Christoph S. Clemen

Accelerated Clearance of Plasmodium-infected Erythrocytes in Sickle Cell Trait and Annexin-A7 Deficiency

The course of malaria does not only depend on the virulence of the parasite Plasmodium but also on properties of host erythrocytes. Here, we show that infection of erythrocytes from human sickle cell trait (HbA/S) carriers with ring stages of P. falciparum led to significantly enhanced PGE2 formation, Ca2+ permeability, annexin-A7 degradation, phosphatidylserine (PS) exposure at the cell surface, and clearance by macrophages. P. berghei-infected erythrocytes from annexin-A7-deficient (annexin-A7-/-) mice were more rapidly cleared than infected wildtype cells. Accordingly, P. berghei-infected annexin-A7-/- mice developed less parasitemia than wildtype mice. The cyclooxygenase inhibitor aspirin decreased erythrocyte PS exposure in infected annexin-A7-/- mice and abolished the differences of parasitemia and survival between the genotypes. Conversely, the PGE2-agonist sulprostone decreased parasitemia and increased survival of wild type mice. In conclusion, PS exposure on erythrocytes results in accelerated clearance of Plasmodium ring stage-infected HbA/S or annexin-A7-/- erythrocytes and thus confers partial protection against malaria in vivo.

Desminopathies: pathology and mechanisms

The intermediate filament protein desmin is an essential component of the extra-sarcomeric cytoskeleton in muscle cells. This three-dimensional filamentous framework exerts central roles in the structural and functional alignment and anchorage of myofibrils, the positioning of cell organelles and signaling events. Mutations of the human desmin gene on chromosome 2q35 cause autosomal dominant, autosomal recessive, and sporadic myopathies and/or cardiomyopathies with marked phenotypic variability. The disease onset ranges from childhood to late adulthood. The clinical course is progressive and no specific treatment is currently available for this severely disabling disease. The muscle pathology is characterized by desmin-positive protein aggregates and degenerative changes of the myofibrillar apparatus. The molecular pathophysiology of desminopathies is a complex, multilevel issue. In addition to direct effects on the formation and maintenance of the extra-sarcomeric intermediate filament network, mutant desmin affects essential protein interactions, cell signaling cascades, mitochondrial functions, and protein quality control mechanisms. This review summarizes the currently available data on the epidemiology, clinical phenotypes, myopathology, and genetics of desminopathies. In addition, this work provides an overview on the expression, filament formation processes, biomechanical properties, post-translational modifications, interaction partners, subcellular localization, and functions of wild-type and mutant desmin as well as desmin-related cell and animal models.

Beyond LGMD1A: myotilin is a component of central core lesions and nemaline rods

Myotilin is a Z-disc protein that binds alpha-actinin, gamma-filamin and F-actin. The essential role of myotilin in skeletal muscle is highlighted by the recent observation that autosomal dominant limb girdle muscular dystrophy type 1A is caused by mutations in the human myotilin gene. We studied the expression and subcellular distribution of myotilin in nemaline myopathy, central core disease, centronuclear myopathy, and myopathies with tubular aggregates. A prominent myotilin immunostaining of nemaline rods and core lesions was detected in all ten cases of nemaline myopathy and five cases of central core disease. This renders myotilin a sensitive, though non-specific marker for these structural lesions. Western blot analysis did not indicate an increased myotilin expression in nemaline myopathy muscle. However, the analysis indicated upregulation of a 75 kDa immunoreactive band, very weak in control muscle but previously detected in limb girdle muscular dystrophy 1A samples. Our findings indicate that myotilin is a core structural molecule in nemaline rods and central core lesions and suggest modification of myotilin in nemaline myopathy, and further support the notion that myotilin may have a key role in the dynamic molecular events mediating myofibril assembly in normal and diseased human skeletal muscle.

CAP2, cyclase-associated protein 2, is a dual compartment protein.

