Bruchpilot, a Protein with Homology to ELKS/CAST, Is Required for Structural Integrity and Function of Synaptic Active Zones in Drosophila

This report integrates knowledge of in situ macromolecular structures and synaptic protein biochemistry to propose a unified hypothesis for the regulation of certain vesicle trafficking events (i.e., docking, priming, Ca2+-triggering, and membrane fusion) that lead to neurotransmitter secretion from specialized “active zones” of presynaptic axon terminals. Advancements in electron tomography, to image tissue sections in 3D at nanometer scale resolution, have led to structural characterizations of a network of different classes of macromolecules at the active zone, called “Active Zone Material’. At frog neuromuscular junctions, the classes of Active Zone Material macromolecules “top-masts”, “booms”, “spars”, “ribs” and “pins” direct synaptic vesicle docking while “pins”, “ribs” and “pegs” regulate priming to influence Ca2+-triggering and membrane fusion. Other classes, “beams”, “steps”, “masts”, and “synaptic vesicle luminal filaments’ likely help organize and maintain the structural integrity of active zones. Extensive studies on the biochemistry that regulates secretion have led to comprehensive characterizations of the many conserved proteins universally involved in these trafficking events. Here, a hypothesis including a partial proteomic atlas of Active Zone Material is presented which considers the common roles, binding partners, physical features/structure, and relative positioning in the axon terminal of both the proteins and classes of macromolecules involved in the vesicle trafficking events. The hypothesis designates voltage-gated Ca2+ channels and Ca2+-gated K+ channels to ribs and pegs that are connected to macromolecules that span the presynaptic membrane at the active zone. SNARE proteins (Syntaxin, SNAP25, and Synaptobrevin), SNARE-interacting proteins Synaptotagmin, Munc13, Munc18, Complexin, and NSF are designated to ribs and/or pins. Rab3A and Rabphillin-3A are designated to top-masts and/or booms and/or spars. RIM, Bassoon, and Piccolo are designated to beams, steps, masts, ribs, spars, booms, and top-masts. Spectrin is designated to beams. Lastly, the luminal portions of SV2 are thought to form the bulk of the observed synaptic vesicle luminal filaments. The goal here is to help direct future studies that aim to bridge Active Zone Material structure, biochemistry, and function to ultimately determine how it regulates the trafficking events in vivo that lead to neurotransmitter secretion.

Download Full-text

Codon harmonization reduces amino acid misincorporation in bacterially expressed P. falciparum proteins and improves their immunogenicity

AMB Express ◽

10.1186/s13568-019-0890-6 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 1

Author(s):

Neeraja Punde ◽

Jennifer Kooken ◽

Dagmar Leary ◽

Patricia M. Legler ◽

Evelina Angov

Keyword(s):

Protein Structure ◽

Amino Acid ◽

Codon Usage ◽

Dna Sequences ◽

Structural Integrity ◽

Host Cells ◽

Loss Of Function ◽

Species Specific ◽

And Function ◽

The Impact

Abstract Codon usage frequency influences protein structure and function. The frequency with which codons are used potentially impacts primary, secondary and tertiary protein structure. Poor expression, loss of function, insolubility, or truncation can result from species-specific differences in codon usage. “Codon harmonization” more closely aligns native codon usage frequencies with those of the expression host particularly within putative inter-domain segments where slower rates of translation may play a role in protein folding. Heterologous expression of Plasmodium falciparum genes in Escherichia coli has been a challenge due to their AT-rich codon bias and the highly repetitive DNA sequences. Here, codon harmonization was applied to the malarial antigen, CelTOS (Cell-traversal protein for ookinetes and sporozoites). CelTOS is a highly conserved P. falciparum protein involved in cellular traversal through mosquito and vertebrate host cells. It reversibly refolds after thermal denaturation making it a desirable malarial vaccine candidate. Protein expressed in E. coli from a codon harmonized sequence of P. falciparum CelTOS (CH-PfCelTOS) was compared with protein expressed from the native codon sequence (N-PfCelTOS) to assess the impact of codon usage on protein expression levels, solubility, yield, stability, structural integrity, recognition with CelTOS-specific mAbs and immunogenicity in mice. While the translated proteins were expected to be identical, the translated products produced from the codon-harmonized sequence differed in helical content and showed a smaller distribution of polypeptides in mass spectra indicating lower heterogeneity of the codon harmonized version and fewer amino acid misincorporations. Substitutions of hydrophobic-to-hydrophobic amino acid were observed more commonly than any other. CH-PfCelTOS induced significantly higher antibody levels compared with N-PfCelTOS; however, no significant differences in either IFN-γ or IL-4 cellular responses were detected between the two antigens.

