Systematic Analysis of Post-Translational Modifications for Increased Longevity of Biotherapeutic Proteins

Protein-based therapeutics (PPTs) are drugs used to treat a variety of different conditions in the human body by alleviating enzymatic deficiencies, augmenting other proteins and drugs, modulating signal pathways, and more. However, many PPTs struggle from a short half-life due to degradation caused by irreversible protein aggregation in the bloodstream. Currently, the most researched strategies for improving the efficiency and longevity of PPTs are post-translational modifications (PTMs). The goal of our research was to determine which type of PTM increases longevity the most for each of three commonly-used therapeutic proteins by comparing the docking scores (DS) and binding free energies (BFE) from protein aggregation and reception simulations. DS and BFE values were used to create a quantitative index that outputs a relative number from −1 to 1 to show reduced performance, no change, or increased performance. Results showed that methylation was the most beneficial for insulin (p < 0.1) and human growth hormone (p < 0.0001), and both phosphorylation and methylation were somewhat optimal for erythropoietin (p < 0.1 and p < 0.0001, respectively). Acetylation consistently provided the worst benefits with the most negative indices, while methylation had the most positive indices throughout. However, PTM efficacy varied between PPTs, supporting previous studies regarding how each PTM can confer different benefits based on the unique structures of recipient proteins.

Aberrant post-translational protein modifications in the pathogenesis of alcohol-induced liver injury

It is likely that the majority of proteins will undergo post-translational modification, be it enzymatic or non-enzymatic. These modified protein(s) regulate activity, localization and interaction with other cellular molecules thereby maintaining cellular hemostasis. Alcohol exposure significantly alters several of these post-translational modifications leading to impairments of many essential physiological processes. Here, we present new insights into novel modifications following ethanol exposure and their role in the initiation and progression of liver injury. This critical review condenses the proceedings of a symposium at the European Society for the Biomedical Research on Alcoholism Meeting held September 12-15, 2015, in Valencia, Spain.

Post-translational modifications of hepatitis C viral proteins and their biological significance

Replication of hepatitis C virus(HCV)depends on the interaction of viral proteins with various host cellular proteins and signalling pathways.Similar to cellular proteins,post-translational modifications(PTMs)of HCV proteins are essential for proper protein function and regulation,thus,directly affecting viral life cycle and the generation of infectious virus particles.Cleavage of the HCV polyprotein by cellular and viral proteases into more than 10 proteins represents an early protein modification step after translation of the HCV positivestranded RNA genome.The key modifications include the regulated intramembranous proteolytic cleavage of core protein,disulfide bond formation of core,glycosylation of HCV envelope proteins E1 and E2,methylation of nonstructural protein 3(NS3),biotinylation of NS4A,ubiquitination of NS5B and phosphorylation of core and NS5B.Other modifications like ubiquitination of core and palmitoylation of core and NS4B proteins have been reported as well.For some modifications such as phosphorylation of NS3 and NS5A and acetylation of NS3,we have limited understanding of their effects on HCV replication and pathogenesis while the impact of other modifications is far from clear.In this review,we summarize the available information on PTMs of HCV proteins and discuss their relevance to HCV replication and pathogenesis.

Protein post-translational modifications after spinal cord injury

Deficits in intrinsic neuronal capacities in the spinal cord,a lack of growth support,and suppression of axonal outgrowth by inhibitory molecules mean that spinal cord injury almost always has devastating consequences.As such,one of the primary targets for the treatment of spinal cord injury is to develop strategies to antagonize extrinsic or intrinsic axonal growth-inhibitory factors or enhance the factors that support axonal growth.Among these factors,a series of individual protein level disorders have been identified during the generation of axons following spinal cord injury.Moreover,an increasing number of studies have indicated that post-translational modifications of these proteins have important implications for axonal growth.Some researchers have discovered a variety of post-translational modifications after spinal cord injury,such as tyrosination,acetylation,and phosphorylation.In this review,we reviewed the post-translational modifications for axonal growth,functional recovery,and neuropathic pain after spinal cord injury,a better understanding of which may elucidate the dynamic change of spinal cord injury-related molecules and facilitate the development of a new therapeutic strategy for spinal cord injury.

