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.

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.

Stem cell-based ischemic stroke therapy:Novel modifications and clinical challenges

Ischemic stroke(IS)causes severe disability and high mortality worldwide.Stem cell(SC)therapy exhibits unique therapeutic potential for IS that differs from current treatments.SC’s cell homing,differentiation and paracrine abilities give hope for neuroprotection.Recent studies on SC modification have enhanced therapeutic effects for IS,including gene transfection,nanoparticle modification,biomaterial modification and pretreatment.Thesemethods improve survival rate,homing,neural differentiation,and paracrine abilities in ischemic areas.However,many problems must be resolved before SC therapy can be clinically applied.These issues include production quality and quantity,stability during transportation and storage,as well as usage regulations.Herein,we reviewed the brief pathogenesis of IS,the“multi-mechanism”advantages of SCs for treating IS,various SC modification methods,and SC therapy challenges.We aim to uncover the potential and overcome the challenges of using SCs for treating IS and convey innovative ideas for modifying SCs.

Implications of electrode modifications in aqueous organic redox flow batteries

Aqueous organic redox flow batteries(RFBs)exhibit favorable characteristics,such as tunability,multielectron transfer capability,and stability of the redox active molecules utilized as anolytes and catholytes,making them very viable contenders for large-scale grid storage applications.Considerable attention has been paid on the development of efficient redox-active molecules and their performance optimization through chemical substitutions at various places on the backbone as part of the pursuit for high-performance RFBs.Despite the fact that electrodes are vital to optimal performance,they have not garnered significant attention.Limited research has been conducted on the effects of electrode modifications to improve the performance of RFBs.The primary emphasis has been given on the impact of electrode engineering to augment the efficiency of aqueous organic RFBs.An overview of electron transfer at the electrode-electrolyte interface is provided.The implications of electrode modification on the performance of redox flow batteries,with a particular focus on the anodic and cathodic half-cells separately,are then discussed.In each section,significant discrepancies surrounding the effects of electrode engineering are thoroughly examined and discussed.Finally,we have presented a comprehensive assessment along with our perspectives on the future trajectory.

Variations of Atmospheric ELF/VLF Radio Noise Due to Seismogenic Modifications in Tropospheric Conductivity

We suggest a possible explanation of the influence of pre-seismic activity on the registration rate of natural ELF(extremely low frequency)/VLF(very low frequency) pulses and the changes of their characteristics. The main idea is as follows. The distribution of the electric field around a thundercloud depends on the conductivity profile of the atmosphere. Quasi-static electric fields of a thundercloud decrease in those tropospheric regions where an increase of air conductivity is generated by pre-seismic activities due to emanation of radioactive gas and water into the lower atmosphere. The electric field becomes reduced in the lower troposphere, and the probability decreases of the cloud-to-ground (CG) strokes in such “contaminated” areas. Simultaneously, the electric field grows inside and above the thunderclouds, and hence, we anticipate a growth in the number of horizontal and tilted inter-cloud (or intra-cloud) (both termed as IC discharges) strokes. Spatial orientation of lightning strokes reduces vertical projection of their individual amplitudes, while the rate (median number strokes per a unit time) of discharges grows. We demonstrate that channel tilt of strokes modifies the spectral content of ELF/VLF radio noise and changes the rate of detected pulses during the earthquake preparation phase.

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.

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 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.

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.

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.

Epigenetic modifications and metabolic memory in diabetic retinopathy:beyond the surface

Epigenetics focuses on DNA methylation,histone modification,chromatin remodeling,noncoding RNAs,and other gene regulation mechanisms beyond the DNA sequence.In the past decade,epigenetic modifications have drawn more attention as they participate in the development and progression of diabetic retinopathy despite tight control of glucose levels.The underlying mechanisms of epigenetic modifications in diabetic retinopathy still urgently need to be elucidated.The diabetic condition facilitates epigenetic changes and influences target gene expression.In this review,we summarize the involvement of epigenetic modifications and metabolic memory in the development and progression of diabetic retinopathy and propose novel insights into the treatment of diabetic retinopathy.

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.

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.

