Structural insights into lethal contractural syndrome type 3 (LCCS3) caused by a missense mutation of PIP5Kγ.

Signaling molecule phosphatidylinositol 4,5-bisphosphate is produced primarily by phosphatidylinositol 4-phosphate 5-kinase (PIP5K). PIP5K is essential for the development of the human neuronal system, which has been exemplified by a recessive genetic disorder, lethal congenital contractural syndrome type 3, caused by a single aspartate-to-asparagine mutation in the kinase domain of PIP5Kγ. So far, the exact role of this aspartate residue has yet to be elucidated. In this work, we conducted structural, functional and computational studies on a zebrafish PIP5Kα variant with a mutation at the same site. Compared with the structure of the wild-type (WT) protein in the ATP-bound state, the ATP-associating glycine-rich loop of the mutant protein was severely disordered and the temperature factor of ATP was significantly higher. Both observations suggest a greater degree of disorder of the bound ATP, whereas neither the structure of the catalytic site nor the Km toward ATP was substantially affected by the mutation. Microsecond molecular dynamics simulation revealed that negative charge elimination caused by the mutation destabilized the involved hydrogen bonds and affected key electrostatic interactions in the close proximity of ATP. Taken together, our data indicated that the disease-related aspartate residue is a key node in the interaction network crucial for effective ATP binding. This work provides a paradigm of how a subtle but critical structural perturbation caused by a single mutation at the ATP-binding site abolishes the kinase activity, emphasizing that stabilizing substrate in a productive conformational state is crucial for catalysis.

Hereditary inclusion-body myopathies.

The term hereditary inclusion-body myopathies (HIBMs) defines a group of rare muscle disorders with autosomal recessive or dominant inheritance and presence of muscle fibers with rimmed vacuoles and collection of cytoplasmic or nuclear 15-21 nm diameter tubulofilaments as revealed by muscle biopsy. The most common form of HIBM is due to mutations of the GNE gene that codes for a rate-limiting enzyme in the sialic acid biosynthetic pathway. This results in abnormal sialylation of glycoproteins that possibly leads to muscle fiber degeneration. Mutations of the valosin containing protein are instead responsible for hereditary inclusion-body myopathy with Paget's disease of the bone and frontotemporal dementia (IBMPFD), with these three phenotypic features having a variable penetrance. IBMPFD probably represents a disorder of abnormal cellular trafficking of proteins and maturation of the autophagosome. HIBM with congenital joint contractures and external ophthalmoplegia is due to mutations of the Myosin Heavy Chain IIa gene that exerts a pathogenic effect through interference with filament assembly or functional defects in ATPase activity. This review illustrates the clinical and pathologic characteristics of HIBMs and the main clues available to date concerning the possible pathogenic mechanisms and therapeutic perspectives of these disorders. This article is part of a Special Issue entitled: Neuromuscular Diseases: Pathology and Molecular Pathogenesis.

Human ZMPSTE24 disease mutations: residual proteolytic activity correlates with disease severity.

The zinc metalloprotease ZMPSTE24 plays a critical role in nuclear lamin biology by cleaving the prenylated and carboxylmethylated 15-amino acid tail from the C-terminus of prelamin A to yield mature lamin A. A defect in this proteolytic event, caused by a mutation in the lamin A gene (LMNA) that eliminates the ZMPSTE24 cleavage site, underlies the premature aging disease Hutchinson-Gilford Progeria Syndrome (HGPS). Likewise, mutations in the ZMPSTE24 gene that result in decreased enzyme function cause a spectrum of diseases that share certain features of premature aging. Twenty human ZMPSTE24 alleles have been identified that are associated with three disease categories of increasing severity: mandibuloacral dysplasia type B (MAD-B), severe progeria (atypical 'HGPS') and restrictive dermopathy (RD). To determine whether a correlation exists between decreasing ZMPSTE24 protease activity and increasing disease severity, we expressed mutant alleles of ZMPSTE24 in yeast and optimized in vivo yeast mating assays to directly compare the activity of alleles associated with each disease category. We also measured the activity of yeast crude membranes containing the ZMPSTE24 mutant proteins in vitro. We determined that, in general, the residual activity of ZMPSTE24 patient alleles correlates with disease severity. Complete loss-of-function alleles are associated with RD, whereas retention of partial, measureable activity results in MAD-B or severe progeria. Importantly, our assays can discriminate small differences in activity among the mutants, confirming that the methods presented here will be useful for characterizing any new ZMPSTE24 mutations that are discovered.

