Peroxisome biology and peroxisomal disorders

Peroxisomal disorders are caused by defects in peroxisome functions. Peroxisome biogenesis disorders (PBDs) include the Zellweger syndrome spectrum and rhizomelic chondrodysplasia punctata type 1. The Zellweger syndrome spectrum is a continuum of disorder characterized by developmental brain defects, skeletal and craniofacial dysmorphism, liver dysfunction, retinopathy, and sensorineural hearing loss.

Many questions relating to biogenesis, function and pathophysiology of peroxisomes, the T-tubule sys­tem and the endo­membrane system are still unanswered. Following up on the ongoing and recently com­pleted work on the intracellular transport of membrane proteins, the function of readthrough pro­teins, and on the role of peroxisomes in the heart and in neuronal tissue cells, we are focussing our interest on fhe functin and biogenesis of per­oxi­so­mes and the T-tubule system.


We recently completed the first super-resolution microscopy study of peroxisomes in wild-type human fibro­blasts and cells derived from patients with a peroxisomal biogenesis disorder (Figure) (Soliman et al. 2017)



STED micros­copy re­veals the sub-diffraction mor­pho­logy of per­oxisomes.  Two-color STED of per­oxi­some mem­brane and matrix. Im­mu­no­fluor­escence on human skin fibro­blasts with anti-PMP70 (per­oxi­some mem­brane) and anti-ACAA1 (ace­tyl-CoA acyltransferase1, ma­trix). (Figure from Soliman et al. 2018).urrently, we are studying peroxisome function in the heart (using stem cell and mouse models) and in the brain.

Translational readthrough and readthrough therapy

FTR (functional translational readthrough) is a gene regulatory process in which stop codons are inter­pre­ted as sense codons during translation (Figure). By developing novel bioinformatics and screening tech­niques, we co-discovered the first hu­man genes regulated by FTR: Lactate and ma­late de­hydrogenase (LDH and MDH) are ex­ten­ded by several amino acids by FTR [Schueren et al. Hofhus Et al. Schueren and Thoms]. The ex­tensions (‘x’) con­tain functional targeting signals for peroxisomes.




Endogenous trans­lational read­through – Ex­ploring a new world of pro­teins. LDHBx, the ex­ten­ded form of LDHB, is ge­ne­ra­ted by trans­lational read­through (TR) of the first stop co­don during trans­lation of the normal mRNA into pro­te­in. The inter­pre­ta­tion of the stop codon as a sense co­don leads to the pro­long­ation of trans­lation until the next in-frame stop codon is reached. This leads to the C-ter­mi­nal addition of seven amino acids to the normal LDHB. The extension (between StopTR and Stop) har­bors a per­oxi­so­mal tar­ge­ting signal (PTS). Tetra­mers, which comprise the conventional subunits LDHB and/or LDHA together with a read­through subunit LDHBx, can be trans­ported into peroxisomes by the per­oxi­somal import machinery (piggy­back im­port). The same me­chanism com­bining readthrough and a hidden PTS1 after the first stop codon leads to the peroxisomal import of dimeric MDH1. The NCBI has adopted the definition of the x-nomen­clature for read­through proteins.

Based this FTR mechanism, novel peroxisomal redox shuttles may exist (lactate-pyruvate, and aspar­tate-malate shuttles). Readthrough-dependent metabolite shuttles can explain import of redox equivalents into the peroxisome and their export with mitochondria as the final destination.

The physiological readthrough is stimu­lated by a nucleotide consensus motif, which is also present in other genes that may be regulated by FTR.

LDHBx and MDH1x are only two of more than 50 new proteins that we have discovered to receive readthrough-extensions by a very leaky stop codon nucleotide context.

One goal of our research is to functionally and structurally understand the re­gu­la­tion of trans­lational readthrough.