Succinylation modification:a potential therapeutic target in stroke

Stroke is a leading cause of mortality and disability worldwide.Ischemic cell death triggered by the compromised supply of blood oxygen and glucose is one of the major pathophysiology of strokeinduced brain injury.Impaired mitochondrial energy metabolism is observed minutes after stroke and is closely associated with the progression of neuropathology.Recently,a new type of posttranslational modification,known as lysine succinylation,has been recognized to play a significant role in mitochondrial energy metabolism after ischemia.However,the role of succinylation modification in cell metabolism after stroke and its regulation are not well understood.We aimed to review the effects of succinylation on energy metabolism,reactive oxygen species generation,and neuroinflammation,as well as Sirtuin 5 mediated desuccinylation after stroke.We also highlight the potential of targeting succinylation/desuccinylation as a promising strategy for the treatment of stroke.The succinylation level is dynamically regulated by the nonenzymatic or enzymatic transfer of a succinyl group to a protein on lysine residues and the removal of succinyl catalyzed by desuccinylases.Mounting evidence has suggested that succinylation can regulate the metabolic pathway through modulating the activity or stability of metabolic enzymes.Sirtuins,especially Sirtuin 5,are characterized for their desuccinylation activity and have been recognized as a critical regulator of metabolism through desuccinylating numerous metabolic enzymes.Imbalance between succinylation and desuccinylation has been implicated in the pathophysiology of stroke.Pharmacological agents that enhance the activity of Sirtuin 5 have been employed to promote desuccinylation and improve mitochondrial metabolism,and neuroprotective effects of these agents have been observed in experimental stroke studies.However,their therapeutic efficacy in stroke patients should be validated.

Successful lifestyle modifications may underlie umbilical cordmesenchymal stromal cell effects in type 2 diabetes mellitus

Type 2 diabetes mellitus(T2DM)is a lifelong condition and a grave threat to human health.Innovative efforts to relieve its detrimental effects are acutely needed.The sine qua non in T2DM management is consistent adherence to a prudent lifestyle and nutrition,combined with aerobic and resistance exercise regimens,together repeatedly shown to lead to complete reversal and even longterm remission.Non-adherence to the above lifestyle adjustments condemns any treatment effort and ultimately the patient to a grim fate.It is thus imperative that every study evaluating the effects of innovative interventions in T2DM objectively compares the novel treatment modality to lifestyle modifications,preferably through double-blind controlled randomization,before claiming efficacy.

Genetically modified non-human primate models for research on neurodegenerative diseases

Neurodegenerative diseases(NDs)are a group of debilitating neurological disorders that primarily affect elderly populations and include Alzheimer's disease(AD),Parkinson's disease(PD),Huntington's disease(HD),and amyotrophic lateral sclerosis(ALS).Currently,there are no therapies available that can delay,stop,or reverse the pathological progression of NDs in clinical settings.As the population ages,NDs are imposing a huge burden on public health systems and affected families.Animal models are important tools for preclinical investigations to understand disease pathogenesis and test potential treatments.While numerous rodent models of NDs have been developed to enhance our understanding of disease mechanisms,the limited success of translating findings from animal models to clinical practice suggests that there is still a need to bridge this translation gap.Old World nonhuman primates(NHPs),such as rhesus,cynomolgus,and vervet monkeys,are phylogenetically,physiologically,biochemically,and behaviorally most relevant to humans.This is particularly evident in the similarity of the structure and function of their central nervous systems,rendering such species uniquely valuable for neuroscience research.Recently,the development of several genetically modified NHP models of NDs has successfully recapitulated key pathologies and revealed novel mechanisms.This review focuses on the efficacy of NHPs in modeling NDs and the novel pathological insights gained,as well as the challenges associated with the generation of such models and the complexities involved in their subsequent analysis.

Enhanced Electrochemical Properties and Optimized Li^(+)Transmission Pathways of PEO/LLZTO-Based Composite Electrolytes Modified by Supramolecular Combination