Different levels of COX-1 and COX-2 enzymes in synoviocytes and chondrocytes during joint contracture formation.

OBJECTIVE: To measure the levels of prostaglandin endoperoxide H synthase (PGHS) isozymes (or cyclooxygenase, COX) in vivo during the development of joint contractures secondary to immobilization in rats. METHODS: Rats had one knee joint immobilized for up to 32 weeks. Three groups were compared: 47 rats had knee joints immobilized, 38 animals had sham surgery, and 13 unoperated animals served as controls. Levels of PGHS-1 and PGHS-2 enzymes were characterized in the chondrocytes and synoviocytes of the knee joint by immunohistochemistry. Immunostaining intensity was quantified by microscopy using conventional analysis. RESULTS: PGHS-1 level was lower in synoviocytes of the anterior capsule compared with shams (1.3 vs 2.0; p < 0.05). PGHS-2 level was also lower in synoviocytes of the posterior capsule (1.8 vs 2.3; p < 0.05), but higher in chondrocytes at the anterior aspect of the tibia compared with shams (1.6 vs 0.8; p < 0.05). PGHS-2 staining was increased in chondrocytes at the posterior, opposed, and anterior aspects of the tibia compared with controls (1.1, 0.6, 0.8 vs 0.2, 0.1, 0.2, respectively; all p < 0.05). CONCLUSION: Immobility induced joint contractures are characterized by a contrasting cellular pattern of PGHS enzyme levels: decreased in the synovium and increased in the chondrocytes. These findings suggest that chondrocytic PGHS isoenzymes are important in cartilage degradation of contractured joints.

Myoadenylate deaminase deficiency in children.

Myoadenylate deaminase (MADA) is an enzyme which participates in the purine nucleotide cycle necessary for energy production in human skeletal muscle. Approximately 35 patients with deficiency of this enzyme have been reported; one-half experienced their initial difficulties in childhood. Children with "primary" MADA deficiency typically have symptoms including muscle cramps, stiffness, and post-exercise myalgia and weakness. In "secondary" MADA deficiency, the clinical findings have been variable with delayed motor development, hypotonia, cardiomyopathy, delayed speech development, and generalized weakness. In most cases creatine kinase determinations, nerve conduction velocity studies, and routine muscle histopathology have been normal. Diagnosis has been established by demonstrating an absence of MADA activity by either direct muscle enzyme assay or histochemical staining. In this report we describe a 12-year-old boy with primary MADA deficiency and contrast his symptoms with those of previously described pediatric patients.

[Clinical and experimental studies on muscle contracture].

Concerning muscle contracture, etiology, diagnosis and treatment still remain a problem. The author examined diagnosis and treatment clinically and examined the influence of muscle fiber after intramuscular injection of some drugs. Clinical and experimental results were as follows. 1) In the diagnosis of quadriceps muscle contracture, the measurement of buttock elevation phenomenon varies according to the examiner, and in healthy people, the same phenomenon may occur. Hence, the necessity of not over-estimating the phenomenon itself should be emphasized. 2) Adult cases with deltoid muscle contracture are accompanied by pain. 3) Intramuscular injection of chloramphenicol sol on rabbit brought remarkable muscle lesion (fibrosis, lowering of phosphorylase activity) one year after ten consecutive daily injections. Intramuscular injection of chloramphenicol sol causes irreversible lesion in the muscle and this is one of the main causes of etiology of muscle contracture.