Human T-lymphotropic virus proteins and post-translational modification pathways

Cell life from the cell cycle to the signaling transduction and response to stimuli is finely tuned by protein post-translational modifications(PTMs).PTMs alter the conformation,the stability,the localization,and hence the pattern of interactions of the targeted protein.Cell pathways involve the activation of enzymes,like kinases,ligases and transferases,that,once activated,act on many proteins simultaneously,altering the state of the cell and triggering the processes they are involved in.Viruses enter a balanced system and hijack the cell,exploiting the potential of PTMs either to activate viral encoded proteins or to alter cellular pathways,with the ultimate consequence to perpetuate through their replication.Human T-lymphotropic virus type 1(HTLV-1)is known to be highly oncogenic and associates with adult T-cell leukemia/lymphoma,HTLV-1-associated myelopathy/tropical spastic paraparesis and other inflammatory pathological conditions.HTLV-1 protein activity is controlled by PTMs and,in turn,viral activity is associated with the modulation of cellular pathways based on PTMs.More knowledge is acquired about the PTMs involved in the activation of its proteins,like Tax,Rex,p12,p13,p30,HTLV-I basic leucine zipper factorand Gag.However,more has to be understood at the biochemical level in order to counteract the associated fatal outcomes.This review will focus on known PTMs that directly modify HTLV-1 components and on enzymes whose activity is modulated by viral proteins.

Complex interactomes and post-translational modifications of the regulatory proteins HABP4 and SERBP1 suggest pleiotropic cellular functions

The 57 kDa antigen recognized by the Ki-1 antibody,is also known as intracellular hyaluronic acid binding protein 4 and shares 40.7%identity and 67.4%similarity with serpin mRNA binding protein 1,which is also named CGI-55,or plasminogen activator inhibitor type-1-RNA binding protein-1,indicating that they might be paralog proteins,possibly with similar or redundant functions in human cells.Through the identification of their protein interactomes,both regulatory proteins have been functionally implicated in transcriptional regulation,mRNA metabolism,specifically RNA splicing,the regulation of mRNA stability,especially,in the context of the progesterone hormone response,and the DNA damage response.Both proteins also show a complex pattern of post-translational modifications,involving Ser/Thr phosphorylation,mainly through protein kinase C,arginine methylation and SUMOylation,suggesting that their functions and locations are highly regulated.Furthermore,they show a highly dynamic cellular localization pattern with localizations in both the cytoplasm and nucleus as well as punctuated localizations in both granular cytoplasmic protein bodies,upon stress,and nuclear splicing speckles.Several reports in the literature show altered expressions of both regulatory proteins in a series of cancers as well as mutations in their genes that may contribute to tumorigenesis.This review highlights important aspects of the structure,interactome,post-translational modifications,sub-cellular localization and function of both regulatory proteins and further discusses their possible functions and their potential as tumor markers in different cancer settings.

Computer-Assisted analysis of subcellular localization signals and post-translational modifications of human prion proteins

In the present work, computational analyses were applied to study the subcellular localiza-tion and posttranslational modifications of hu-man prion proteins (PrPs). The tentative location of prion protein was determined to be in the nu-cleolus inside the nucleus by the following bio-informatics tools: Hum-PLoc, Euk-PLoc and Nuc-PLoc. Based on our results signal peptides with average of 22 base pairs in N-terminal were identified in human PrPs. This theoretical study demonstrates that PrP is post-translationally modified by: 1) attachment of two N-linked complex carbohydrate moieties (N181 and N197), 2) attachmet of glycosylphosphatidylinositol (GPI) at serine 230 and 3) formation of two di-sulfide bonds between “6–22” and “179–214” cysteines. Furthermore, ten protein kinase phosphorylation sites were predicted in human PrP. The above-noted phosphorylation was car-ried out by PKC and CK2. By using bioinfor-matics tools, we have shown that computation-ally human PrPs locate particularly into the nu-cleolus.