Download Full-text

Clinical Relevance of Elastin in the Structure and Function of Skin

Aesthetic Surgery Journal Open Forum ◽

10.1093/asjof/ojab019 ◽

2021 ◽

Author(s):

Leslie Baumann ◽

Eric F Bernstein ◽

Anthony S Weiss ◽

Damien Bates ◽

Shannon Humphrey ◽

...

Keyword(s):

Wound Healing ◽

Clinical Relevance ◽

Structural Integrity ◽

Elastic Fiber ◽

Subcutaneous Fat ◽

Structure And Function ◽

Fiber Formation ◽

Elastic Fibers ◽

Superficial Dermis ◽

And Function

Abstract Elastin is the main component of elastic fibers, which provide stretch, recoil, and elasticity to the skin. Normal levels of elastic fiber production, organization, and integration with other cutaneous extracellular matrix proteins, proteoglycans, and glycosaminoglycans are integral to maintaining healthy skin structure, function, and youthful appearance. Although elastin has very low turnover, its production decreases after individuals reach maturity and it is susceptible to damage from many factors. With advancing age and exposure to environmental insults, elastic fibers degrade. This degradation contributes to the loss of the skin’s structural integrity; combined with subcutaneous fat loss, this results in looser, sagging skin, causing undesirable changes in appearance. The most dramatic changes occur in chronically sun-exposed skin, which displays sharply altered amounts and arrangements of cutaneous elastic fibers, decreased fine elastic fibers in the superficial dermis connecting to the epidermis, and replacement of the normal collagen-rich superficial dermis with abnormal clumps of solar elastosis material. Disruption of elastic fiber networks also leads to undesirable characteristics in wound healing, and the worsening structure and appearance of scars and stretch marks. Identifying ways to replenish elastin and elastic fibers should improve the skin’s appearance, texture, resiliency, and wound-healing capabilities. However, few therapies are capable of repairing elastic fibers or substantially reorganizing the elastin/microfibril network. This review describes the clinical relevance of elastin in the context of the structure and function of healthy and aging skin, wound healing, and scars and introduces new approaches being developed to target elastin production and elastic fiber formation.

Download Full-text

Combined Supplementation of Choline and Docosahexaenoic Acid during Pregnancy Enhances Neurodevelopment of Fetal Hippocampus

Neurology Research International ◽

10.1155/2017/8748706 ◽

2017 ◽

Vol 2017 ◽

pp. 1-9 ◽

Cited By ~ 3

Author(s):

Huban Thomas Rajarethnem ◽

Kumar Megur Ramakrishna Bhat ◽

Malsawmzuali Jc ◽

Siva Kumar Gopalkrishnan ◽

Ramesh Babu Mugundhu Gopalram ◽

...

Keyword(s):

Docosahexaenoic Acid ◽

Structural Integrity ◽

Fetal Brain ◽

Normal Pregnancy ◽

Saline Control ◽

Fetal Brain Development ◽

Essential Nutrient ◽

Combined Supplementation ◽

And Function ◽

The Brain

Choline is an essential nutrient for humans which plays an important role in structural integrity and signaling functions. Docosahexaenoic acid (DHA) is a polyunsaturated fatty acid, highly enriched in cell membranes of the brain. Dietary intake of choline or DHA alone by pregnant mothers directly affects fetal brain development and function. But no studies show the efficacy of combined supplementation of choline and DHA on fetal neurodevelopment. The aim of the present study was to analyze fetal neurodevelopment on combined supplementation of pregnant dams with choline and DHA. Pregnant dams were divided into five groups: normal control [NC], saline control [SC], choline [C], DHA, and C + DHA. Saline, choline, and DHA were given as supplements to appropriate groups of dams. NC dams were undisturbed during entire gestation. On postnatal day (PND) 40, brains were processed for Cresyl staining. Pups from choline or DHA supplemented group showed significant (p<0.05) increase in number of neurons in hippocampus when compared to the same in NC and SC groups. Moreover, pups from C + DHA supplemented group showed significantly higher number of neurons (p<0.001) in hippocampus when compared to the same in NC and SC groups. Thus combined supplementation of choline and DHA during normal pregnancy enhances fetal hippocampal neurodevelopment better than supplementation of choline or DHA alone.

Download Full-text

Antimicrobial mode of action of secretions from the metapleural gland ofMytmecia gulosa(Australian bull ant)

Canadian Journal of Microbiology ◽

10.1139/m95-018 ◽

1995 ◽

Vol 41 (2) ◽

pp. 136-144 ◽

Cited By ~ 23

Author(s):

J. A. Mackintosh ◽

J. E. Trimble ◽

A. J. Beattie ◽

D. A. Veal ◽

M. K. Jones ◽

...