Abstract.Cyclase-associated proteins (CAPs) are evolutionarily conserved proteins with roles in regulating the actin cytoskeleton and in signal transduction. Mammals have two CAP genes encoding the related CAP1 and CAP2. We studied the distribution and subcellular localization of CAP1 and CAP2 using specific antibodies. CAP1 shows a broad tissue distribution, whereas CAP2 is significantly expressed only in brain, heart and skeletal muscle, and skin. CAP2 is found in the nucleus in undifferentiated myoblasts and at the M-line of differentiated myotubes. In PAM212, a mouse keratinocyte cell line, CAP2 is enriched in the nucleus, and sparse in the cytosol. By contrast, CAP1 localizes to the cytoplasm in PAM212 cells. In human skin, CAP2 is present in all living layers of the epidermis localizing to the nuclei and the cell periphery. In in vitro studies, a C-terminal fragment of CAP2 interacts with actin, indicating that CAP2 has the capacity to bind to actin.

Strumpellin is a novel valosin-containing protein binding partner linking hereditary spastic paraplegia to protein aggregation diseases

Mutations of the human valosin-containing protein gene cause autosomal-dominant inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia. We identified strumpellin as a novel valosin-containing protein binding partner. Strumpellin mutations have been shown to cause hereditary spastic paraplegia. We demonstrate that strumpellin is a ubiquitously expressed protein present in cytosolic and endoplasmic reticulum cell fractions. Overexpression or ablation of wild-type strumpellin caused significantly reduced wound closure velocities in wound healing assays, whereas overexpression of the disease-causing strumpellin N471D mutant showed no functional effect. Strumpellin knockdown experiments in human neuroblastoma cells resulted in a dramatic reduction of axonal outgrowth. Knockdown studies in zebrafish revealed severe cardiac contractile dysfunction, tail curvature and impaired motility. The latter phenotype is due to a loss of central and peripheral motoneuron formation. These data imply a strumpellin loss-of-function pathogenesis in hereditary spastic paraplegia. In the human central nervous system strumpellin shows a presynaptic localization. We further identified strumpellin in pathological protein aggregates in inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia, various myofibrillar myopathies and in cortical neurons of a Huntingtons disease mouse model. Beyond hereditary spastic paraplegia, our findings imply that mutant forms of strumpellin and valosin-containing protein may have a concerted pathogenic role in various protein aggregate diseases.

Expression and localisation of annexin VII (synexin) isoforms in differentiating myoblasts.

Annexin VII exists in a 47 kDa and a 51 kDa isoform with the 51 kDa protein being the only isoform present in skeletal muscle. Expression of the 51 kDa isoform during myogenesis and localization was studied in cells after conversion into myogenic cells by transduction with MyoD and in mouse and human myogenic cell lines. MyoD expression in NIH3T3 and C3H10T1/2 fibroblasts led to disappearance of the mRNA specific for the 47kDa isoform and appearance of the 51 kDa isoform-specific mRNA. The overall amount of annexin VII protein was reduced in myogenic converted cells. Both in undifferentiated and differentiated cells annexin VII was localized by immunofluorescence microscopy to punctate structures which were distributed all over the cell. A GFP annexin VII fusion protein showed a similar distribution. Cell fractionation studies indicated that annexin VII is equally distributed between cytosol and membrane fractions in undifferentiated cells, while in differentiated cells it is exclusively present in the membrane fraction. By sucrose gradient centrifugation of postnuclear supernatants we identified two distinct annexin VII-containing membrane populations that cofractionated with caveolin 3- and sorcin-containing membranes.

Primary effusion lymphoma: genomic profiling revealed amplification of SELPLG and CORO1C encoding for proteins important for cell migration

Primary effusion lymphoma (PEL) is associated with Kaposi sarcoma herpesvirus (KSHV) but its pathogenesis is poorly understood. Many KSHV‐associated products can deregulate cellular pathways commonly targeted in cancer. However, KSHV infection alone is insufficient for malignant transformation. PEL also lacks the chromosomal translocations seen in other lymphoma subtypes. We investigated 28 PELs and ten PEL cell lines by 1 Mb resolution array comparative genomic hybridization (CGH) and found frequent gains of 1q21–41 (47%), 4q28.3‐35 (29%), 7q (58%), 8q (63%), 11 (32%), 12 (61%), 17q (29%), 19p (34%), and 20q (34%), and losses of 4q (32%), 11q25 (29%), and 14q32 (63%). Recurrent focal amplification was seen at several regions on chromosomes 7, 8, and 12. High‐resolution chromosome‐specific tile‐path array CGH confirmed these findings, and identified selectin‐P ligand (SELPLG) and coronin‐1C (CORO1C) as the targets of a cryptic amplification at 12q24.11. Interphase FISH and quantitative PCR showed SELPLG/CORO1C amplification (>4 extra copies) and low levels of copy number gain (1–4 extra copies) in 23% of PELs, respectively. Immunohistochemistry revealed strong expression of both SELPLG and coronin‐1C in the majority of PELs, irrespective of their gene dosage. SELPLG is critical for cell migration and chemotaxis, while CORO1C regulates actin‐dependent processes, thus important for cell motility. Their overexpression in PEL is expected to play an important role in its pathogenesis. Copyright