Comprehensive analysis of the gut microbiome and posttranslational modifications elucidates the route involved in microbiota-host interactions

The gut microbiome interacts with the host to maintain body homeostasis,with gut microbial dysbiosis implicated in many diseases.However,the underlying mechanisms of gut microbe regulation of host behavior and brain functions remain unclear.This study aimed to elucidate the influence of gut microbiota on brain functions via post-translational modification mechanisms in the presence or absence of bacteria without any stimulation.We conducted succinylome analysis of hippocampal proteins in germ-free(GF)and specific pathogen-free(SPF)mice and metagenomic analysis of feces from SPF mice.These results were integrated with previously reported hippocampal acetylome and phosphorylome data from the same batch of mice.Subsequent bioinformatics analyses revealed 584 succinylation sites on 455 proteins,including 54 up-regulated succinylation sites on 91 proteins and 99 down-regulated sites on 51 proteins in the GF mice compared to the SPF mice.We constructed a panoramic map of gut microbiota-regulated succinylation,acetylation,and phosphorylation,and identified cross-talk and relative independence between the different types of post-translational modifications in modulating complicated intracellular pathways.Pearson correlation analysis indicated that 13 taxa,predominantly belonging to the Bacteroidetes phylum,were correlated with the biological functions of post-translational modifications.Positive correlations between these taxa and succinylation and negative correlations between these taxa and acetylation were identified in the modulation of intracellular pathways.This study highlights the hippocampal physiological changes induced by the absence of gut microbiota,and proteomic quantification of succinylation,phosphorylation,and acetylation,contributing to our understanding of the role of the gut microbiome in brain function and behavioral phenotypes.

Enhancing m^(6)A modification in the motor cortex facilitates corticospinal tract remodeling after spinal cord injury

Spinal cord injury typically causes corticospinal tract disruption. Although the disrupted corticospinal tract can self-regenerate to a certain degree, the underlying mechanism of this process is still unclear. N6-methyladenosine(m^(6)A) modifications are the most common form of epigenetic regulation at the RNA level and play an essential role in biological processes. However, whether m^(6)A modifications participate in corticospinal tract regeneration after spinal cord injury remains unknown. We found that expression of methyltransferase 14 protein(METTL14) in the locomotor cortex was high after spinal cord injury and accompanied by elevated m^(6)A levels. Knockdown of Mettl14 in the locomotor cortex was not favorable for corticospinal tract regeneration and neurological recovery after spinal cord injury. Through bioinformatics analysis and methylated RNA immunoprecipitation-quantitative polymerase chain reaction, we found that METTL14 regulated Trib2 expression in an m^(6)A-regulated manner, thereby activating the mitogen-activated protein kinase pathway and promoting corticospinal tract regeneration. Finally, we administered syringin, a stabilizer of METTL14, using molecular docking. Results confirmed that syringin can promote corticospinal tract regeneration and facilitate neurological recovery by stabilizing METTL14. Findings from this study reveal that m^(6)A modification is involved in the regulation of corticospinal tract regeneration after spinal cord injury.

Peanut proteins:Extraction,modifications,and applications:A comprehensive review

As naturally sourced proteins,peanut proteins have garnered significant attention from the food industry,owing to their numerous advantages,such as easy extraction,non-pungency,and high bioavailability.Furthermore,peanut proteins are highly digestible in the gastrointestinal tract and boast a high net protein utilization rate,making them an appealing protein source in food products and a promising alternative to animal protein.In this paper,the recent works on the extraction method,modification method,and application of peanut proteins were reviewed.Both advantages and disadvantages of current extraction and modification were discussed.Recently updated information about peanut protein research was summarized.Based on these,the prospection of peanut proteins research was presented,which may be instructive for future research in this field.Future research is still needed for accessible modification methods to develop the functional properties of peanut proteins.

Post-translational modifications of prostaglandin-endoperoxide synthase 2 in colorectal cancer:An update

The biosynthesis of prostanoids is involved in both physiological and pathological processes. The expression of prostaglandin-endoperoxide synthase 2(PTGS2; also known as COX-2) has been traditionally associated to the onset of several pathologies, from inflammation to cardiovascular, gastrointestinal and oncologic events. For this reason, the search of selective PTGS2 inhibitors has been a focus for therapeutic interventions. In addition to the classic non-steroidal anti-inflammatory drugs, selective and specific PTGS2 inhibitors, termed coxibs, have been generated and widely used. PTGS2 activity is less restrictive in terms of substrate specificity than the homeostatic counterpart PTGS1, and it accounts for the elevated prostanoid synthesis that accompanies several pathologies. The main regulation of PTGS2 occurs at the transcription level. In addition to this, the stability of the mRNA is finely regulated through the interaction with several cytoplasmic elements, ranging from specificmicroR NAs to proteins that control mR NA degradation. Moreover, the protein has been recognized to be the substrate for several post-translational modifications that affect both the enzyme activity and the targeting for degradation via proteasomal and non-proteasomal mechanisms. Among these modifications, phosphorylation, glycosylation and covalent modifications by reactive lipidic intermediates and by free radicals associated to the proinflammatory condition appear to be the main changes. Identification of these post-translational modifications is relevant to better understand the role of PTGS2 in several pathologies and to establish a correct analysis of the potential function of this protein in diseases progress. Finally, these modifications can be used as biomarkers to establish correlations with other parameters, including the immunomodulation dependent on molecular pathological epidemiology determinants, which may provide a better frame for potential therapeutic interventions.

