Janiesch, P.C., Kim, J., Mouysset, J., Krause, S., and Hoppe, T. (2007) Nat. Cell. Biol. 9: 379-390.
  • Hoppe, T., Barral, J.M., Springer, W., Epstein, H.F., and Baumeister, R. (2004) Cell 118: 337-349. 
  • Vakkayil K.L. and Hoppe T. (2022). Temperature-Dependent Regulation of Proteostasis and Longevity. Front. Aging 3: 853588.
  • Das A., Thapa P., Santiago U., Shanmugam N., Banasiak K., Dąbrowska K., Nolte H., Szulc N.A., Gathungu R.M., Cysewski D., Krüger M., Dadlez M., Nowotny M., Camacho C.J., Hoppe T., Pokrzywa W. (2022). A heterotypic assembly mechanism regulates CHIP E3 ligase activity. EMBO J. 41: e109566.
  • Balaji V., Müller L., Lorenz R., Kevei E., Zhang W.H., Santiago U., Gebauer J., Llamas E., Vilchez E., Camacho C.J., Pokrzywa W., Hoppe T. (2022). A dimer-monomer switch controls CHIP-dependent substrate ubiquitylation and processing. Molecular
  • 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.
  • Herzog L.K., Kevei É., Marchante R., Böttcher C., Bindesbøll C., Lystad A.H., Pfeiffer A., Gierisch M.E., Salomons F.A., Simonsen A., Hoppe T.*, Dantuma N.P. (2020). The Machado–Joseph disease deubiquitylase ataxin‐3 interacts with LC3C/GABARAP and promotes autophagy. Aging Cell: e13051. (*co-corresponding author)
  • Dantuma N.P., Hoppe T., and Herzog L.K. (2020). The price of longevity. Aging 12.
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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Group Members

Karoline Bendig (office)
Kerstin Walfort (office)
Dr. Alexandra Segref (PostDoc)
Dr. André Franz (PostDoc)
Dr. Nikolaos Charmpilas (PostDoc)
Dr. Franziska Ottens (PostDoc)
Dr. Qiaochu Li (PostDoc)
Karen Bauer (PhD student)
Sotirios Efstathiou (PhD student)
Carl Elias Kutzner (PhD student)
Leonie Müller (PhD student)
Andriana Ntogka (PhD student)
Serena Salman (PhD student)
Isabel Conze (Master student)
Fuateima Niwa (Master student)
Agata Lisowski (Technician)
Gabriele Stellbrink (Technician)
Gaby Vopper (Technician)