α-Actinin 4 and BAT1 interaction with the Cytochrome c promoter upon skeletal muscle differentiation

To identify common regulatory features of nuclear genes encoding mitochondrial proteins we searched for regulatory elements in the Cytochrome c promoter during skeletal muscle differentiation in cell culture. A consensus element with the sequence GCTGCCGCAC-(N4-20)-GGSCGYGGG was found in both rat Cyt c and coxIV promoters. This new sequence element with yet undescribed function, but high abundance in promoters of nuclear genes encoding mitochondrial proteins available from the databases, showed a striking change in protein binding in electromobility shift assays when myoblasts were compared to myotubes. Proteins involved in the observed protein-DNA complexes were isolated from myotubes and identified by MALDI-TOF as BAT1, a DEAD-box protein of yet unknown function, heat shock protein HSP84, and α-actinin 4, a non-muscle isoform of the structural protein α-actinin. α-actinin 4 was found to be preferentially localized in the nucleus upon induction of myogenesis, suggesting a signaling function during muscle differentiation. In conclusion, the analyzed sequence motif may be a new candidate for common regulatory elements specific for nuclear encoded mitochondrial genes, and α-actinin 4 may be involved in their regulation.

Function, expression and localization of annexin A7 in platelets and red blood cells: Insights derived from an annexin A7 mutant mouse

BackgroundAnnexin A7 is a Ca2+- and phospholipid-binding protein expressed as a 47 and 51 kDa isoform, which is thought to be involved in membrane fusion processes. Recently the 47 kDa isoform has been identified in erythrocytes where it was proposed to be a key component in the process of the Ca2+-dependent vesicle release, a process with which red blood cells might protect themselves against an attack by for example complement components.ResultsThe role of annexin A7 in red blood cells was addressed in erythrocytes from anxA7-/- mice. Interestingly, the Ca2+-mediated vesiculation process was not impaired. Also, the membrane organization appeared not to be disturbed as assessed using gradient fractionation studies. Instead, lack of annexin A7 led to an altered cell shape and increased osmotic resistance of red blood cells. Annexin A7 was also identified in platelets. In these cells its loss led to a slightly slower aggregation velocity which seems to be compensated by an increased number of platelets. The results appear to rule out an important role of annexin A7 in membrane fusion processes occurring in red blood cells. Instead the protein might be involved in the organization of the membrane cytoskeleton. Red blood cells may represent an appropriate model to study the role of annexin A7 in cellular processes.ConclusionWe have demonstrated the presence of both annexin A7 isoforms in red blood cells and the presence of the small isoform in platelets. In both cell types the loss of annexin A7 impairs cellular functions. The defects observed are however not compatible with a crucial role for annexin A7 in membrane fusion processes in these cell types.

Hsp27-2D-gel electrophoresis is a diagnostic tool to differentiate primary desminopathies from myofibrillar myopathies

Small heat shock proteins prevent abnormal protein folding and accumulation. We analyzed the expression of hsp27 and αB‐crystallin in skeletal muscle specimens of patients with desminopathies, plectinopathies, myotilinopathy, and other myofibrillar myopathies by means of differential centrifugation, 2D‐gel electrophoresis, Western blotting, and mass spectrometry. Hsp27‐P82 and ‐P15 as well as αB‐crystallin‐P59 and ‐P45 are the major serine phosphorylation isoforms in normal and diseased human skeletal muscle. 2D‐gel‐electrophoresis revealed spots of hsp27 in a range of pH 5.3–6.4 in samples of all skeletal muscle specimens, except for the seven desminopathies. They indicated a shift of the main hsp27‐spot to alkaline pH degrees, which may help to differentiate primary desminopathies from other myopathies with structural pathology of the desmin cytoskeleton.