Protein post-translational modifications in auxin signaling

Protein post-translational modifications(PTMs),such as ubiquitination,phosphorylation,and small ubiquitin-like modifier(SUMO)ylation,are crucial for regulating protein stability,activity,subcellular localization,and binding with cofactors.Such modifications remarkably increase the variety and complexity of proteomes,which are essential for regulating numerous cellular and physiological processes.The regulation of auxin signaling is finely tuned in time and space to guide various plant growth and development.Accumulating evidence indicates that PTMs play critical roles in auxin signaling regulations.Thus,a thorough and systematic review of the functions of PTMs in auxin signal transduction will improve our profound comprehension of the regulation mechanism of auxin signaling and auxin-mediated various processes.This review discusses the progress of protein ubiquitination,phosphorylation,histone acetylation and methylation,SUMOylation,and S-nitrosylation in the regulation of auxin signaling.

Microfluidic Immunoprecipitation for Post-Translational Modified Protein Purification

In this paper, we report an antibody functionalized microimmunopreci- pitation （IX IP） method used for enrich lowabundant post-translational modified （PT~ proteins. The device is fabricated by inert, nontoxic and disposable polydimethylsiloxane （PDMS） using a silane-based chemical modification protocol, which yield antibody- terminated PDMS surfaces. In this study, the IX IP device is specifically designed for the purification of carbonylated protein, a representative example here to illustrate the potential applications for any other PTMs, which could be immuno-tagged by specific antibodies. The test model in vitro oxidized bovine serum albumin （BSA） was first derivitized by dinitrophenylhydrazide （DNPH） and then captured by the anti-DNP immobilized on this Ix lP device. The surface functional group mapping was systematically analyzed and validated by fluorescence microscopy. Quantitative study of DNP-derivatized carbonylated protein capture recovery and elution efficiency of the device was also studied. We also envision that this proteome enrichment Ix IP device can be assembled with other lab-on-a-chip components, such as microelectrophoresis or micro-chromatographic devices for follow-up protein analysis. This selective enrichment of modified proteins greatly facilitates the study of low abundant protein biomarkers discovery.

The pathogenic mechanism of TAR DNA-binding protein 43(TDP-43)in amyotrophic lateral sclerosis

The onset of amyotrophic lateral sclerosis is usually characterized by focal death of both upper and/or lower motor neurons occurring in the motor cortex,basal ganglia,brainstem,and spinal cord,and commonly involves the muscles of the upper and/or lower extremities,and the muscles of the bulbar and/or respiratory regions.However,as the disease progresses,it affects the adjacent body regions,leading to generalized muscle weakness,occasionally along with memory,cognitive,behavioral,and language impairments;respiratory dysfunction occurs at the final stage of the disease.The disease has a complicated pathophysiology and currently,only riluzole,edaravone,and phenylbutyrate/taurursodiol are licensed to treat amyotrophic lateral sclerosis in many industrialized countries.The TAR DNA-binding protein 43 inclusions are observed in 97%of those diagnosed with amyotrophic lateral sclerosis.This review provides a preliminary overview of the potential effects of TAR DNAbinding protein 43 in the pathogenesis of amyotrophic lateral sclerosis,including the abnormalities in nucleoplasmic transport,RNA function,post-translational modification,liquid-liquid phase separation,stress granules,mitochondrial dysfunction,oxidative stress,axonal transport,protein quality control system,and non-cellular autonomous functions(e.g.,glial cell functions and prion-like propagation).

