Calcium level and autophagy defect in GNE mutants of rare neuromuscular disorder.

Rare genetic disorders are low in prevalence and hence there is little or no attention paid to them in the mainstream medical industry. One of the ultra-rare neuromuscular disorders, GNE myopathy is caused due to biallelic mutations in the bifunctional enzyme, GNE (UDP N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase). It catalyses the rate-limiting step in sialic acid biosynthesis.

Altered autophagic flux in GNE mutant cells of Indian origin: Potential drug target for GNE myopathy.

Autophagy phenomenon in the cell maintains proteostasis balance by eliminating damaged organelles and protein aggregates. Imbalance in autophagic flux may cause accumulation of protein aggregates in various neurodegenerative disorders. Regulation of autophagy by either calcium or chaperone play a key role in the removal of protein aggregates from the cell.

Potential small effector molecules restoring cellular defects due to sialic acid biosynthetic enzyme deficiency: Pathological relevance to GNE myopathy.

GNEM (GNE Myopathy) is a rare neuromuscular disease caused due to biallelic mutations in sialic acid biosynthetic GNE enzyme (UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine Kinase). Recently direct or indirect role of GNE in other cellular functions have been elucidated. Hyposialylation of IGF-1R leads to apoptosis due to mitochondrial dysfunction while hyposialylation of β1 integrin receptor leads to altered F-actin assembly, disrupted cytoskeletal organization and slow cell migration.

Understanding pathophysiology of GNE myopathy and current progress towards drug development.

GNE myopathy is a rare genetic neuromuscular disease that is caused due to mutations in the GNE gene responsible for sialic acid biosynthesis. Foot drop is the most common initial symptom observed in GNE myopathy patients. There is slow progressive muscle weakness in the lower and upper extremities while the quadriceps muscles are usually spared.

Bases immediate upstream of the TATAAT box of the sigma 70 promoter of Escherichia coli significantly influence the activity of a model promoter by altering the bending angle of DNA.

Different workers have found different bases of the spacer of the sigma 70 promoter of Escherichia coli to be important, depending on the base sequence of the two hexameric boxes of the naturally occurring promoter they were working on. Besides, there was no clue as to why particular bases worked better than others in particular positions. This necessitated a fresh look at the spacer region of a model promoter comprising all the consensus promoter elements.

Role of HSP70 chaperone in protein aggregate phenomenon of GNE mutant cells: Therapeutic lead for GNE Myopathy.

Limited treatment options and research in understanding the pathomechanisms of rare diseases has raised concerns about their therapeutic development. One such poorly understood ultra-rare neuromuscular disorder is GNE Myopathy (GNEM) which is caused due to mutation in key sialic acid biosynthetic enzyme, GNE. Treatment with sialic acid or its derivatives/precursors slows the disease progression, but curative strategies need to be explored further.

Effect of GNE Mutations on Cytoskeletal Network Proteins: Potential Gateway to Understand Pathomechanism of GNEM.

GNE myopathy is an inherited neuromuscular disorder caused by mutations in GNE (UDP-N-acetylglucosamine 2-epimerase/N-acetyl mannosamine kinase) gene catalyzing the sialic acid biosynthesis pathway. The characteristic features include muscle weakness in upper and lower extremities, skeletal muscle wasting, and rimmed vacuole formation. More than 200 GNE mutations in either epimerase or kinase domain have been reported worldwide.

Functional characterization of GNE mutations prevalent in Asian subjects with GNE myopathy, an ultra-rare neuromuscular disorder.

UDP-GlcNAc 2-epimerase/ManNAc kinase (GNE) is a bifunctional enzyme (N-terminal epimerase and C-terminal Kinase domain) that catalyses the rate limiting step in sialic acid biosynthesis. More than 200 homozygous missense or compound heterozygous mutations in GNE have been reported worldwide to cause a rare neuromuscular disorder, GNE myopathy. It is characterized by a slowly progressive defect in proximal and distal skeletal muscles with patients becoming wheel-chair-bound.

Optimizing Chaperone Removal Strategy from Overexpressed Recombinant Proteins : GNE, a Case Study.