Poly(ethylene oxide)(PEO)and Li_(6.75)La_(3)Zr_(1.75)Ta_(0.25)O_(12)(LLZTO)-based composite polymer electrolytes(CPEs)are considered one of the most promising solid electrolyte systems.However,agglomeration of LLZTO within PEO and lack of Li^(+)channels result in poor electrochemical properties.Herein,a functional supramolecular combination(CD-TFSI)consisting of activeβ-cyclodextrin(CD)supramolecular with self-assembled LiTFSI salt is selected as an interface modifier to coat LLZTO fillers.Benefiting from vast H-bonds formed betweenβ-CD and PEO matrix and/or LLZTO,homogeneous dispersion and tight interface contact are obtained.Moreover,^(6)Li NMR spectra confirm a new Li^(+)transmission pathway from PEO matrix to LLZTO ceramic then to PEO matrix in the as-prepared PEO/LLZTO@CD-TFSI CPEs due to the typical cavity structure ofβ-CD.As a proof,the conductivity is increased from 5.3×10^(-4)S cm^(-1)to 8.7×10^(-4)S cm^(-1)at 60℃,the Li^(+)transference number is enhanced from 0.38 to 0.48,and the electrochemical stability window is extended to 5.1 V versus Li/Li^(+).Li‖LiFePO_(4)CR2032 coin full cells and pouch cells prove the practical application of the as-prepared PEO/LLZTO@CD-TFSI CPEs.This work offers a new strategy of interface modifying LLZTO fillers with functional supramolecular combination to optimize PEO/LLZTO CPEs for solid lithium batteries.

Dysregulation of RNA modification systems in clinical populations with neurocognitive disorders

The study of modified RNA known as epitranscriptomics has become increasingly relevant in our understanding of disease-modifying mechanisms.Methylation of N6 adenosine(m^(6)A)and C5 cytosine(m^(5)C)bases occur on mRNAs,tRNA,mt-tRNA,and rRNA species as well as non-coding RNAs.With emerging knowledge of RNA binding proteins that act as writer,reader,and eraser effector proteins,comes a new understanding of physiological processes controlled by these systems.Such processes when spatiotemporally disrupted within cellular nanodomains in highly specialized tissues such as the brain,give rise to different forms of disease.In this review,we discuss accumulating evidence that changes in the m^(6)A and m^(5)C methylation systems contribute to neurocognitive disorders.Early studies first identified mutations within FMR1 to cause intellectual disability Fragile X syndromes several years before FMR1 was identified as an m^(6)A RNA reader protein.Subsequently,familial mutations within the m^(6)A writer gene METTL5,m^(5)C writer genes NSUN2,NSUN3,NSUN5,and NSUN6,as well as THOC2 and THOC6 that form a protein complex with the m^(5)C reader protein ALYREF,were recognized to cause intellectual development disorders.Similarly,differences in expression of the m^(5)C writer and reader effector proteins,NSUN6,NSUN7,and ALYREF in brain tissue are indicated in individuals with Alzheimer's disease,individuals with a high neuropathological load or have suffered traumatic brain injury.Likewise,an abundance of m^(6)A reader and anti-reader proteins are reported to change across brain regions in Lewy bodies diseases,Alzheimer's disease,and individuals with high cognitive reserve.m^(6)A-modified RNAs are also reported significantly more abundant in dementia with Lewy bodies brain tissue but significantly reduced in Parkinson's disease tissue,whilst modified RNAs are misplaced within diseased cells,particularly where synapses are located.In parahippocampal brain tissue,m^(6)A modification is enriched in transcripts associated with psychiatric disorders including conditions with clear cognitive deficits.These findings indicate a diverse set of molecular mechanisms are influenced by RNA methylation systems that can cause neuronal and synaptic dysfunction underlying neurocognitive disorders.Targeting these RNA modification systems brings new prospects for neural regenerative therapies.

Current Strategies of Surface Modifications to Polyurethane Biomaterials for Vascular Grafts

As the number of patients suffering from cardiovascular diseases and peripheral vascular diseases rises,the constraints of autologous transplantation remain unavoidable.As a result,artificial vascular grafts must be developed.Adhesion of proteins,platelets and bacteria on implants can result in stenosis,thrombus formation,and postoperative infection,which can be fatal for an implantation.Polyurethane,as a commonly used biomaterial,has been modified in various ways to deal with the adhesions of proteins,platelets,and bacteria and to stimulate endothelium adhesion.In this review,we briefly summarize the mechanisms behind adhesions,overview the current strategies of surface modifications of polyurethane biomaterials used in vascular grafts,and highlight the challenges that need to be addressed in future studies,aiming to gain a more profound understanding of how to develop artificial polyurethane vascular grafts with an enhanced implantation success rate and reduced side effect.