Keyword(s):

Candida Albicans ◽

Antimicrobial Agents ◽

Structural Integrity ◽

Phospholipid Bilayer ◽

Active Components ◽

Cytoplasmic Matrix ◽

Metapleural Glands ◽

Total Leakage ◽

Membrane Bound ◽

And Function

Secretions from exocrine metapleural glands of Myrmecia gulosa (Australian bull ant) exhibit broad-spectrum antimicrobial activity. Treatment of the yeast Candida albicans with metapleural secretion resulted in the rapid and total leakage of K+ions from cells within 10 min. Ultrastructural analysis of the bacteria Bacillus cereus, Escherichia coli, and Pseudomonas aeruginosa, and cells and protoplasts of Candida albicans demonstrated gross damage of the cell membrane and aggregation of the cytoplasmic matrix of treated cells. Degradation of membrane-bound organelles was also observed in Candida albicans. The antimicrobially active components of metapleural secretions were nonpolar and interacted with the phospholipid bilayer, causing damage to the structural integrity of liposomes and the release of carboxyfluorescein. The data suggest that the antimicrobial agents in metapleural secretion act primarily by disrupting the structure and function of the phospholipid bilayer of the cytoplasmic membrane.Key words: ant metapleural secretion, antimicrobial, Candida albicans, cytoplasmic membrane.

Download Full-text

Abstract 126: Peroxisomal Proliferator Activated Receptor Alpha Overexpression in Hypertrophied Cardiomyocytes Restores Mitochondrial Integrity and Function

Circulation Research ◽

10.1161/res.121.suppl_1.126 ◽

2017 ◽

Vol 121 (suppl_1) ◽

Author(s):

Sagartirtha Sarkar ◽

Santanu Rana

Keyword(s):

Energy Demand ◽

Cardiac Tissue ◽

Structural Integrity ◽

Heart Function ◽

Ros Generation ◽

Peroxisome Proliferator ◽

Peroxisome Proliferator Activated Receptor ◽

Atp Production ◽

Receptor Alpha ◽

And Function

Cardiac tissue engineering is an interdisciplinary field that engineers modulation of viable molecular milieu to restore, maintain or improve heart function. Myocardial workload (energy demand) and energy substrate availability (supply) are in continual flux to maintain specialized cellular processes, yet the heart has a limited capacity for substrate storage and utilization during pathophysiological conditions. Damage to heart muscle, acute or chronic, leads to dysregulation of cardiac metabolic processes associated with gradual but progressive decline in mitochondrial respiratory pathways resulting in diminished ATP production. The Peroxisome Proliferator Activated Receptor Alpha ( PPARα ) is known to regulate fatty acid to glucose metabolic balance as well as mitochondrial structural integrity. In this study, a non-canonical pathway of PPARα was analyzed by cardiomyocyte targeted PPARα overexpression during cardiac hypertrophy that showed significant downregulation in p53 acetylation as well as GSK3β activation levels. Targeted PPARα overexpression during hypertrophy resulted in restoration of mitochondrial structure and function along with significantly improved mitochondrial ROS generation and membrane potential. This is the first report of myocyte targeted PPARα overexpression in hypertrophied myocardium that results in an engineered heart with significantly improved function with increased muscle mitochondrial endurance and reduced mitochondrial apoptotic load, thus conferring a greater resistance to pathological stimuli within cardiac microenvironment.

Download Full-text

Recent advances in histone glycation: emerging role in diabetes and cancer

Glycobiology ◽

10.1093/glycob/cwab011 ◽

2021 ◽

Author(s):

Abdul Rouf Mir ◽

Safia Habib ◽

Moin Uddin

Keyword(s):