In the last two decades, numerous innovative advances, strategies and protocols have been developed and optimized to improve the quality and quantity of soluble recombinant protein production in E. coli. One of the major challenges being the coelution of chaperone proteins along with desired recombinant protein of interest.

Elucidation of ER stress and UPR pathway in sialic acid-deficient cells: Pathological relevance to GNEM.

Accumulation of misfolded proteins in endoplasmic reticulum (ER) generates a stress condition in the cell. The cell combats ER stress by activating unfolded protein response (UPR) and ERAD (ER stress-associated degradation) pathway. Failure to restore favorable folding environment results in cell dysfunction and apoptosis.

Generation and Characterization of a Skeletal Muscle Cell-Based Model Carrying One Single Gne Allele: Implications in Actin Dynamics.

UDP-N-Acetyl glucosamine-2 epimerase/N-acetyl mannosamine kinase (GNE) catalyzes key enzymatic reactions in the biosynthesis of sialic acid. Mutation in GNE gene causes GNE myopathy (GNEM) characterized by adult-onset muscle weakness and degeneration. However, recent studies propose alternate roles of GNE in other cellular processes beside sialic acid biosynthesis, particularly interaction of GNE with α-actinin 1 and 2.

Altered Actin Dynamics in Cell Migration of GNE Mutant Cells.

Cell migration is an essential cellular process that requires coordination of cytoskeletal dynamics, reorganization, and signal transduction. The actin cytoskeleton is central in maintaining the cellular structure as well as regulating the mechanisms of cell motility. Glycosylation, particularly sialylation of cell surface proteins like integrins, regulates signal transduction from the extracellular matrix to the cytoskeletal network.

The Inherited Neuromuscular Disorder GNE Myopathy: Research to Patient Care.

Inherited neuromuscular diseases are a heterogeneous group of rare diseases for which the low general awareness leads to frequent misdiagnosis. Advances in DNA sequencing technologies are changing this situation, and it is apparent that these diseases are not as rare as previously thought. Knowledge of the pathogenic variants in patients is helping in research efforts to develop new therapies.

Fighting the Cause of Alzheimer's and GNE Myopathy.

Age is the common risk factor for both neurodegenerative and neuromuscular diseases. Alzheimer disease (AD), a neurodegenerative disorder, causes dementia with age progression while GNE myopathy (GNEM), a neuromuscular disorder, causes muscle degeneration and loss of muscle motor movement with age. Individuals with mutations in presenilin or amyloid precursor protein (APP) gene develop AD while mutations in GNE (UDP -acetylglucosamine 2 epimerase/-acetyl Mannosamine kinase), key sialic acid biosynthesis enzyme, cause GNEM.

Role of IGF-1R in ameliorating apoptosis of GNE deficient cells.

Sialic acids (SAs) are nine carbon acidic amino sugars, found at the outermost termini of glycoconjugates performing various physiological and pathological functions. SA synthesis is regulated by UDP-GlcNAc 2-epimerase/ManNAc kinase (GNE) that catalyzes rate limiting steps. Mutations in GNE result in rare genetic disorders, GNE myopathy and Sialuria.

EhRho1 regulates phagocytosis by modulating actin dynamics through EhFormin1 and EhProfilin1 in Entamoeba histolytica.

The protist parasite Entamoeba histolytica causes amoebiasis, a major public health problem in developing countries and a major cause of morbidity and mortality. Invasive infection in amoebiasis mostly affects intestinal epithelial cell lining but can also involve other organs, such as liver, lungs, or brain. Phagocytosis is an essential mode of nutrition in amoeba and has often been associated with virulence behaviour of E.

Mutation in GNE Downregulates Peroxiredoxin IV Altering ER Redox Homeostasis.

GNE myopathy is a rare neuromuscular genetic disorder characterized by early adult onset and muscle weakness due to mutation in sialic acid biosynthetic enzyme, UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE). More than 180 different GNE mutations are known all over the world with unclear pathomechanism. Although hyposialylation of glycoproteins is speculated to be the major cause, but cellular mechanism leading to loss of muscle mass has not yet been deciphered.