Translational readthrough. Competition of near-cognate tRNA (nc-tRNA) and release factor bind­ing at the A-site of the ribosome is the basis of trans­lational readthrough. In prokaryotes and eukaryotes, ter­mi­nation/release factors (eRF) mediate the ter­mi­nation of translation. By binding of nc-tRNA in place of eRF, the stop codon is read-through and an ex­tended pro­tein is formed. Normally, the equilibrium be­tween re­lease factor binding and (mis)incorporation of nc-tRNAs is >99.9% on the side of release factor bind­ing and termination. Several conditions can shift this equi­librium: (1) We have identified stop codon contexts (SCCs), defined as stop codon with neigh­boring nu­cle­o­tides that drastically shift the equilibrium towards nc-tRNA binding and continuation of translation so that the readthrough probability is increased by one or two orders of magnitude. This endogenous mechanism is universally conserved. (2) Amino­glycosides and other low mo­lecular weight substances can bind to the small subunit of the ribosome and increase readthrough pro­ba­bi­li­ty. This is a viable approach in the therapy of diseases caused by premature stop codon mutations.

Dysferlin and dysferlinopathies

Dysferlinopathies belong to the diverse group of muscular dystrophies and are caused by the absence of Dysferlin protein in skeletal muscle. The main two forms are Miyoshi myopathy (MM) and limb girdle muscular dystrophy type 2B (LGMD 2B). These diseases are characterized by progressive muscular weakness and wasting, typically starting in the second decade of life. Interestingly, about half of the Dysferlin-deficient patients show an unusual disease phenotype by displaying increased levels of fitness before the onset of symptoms, which is not seen in any other form of muscular dystrophy. So far, no curative treatment is available.

The 230 kDa protein Dysferlin is composed of a C-terminal transmembrane domain and seven C2 domains. C2 domains are often associated with membrane binding and tubulating functions.​ Dysferlin has been shown to localize to the plasma membrane and the T-Tubule system in skeletal muscle and is known to act on intracellular membranes and to play a role in membrane repair during muscle regeneration.

The T-tubule system is an extensive network formed by invaginations of the plasma mem­brane ac­counting for about 80% of the plasma membrane surface of striated muscle. Employing a wide va­riety of methods from in vitro reconstitution in liposomes, through cell models like human myo­blasts, to mouse models, we could show that Dysferlin tubulates liposomes, generates a T-tubule-like mem­brane sys­tem in non-muscle cells, and links the re­cruitment of phosphatidylinositol 4,5-bis­phosphate to the bio­gene­sis of the T-tubule system (Hofhuis et al. 2017). Pathogenic mutants interfere with all of these functions, in­di­cating that muscular wasting and dystrophy are caused by the Dysferlin mutants’ in­ability to form a functional T-tubule membrane system.

Dysferlin is required for T-tubule formation in vivo (and in vitro). The multi-C2 domain protein dysferlin localizes to the plasma membrane and the T-tubule system in skeletal muscle, however, its physiological mode of action is unknown. Mutations in the DYSF gene lead to autosomal recessive limb-girdle muscular dys­trophy type 2B (LDMD2B). Tubule staining, using DiIC16(3), of human wild-type and dysferlin-deficient myoblasts, after differentiation in culture, shows compact tubular aggregates in dysferlin-deficient myoblasts that were not observed in control fibres. Scale bar: 10 µm. Figure from Hofhuis et al 2017.

Far less is known about the role and function of Dysferlin in the heart as dysferlinopathies are mainly considered as skeletal muscle diseases and only few patients suffer from cardiomyopathies. However, we showed that also in the heart, Dysferlin executes an important role in maintenance of the tubular system (TATS) (Hofhuis et al. 2020). By analysing Dysferlin expression and localization in healthy rat and mouse cardiomyocytes and Dysferlin-deficient mouse cardiomyocytes we were able to show that the protein localizes to the TATS in the heart and expression is upregulated during development. Absence of Dysferlin leads to massive structural reorganization and loss of transverse TATS structures resulting in defective calcium homeostasis and arrhythmia susceptibility that may have functional consequences in dysferlinopathy patients.