Structural Integrity ◽

Cell Function ◽

Nuclear Proteins ◽

Chromatin Interaction ◽

Epigenetic Landscape ◽

Histone Proteins ◽

Nucleosome Dynamics ◽

Circulating Antibodies ◽

And Function ◽

By Products

Abstract Ever increasing information on genome and proteome has offered fascinating details and new opportunities to understand the molecular biology. It is now known that histone proteins surrounding the DNA play a crucial role in the chromatin structure and function. Histones undergo a plethora of posttranslational enzymatic modifications that influence nucleosome dynamics and affect DNA activity. Earlier research offered insights into the enzymatic modifications of histones; however, attention has been diverted to histone modifications induced by by-products of metabolism without enzymatic engagement in the last decade. Nonenzymatic modifications of histones are believed to be crucial for epigenetic landscape, cellular fate and for role in human diseases. Glycation of histone proteins constitutes the major non enzymatic modifications of nuclear proteins that have implications in diabetes and cancer. It has emerged that glycation damages nuclear proteins, modifies amino acids of histones at crucial locations, generates adducts affecting histone chromatin interaction, develops neo-epitopes inducing specific immune response and impacts cell function. Presence of circulating antibodies against glycated histone proteins in diabetes and cancer has shown immunological implications with diagnostic relevance. These crucial details make histone glycation an attractive focus for investigators. This review article, therefore, makes an attempt to exclusively summarize the recent researches in histone glycation, its impact on structural integrity of chromatin and elaborates on their role in diabetes and cancer. The work offers insights for future scientists who investigate the link between metabolism, biomolecular structures, glycobiology, histone–DNA interactions in relation to diseases in humans.

Download Full-text

Non-inflammatory myopathies

Oxford Textbook of Rheumatology ◽

10.1093/med/9780199642489.003.0126 ◽

2013 ◽

pp. 1033-1040

Author(s):

Mark Roberts

Keyword(s):

Structural Integrity ◽

Genetic Disorders ◽

Inflammatory Myopathies ◽

Muscle Enzyme ◽

Diagnostic Odyssey ◽

Diagnostic Confusion ◽

Myopathic Change ◽

Future Treatments ◽

And Function ◽

Inflammatory Changes

Non-inflammatory myopathies (NIM) are a diverse group of genetic disorders, characterized by neuromuscular weakness, fatigue, muscle wasting, and pain, due to inherited defects in proteins critical in the structural integrity and function of muscle fibre or in enzymes involved in energy production in this most metabolic tissue. The shared clinical and laboratory features (including elevated muscle enzyme levels, myopathic change on electromyography, and even inflammatory changes on muscle biopsy) of myositis and NIM frequently cause diagnostic confusion. Failure to distinguish these disorders will result in unnecessary immunosuppression, lack of screening for cardiorespiratory and other associations of NIM, and a missed opportunity for genetic counselling and potential future treatments. A strong index of suspicion is required in all patients presenting with neuromuscular syndromes if a long diagnostic odyssey is to be avoided. A clinically focused multidisciplinary approach, with a working knowledge of subtypes of NIM, is outlined in this chapter.

Download Full-text

Effect of salbutamol on neuromuscular junction function and structure in a mouse model of DOK7 congenital myasthenia

Human Molecular Genetics ◽

10.1093/hmg/ddaa116 ◽

2020 ◽

Vol 29 (14) ◽

pp. 2325-2336 ◽

Cited By ~ 2

Author(s):

Richard G Webster ◽

An E Vanhaesebrouck ◽

Susan E Maxwell ◽

Judith A Cossins ◽

Weiwei Liu ◽

...

Keyword(s):

Weight Gain ◽

Neuromuscular Junction ◽

Mouse Model ◽

Structural Integrity ◽

Ex Vivo ◽

Adrenergic Agonists ◽

Mice Model ◽

Synaptic Structure ◽

Perinatal Lethality ◽

And Function

Abstract Congenital myasthenic syndromes (CMS) are characterized by fatigable muscle weakness resulting from impaired neuromuscular transmission. β2-adrenergic agonists are an effective treatment for DOK7-CMS. DOK7 is a component within the AGRN-LRP4-MUSK-DOK7 signalling pathway that is key for the formation and maintenance of the synaptic structure of the neuromuscular junction (NMJ). The precise mechanism of action of β2-adrenergic agonists at the NMJ is not fully understood. In this study, we investigated whether β2-adrenergic agonists improve both neurotransmission and structural integrity of the NMJ in a mouse model of DOK7-CMS. Ex-vivo electrophysiological techniques and microscopy of the NMJ were used to study the effect of salbutamol, a β2-adrenergic agonist, on synaptic structure and function. DOK7-CMS model mice displayed a severe phenotype with reduced weight gain and perinatal lethality. Salbutamol treatment improved weight gain and survival in DOK7 myasthenic mice. Model animals had fewer active NMJs, detectable by endplate recordings, compared with age-matched wild-type littermates. Salbutamol treatment increased the number of detectable NMJs during endplate recording. Correspondingly, model mice had fewer acetylcholine receptor-stained NMJs detected by fluorescent labelling, but following salbutamol treatment an increased number were detectable. The data demonstrate that salbutamol can prolong survival and increase NMJ number in a severe model of DOK7-CMS.

Download Full-text