EhRho1 regulates plasma membrane blebbing through PI3 kinase in Entamoeba histolytica.

The protozoan parasite Entamoeba histolytica causes amoebiasis, a major public health problem in developing countries. Motility of E. histolytica is important for its pathogenesis.

GNE Myopathy and Cell Apoptosis: A Comparative Mutation Analysis.

In a number of genetic disorders such as GNE myopathy, it is not clear how mutations in target genes result in disease phenotype. GNE myopathy is a progressive neuro-degenerative disorder associated with homozygous or compound heterozygous missense mutations in either epimerase or kinase domain of UDP-GlcNAc 2-epimerase/ManNAc kinase (GNE). This bifunctional enzyme catalyses the rate limiting step in sialic acid biosynthesis.

Optimization of culture parameters and novel strategies to improve protein solubility.

The production of recombinant proteins, in soluble form in a prokaryotic expression system, still remains a challenge for the biotechnologist. Innovative strategies have been developed to improve protein solubility in various protein overexpressing hosts. In this chapter, we would focus on methods currently available and amenable to "desired modifications," such as (a) the use of molecular chaperones; (b) the optimization of culture conditions; (c) the reengineering of a variety of host strains and vectors with affinity tags; and (d) optimal promoter strengths.

Expression and secretion of wild type and mutant GNE proteins in Dictyostelium discoideum.

GNE (UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase) is a bifunctional enzyme which catalyzes the conversion of UDP-GlcNAc to ManNAc and ManNAc to ManNAc 6-phosphate, key steps in the sialic acid biosynthesis. Mutations in GNE lead to a neuromuscular disorder, Hereditary Inclusion Body Myopathy (HIBM). A major limitation in understanding the function of GNE is lack of recombinant full length GNE (rGNE) protein for detailed biophysical and structural characterization.

Role of UDP-N-acetylglucosamine2-epimerase/N-acetylmannosamine kinase (GNE) in β1-integrin-mediated cell adhesion.

Hereditary inclusion body myopathy (GNE myopathy) is a neuromuscular disorder due to mutation in key sialic acid biosynthetic enzyme, GNE. The pathomechanism of the disease is poorly understood as GNE is involved in other cellular functions beside sialic acid synthesis. In the present study, a HEK293 cell-based model system has been established where GNE is either knocked down or over-expressed along with pathologically relevant GNE mutants (D176V and V572L).

Endosomal-sorting complexes required for transport (ESCRT) pathway-dependent endosomal traffic regulates the localization of active Src at focal adhesions.

Active Src localization at focal adhesions (FAs) is essential for cell migration. How this pool is linked mechanistically to the large pool of Src at late endosomes (LEs)/lysosomes (LY) is not well understood. Here, we used inducible Tsg101 gene deletion, TSG101 knockdown, and dominant-negative VPS4 expression to demonstrate that the localization of activated cellular Src and viral Src at FAs requires the endosomal-sorting complexes required for transport (ESCRT) pathway.

Human spleen tyrosine kinase (Syk) recombinant expression systems for high-throughput assays.

Spleen tyrosine kinase (Syk) is an important non-receptor tyrosine kinase and its aberrant regulation is associated with a variety of allergic disorders and autoimmune diseases. To identify small molecule inhibitors of Syk in high-throughput assays, recombinant Syk protein is needed in bulk quantity. We studied the expression of recombinant human Syk in three heterologous systems: E.

Dictyostelium discoideum--a promising expression system for the production of eukaryotic proteins.

In general, four different expression systems, namely, bacterial, yeast, baculovirus, and mammalian, are widely used for the overproduction of biochemical enzymes and therapeutic proteins. Clearly, bacterial expression systems offer ease of maneuverability with respect to large-scale production of recombinant proteins, while, a baculovirus expression system ensures proper protein modifications, processing, and refolding of complex proteins. Despite these advantages, mammalian cells remain the preferred host for many eukaryotic proteins of pharmaceutical importance, particularly, those requiring post-translational modifications.