Brain Signaling of Indispensable Amino Acid Deficiency

Our health requires continual protein synthesis for maintaining and repairing tissues. For protein synthesis to function, all the essential (indispensable) amino acids (IAAs) must be available in the diet, along with those AAs that the cells can synthesize (the dispensable amino acids). Here we review studies that have shown the location of the detector for IAA deficiency in the brain, specifically for recognition of IAA deficient diets (IAAD diets) in the anterior piriform cortex (APC), with subsequent responses in downstream brain areas. The APC is highly excitable, which makes is uniquely suited to serve as an alarm for reductions in IAAs. With a balanced diet, these neurons are kept from over-excitation by GABAergic inhibitory neurons. Because several transporters and receptors on the GABAergic neurons have rapid turnover times, they rely on intact protein synthesis to function. When an IAA is missing, its unique tRNA cannot be charged. This activates the enzyme General Control Nonderepressible 2 (GCN2) that is important in the initiation phase of protein synthesis. Without the inhibitory control supplied by GABAergic neurons, excitation in the circuitry is free to signal an urgent alarm. Studies in rodents have shown rapid recognition of IAA deficiency by quick rejection of the IAAD diet.

HDfleX: Software for flexible high structural resolution of hydrogen/deuterium-exchange mass spectrometry data

Hydrogen/deuterium-exchange mass spectrometry (HDX-MS) experiments on protein structures can be performed at three levels: (1) by enzymatically digesting labelled proteins and analyzing the peptides (bottom-up), (2) by further fragmenting peptides following digestion (middle-down), and (3) by fragmenting the intact labelled protein (top-down), using soft gas-phase fragmentation methods, such as electron transfer dissociation (ETD). However, to the best of our knowledge, the software packages currently available for the analysis of HDX-MS data do not enable the peptide- and ETD-levels to be combined - they can only be analyzed separately. Thus, we developed HDfleX - a standalone application for the analysis of flexible high structural resolution of HDX-MS data, which allows data at any level of structural resolution (intact protein, peptide, fragment) to be merged. HDfleX features rapid experimental data fitting, robust statistical significance analyses and optional methods for theoretical intrinsic calculations and a novel empirical correction for comparison between solution conditions.

Brain Signaling of Indispensable Amino Acid Deficiency

Our health requires continual protein synthesis for maintaining and repairing tissues. For protein synthesis to function, all the essential (indispensable) amino acids (IAA) that must be available in the diet, along with those AAs that the cells can synthesize, the dispensable amino acids. Here we review studies that have shown the location of the detector for IAA deficiency in the brain, specifically for recognition of IAA deficient diets (IAAD diets) in the anterior piriform cortex (APC), with subsequent responses in downstream brain areas. The APC is highly excitable, uniquely suited to serve as an alarm for reductions in IAAs. With a balanced diet, these neurons are kept from over-excitation by GABAergic inhibitory neurons. Because several transporters and receptors on the GABAergic neurons have rapid turnover times, they rely on intact protein synthesis to function. When an IAA is missing, its unique tRNA cannot be charged. This activates the enzyme General Control Nonderepressible 2 (GCN2) that is important in the initiation phase of protein synthesis. Without the inhibitory control supplied by GABAergic neurons, excitation in the circuitry is free to signal an urgent alarm. Studies in rodents have shown rapid recognition of IAA deficiency by quick rejection of the IAAD diet.

Recombinant production of a functional SARS-CoV-2 spike receptor binding domain in the green algae Chlamydomonas reinhardtii

Recombinant production of viral proteins can be used to produce vaccine antigens or reagents to identify antibodies in patient serum. Minimally, these proteins must be correctly folded and have appropriate post-translation modifications. Here we report the production of the SARS-CoV-2 spike protein Receptor Binding Domain (RBD) in the green algae Chlamydomonas. RBD fused to a fluorescent reporter protein accumulates as an intact protein when targeted for ER-Golgi retention or secreted from the cell, while a chloroplast localized version is truncated. The ER-retained RBD fusion protein was able to bind the human ACE2 receptor, the host target of SARS-CoV-2, and was specifically out-competed by mammalian cell-produced recombinant RBD, suggesting that the algae produced proteins are sufficiently post-translationally modified to act as authentic SARS-CoV-2 antigens. Because algae can be grown at large scale very inexpensively, this recombinant protein may be a low cost alternative to other expression platforms.

Multitasking in the gut: the X-ray structure of the multidomain BbgIII from Bifidobacterium bifidum offers possible explanations for its alternative functions

β-Galactosidases catalyse the hydrolysis of lactose into galactose and glucose; as an alternative reaction, some β-galactosidases also catalyse the formation of galactooligosaccharides by transglycosylation. Both reactions have industrial importance: lactose hydrolysis is used to produce lactose-free milk, while galactooligosaccharides have been shown to act as prebiotics. For some multi-domain β-galactosidases, the hydrolysis/transglycosylation ratio can be modified by the truncation of carbohydrate-binding modules. Here, an analysis of BbgIII, a multidomain β-galactosidase from Bifidobacterium bifidum, is presented. The X-ray structure has been determined of an intact protein corresponding to a gene construct of eight domains. The use of evolutionary covariance-based predictions made sequence docking in low-resolution areas of the model spectacularly easy, confirming the relevance of this rapidly developing deep-learning-based technique for model building. The structure revealed two alternative orientations of the CBM32 carbohydrate-binding module relative to the GH2 catalytic domain in the six crystallographically independent chains. In one orientation the CBM32 domain covers the entrance to the active site of the enzyme, while in the other orientation the active site is open, suggesting a possible mechanism for switching between the two activities of the enzyme, namely lactose hydrolysis and transgalactosylation. The location of the carbohydrate-binding site of the CBM32 domain on the opposite site of the module to where it comes into contact with the catalytic GH2 domain is consistent with its involvement in adherence to host cells. The role of the CBM32 domain in switching between hydrolysis and transglycosylation modes offers protein-engineering opportunities for selective β-galactosidase modification for industrial purposes in the future.