Impact of hot alkali modification conditions on secondary structure of peanut protein and embedding rate of curcumin

This study aimed to modify isolated and extracted peanut protein with hot alkali to study the impact of pH,heating temperature,processing time and other alkali liquor conditions on the molecular structure of the peanut.Curcumin was loaded in modified peanut protein.The results of the study are as follows:Within the alkaline range of 8<pH<12,the percentage of amino acid residue(AAR)and-turns first increased and then decreased with the increasing pH,and the percentage of AAR reached a maximum 5.21±0.33%when the pH was 11(p<0.01).The percentage of˛-helices andβ-sheets gradually decreased with increasing pH,while that of random coils gradually increased with increasing pH,reaching a maximum 11.24±0.87%when the pH was 11(p<0.05).Within the range of the heating temperature 75℃<T<95℃,the percentage of random coils andβ-sheets gradually increased with increasing heating temperature,while that of-helices and AAR gradually decreased with increasing heating temperature;they remained unchanged when the heating temperature was 90℃,and then decreased to(10.41±1.18%;p<0.01)and(4.02±2.12%;p<0.01),respectively.Within the range of 5 min<t<20 min,the percentage of random coils and AAR gradually increased with increasing heating time,while the percentage ofα-helices decreased from 11.83±1.04%to 10.75±2.34%with increased heating time(p<0.01).The optimum conditions for hot alkali modification of peanut protein as followed:heating temperature of 90℃,heating time of 20 min and a pH of alkali liquor of 11.Under these optimum conditions,the embedding rate of curcumin by the modified protein can reach 88.32±1.29%.

Lipid accumulation and protein modifications of Bruch’s membrane in age-related macular degeneration

Age-related macular degeneration(AMD)is a progressive retinal disease,which is the leading cause of blindness in western countries.There is an urgency to establish new therapeutic strategies that could prevent or delay the progression of AMD more efficiently.Until now,the pathogenesis of AMD has remained unclear,limiting the development of the novel therapy.Bruch’s membrane(BM)goes through remarkable changes in AMD,playing a significant role during the disease course.The main aim of this review is to present the crucial processes that occur at the level of BM,with special consideration of the lipid accumulation and protein modifications.Besides,some therapies targeted at these molecules and the construction of BM in tissue engineering of retinal pigment epithelium(RPE)cells transplantation were listed.Hopefully,this review may provide a reference for researchers engaged in pathogenesis or management on AMD.

Characterization of physicochemical and immunogenic properties of allergenic proteins altered by food processing:a review

Food allergens are mainly naturally-occurring proteins with immunoglobulin E(IgE)-binding epitopes.Understanding the structural and immunogenic characteristics of allergenic proteins is essential in assessing whether and how food processing techniques reduce allergenicity.We here discuss the impacts of food processing technologies on the modification of physicochemical,structural,and immunogenic properties of allergenic proteins.Detection techniques for characterizing changes in these properties of food allergens are summarized.Food processing helps to reduce allergenicity by aggregating or denaturing proteins,which masks,modifies,or destroys antigenic epitopes,whereas,it cannot eliminate allergenicity completely,and sometimes even improves allergenicity by exposing new epitopes.Moreover,most food processing techniques have been tested on purified food allergens rather than food products due to potential interference of other food components.We provide guidance for further development of processing operations that can decrease the allergenicity of allergenic food proteins without negatively impacting the nutritional profile.

2007 International Symposium on Protein Modification and Degradation in Beijing(SPMDB2007)

Presidents of Symposium Depei Liu,President of Chinese Academy of Medical Sciences Zhu Chen,Vice President of Chinese Academy ofSciences.

Effect of Electrode Surface Modification by Sulfide on QCM Based Protein Biosensor

The rapid development of surface sensitive biosensor technologies requires optimum control of surface modification to provide reliable and reproducible results. With the aim to assemble a quartz crystal microbalance (QCM)-based protein biosensor, we focus our attention on sulfide receptor and its integration with the surface of the electrode. Here, we present different surface modification processing time to allow sulfide molecules to be immobilized to gold coated sensor for QCM sensing. The optimum surface modification processing time is also obtained by bovine serum albumin (BSA) binding measurement.

Chemical Modification of Food Proteins

Acylation has been shown to be an effective toolfor improving surface functional properties of plant proteins.Soy bean protein has been extensively modified throughchemical and enzvmatic treatments.Their effectiveness lies intheir high nutritional value and low cost,which promotetheir use as ingredients for the formulation of food products.This paper reports a complete review of chemical modificationof various proteins from plant and animal sources,The nutri-tive and toxicological aspects through in vitro and in vivotests are also described.