High Throughput Intact Protein Analysis Using Infrared Matrix-Assisted Laser Desorption Electrospray Ionization Mass Spectrometry

Mass spectrometry (MS) is the primary analytical tool used to characterize proteins within the biopharmaceutical industry. Electrospray ionization (ESI) coupled to liquid chromatography is the gold standard technique for intact protein analysis. However, speed limitations prevent analysis of large sample numbers (>1000) in a day. Infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) MS, an ambient ionization MS technology, has recently been established as a platform for high throughput small molecule analysis. Here we report the application of such system for intact protein analysis. Up to 150 kDa proteins are detectable on a high throughput IR-MALDESI-MS system and we have evaluated how matrix affects signal. The system can analyze up to 22 protein samples in a second. Protein autophosphorylation, compound binding kinetics and compound modifications to a probe protein are demonstrated as applications. Top-down protein sequencing was conducted to identify a cysteine modification site. Two plate-based high throughput sample cleanup methods were coupled to IR-MALDESI-MS to enable analysis of samples containing high amounts of salts and buffers without compromising speed.

Term infant formula supplemented with milk-derived oligosaccharides shifts the gut microbiota closer to that of human milk-fed infants and improves intestinal immune defense: A randomized controlled trial

Background Bovine milk-derived oligosaccharides (MOS) containing primarily galacto-oligosaccharides with inherent levels of sialylated oligosaccharides can be added to infant formula to enhance the oligosaccharide profile. Objective To investigate the effects of a MOS-supplemented infant formula on gut microbiota and intestinal immunity. Methods In a double-blind, randomized, controlled trial, healthy-term formula-fed infants aged 21–26 days either received an intact protein cow's milk-based formula (control group, CG, n = 112) or the same formula containing 7.2 g MOS/L (experimental group, EG, n = 114) until age 6 months. Exclusively human milk-fed infants (HFI, n = 70) from an observational study served as reference. Fecal samples collected at baseline, 2.5 and 4 months of age were assessed for microbiota (16S ribosomal ribonucleic acid—based approaches), metabolites and biomarkers of gut health and immune response. Results At age 2.5 and 4 months, redundancy analysis (P = 0.002) and average phylogenetic distance (P < 0.05) showed that the overall microbiota composition in EG was different from CG and closer to that of HFI. Similarly, EG caesarean-born infants were different from CG caesarean- or vaginally-born infants and approaching HFI vaginally-born infants. Relative bifidobacteria abundance was higher in EG vs. CG (P < 0.05) approaching HFI. At age 4 months, counts of Clostridioides difficile and Clostridium perfringens were ∼90% (P < 0.001) and ∼65% (P < 0.01) lower in EG vs. CG, respectively. Mean (95%CI) fecal secretory immunoglobulin A (IgA) in EG was twice that of CG [70 (57,85) vs. 34 (28,42) mg/g, P < 0.001] and closer to HFI. Fecal oral polio vaccine-specific IgA was ∼50% higher in EG vs. CG (P = 0.065). Compared to CG, EG and HFI had lower fecal calcium excretion (by ∼30%) and fecal pH (P < 0.001), and higher lactate concentration (P < 0.001). Conclusions Infant formula with MOS shifts the gut microbiota and metabolic signature closer to that of HFI, has a strong bifidogenic effect, reduces fecal pathogens, and improves intestinal immune response.

Investigation of sequon engineering for improved O-glycosylation by the human polypeptide N-acetylgalactosaminyl transferase T2 isozyme and two orthologues

We have been developing bacterial expression systems for human mucin-type O-glycosylation on therapeutic proteins, which is initiated by the addition of α-linked GalNAc to serine or threonine residues by enzymes in the GT-27 family of glycosyltransferases. Substrate preference across different isoforms of this enzyme is influenced by isoform-specific amino acid sequences at the site of glycosylation, which we have exploited to engineer production of Core 1 glycan structures in bacteria on human therapeutic proteins. Using RP-HPLC with a novel phenyl bonded phase to resolve intact protein glycoforms, the effect of sequon mutation on O-glycosylation initiation was examined through in vitro modification of the naturally O-glycosylated human interferon α-2b, and a sequon engineered human growth hormone. As part of the development of our glycan engineering in the bacterial expression system we are surveying various orthologues of critical enzymes to ensure complete glycosylation. Here we present an in vitro enzyme kinetic profile of three related GT-27 orthologues on natural and engineered sequons in recombinant human interferon α2b and human growth hormone where we show a significant change in kinetic properties with the amino acid changes. It was found that optimizing the protein substrate amino acid sequence using Isoform Specific O-Glycosylation Prediction (ISOGlyP, http://isoglyp.utep.edu/index.php) resulted in a measurable increase in kcat/KM, thus improving glycosylation efficiency. We showed that the Drosophila orthologue showed superior activity with our human growth hormone designed sequons compared with the human enzyme.