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.

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.

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.

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.

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.

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.

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.

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.

Post-translational regulation of inflammasomes

Infiammasomes play essential roles in immune protection against microbial infections. However, excessive inflammation is implicated in various human diseases, including autoinflammatory syndromes, diabetes, multiple sclerosis, cardiovascular disorders and neurodegenerative diseases. Therefore, precise regulation of inflammasome activities is critical for adequate immune protection while limiting collateral tissue damage. In this review, we focus on the emerging roles of post-translational modifications （PTMs） that regulate activation of the NLRP3, NLRP1, NLRC4, AIM2 and IFI16 inflammasomes. We anticipate that these types of PTMs will be identified in other types of and less well-characterized inflammasomes. Because these highly diverse and versatile PTMs shape distinct inflammatory responses in response to infections and tissue damage, targeting the enzymes involved in these PTMs will undoubtedly offer opportunities for precise modulation of inflammasome activities under various pathophysiological conditions.

Post-translational inhibitory regulation of acid invertase induced by fructose and glucose in developing apple fruit

Acid invertase (EC 3.2.1.26) is one of the key enzymes involved in the carbohydrate sink-organ development and the sink strength modulation in crops. The experiment conducted with 'Starkrimson' apple (Malus domestica Borkh) fruit showed that, during the fruit development, the activity of acid invertase gradually declined concomitantly with the progressive accumulation of fructose, glucose and sucrose, while Western blotting assay of acid invertase detected a 30 ku peptide of which the immuno-signal intensity increased during the fruit development. The immuno-localization via immunogold electron microscopy showed that, on the one hand, acid invertase was mainly located on the flesh cell wall with numbers of the immunosignals present in the vacuole at the late stage of fruit development; and on the other hand, the amount of acid invertase increased during fruit development, which was consistent with the results of Western blotting. The in vivo pre-incubation of fruit discs with soluble sugars showed that the activity of extractible acid invertase was inhibited by fructose or glucose, while Western blotting did not detect any changes in apparent quantity of the enzyme nor other peptides than 30 ku one. So it is considered that fructose and glucose induced the post-translational or translocational inhibitory regulation of acid invertase in developing apple fruit. The mechanism of the post-translational inhibition was shown different from both the two previously reported ones that proposed either the inhibition by hexose products in the in vitro chemical reaction equilibrium system or the inhibition by the proteinaceous inhibitors. It was hypothesized that fructose and glucose might induce acid invertase inhibition by modulating the expression of some inhibition-related genes or some structural modification of acid invertase.

Autophagy in plants: Physiological roles and post-translational regulation

In eukaryotes, autophagy helps maintain cellular homeostasis by degrading and recycling cytoplasmic materials via a tightly regulated pathway.Over the past few decades, significant progress has been made towards understanding the physiological functions and molecular regulation of autophagy in plant cells. Increasing evidence indicates that autophagy is essential for plant responses to several developmental and environmental cues, functioning in diverse processes such as senescence, male fertility, root meristem maintenance, responses to nutrient starvation,and biotic and abiotic stress. Recent studies have demonstrated that, similar to nonplant systems,the modulation of core proteins in the plant autophagy machinery by posttranslational modifications such as phosphorylation, ubiquitination,lipidation, S-sulfhydration, S-nitrosylation, and acetylation is widely involved in the initiation and progression of autophagy. Here, we provide an overview of the physiological roles and posttranslational regulation of autophagy in plants.

Applications of post-translational modifications of FoxO family proteins in biological functions

The functions of the FoxO family proteins,in particular their transcriptional activities,are modulated by post-translational modifi-cations(PTMs),including phosphorylation,acetylation,ubiquitination,methylation and glycosylation.These PTMs occur in response to different cellular stresses,which in turn regulate the subcellular localization of FoxO family proteins,as well as their half-life,DNA binding,transcriptional activity and ability to interact with other cellular proteins.In this review,we summarize the role of PTMs of FoxO family proteins in linking their biological and functional relevance with various diseases.

Histone post-translational modification and the DNA damage response

DNA is highly vulnerable to spontaneous and environmental timely damage in living cells.DNA damage may cause genetic instability and increase cancer risk if the damages are not repaired timely and efficiently.Human cells possess several DNA damage response(DDR)mechanisms to protect the integrity of their genome.Clarification of the mechanisms under-lying the DNA damage response following lethal damage will facilitate the identification of therapeutic targets for cancers.Histone post-translational modifications(PTMs)have been indicated to play different roles in the repair of DNA damage.In this context,histone PTMs regulate recruitment of downstream effectors,and facilitate appropriate repair response.This review outlines the current understanding of different histone PTMs in response to DNA dam-age repair,besides,enumerates the role of new type PTMs such as histone succinylation and crotonylation in regulating DNA damage repair processes.

Post-translational modification of Parkin and its research progress in cancer

Clinical practice has shown that Parkin is the major causative gene found in an autosomal recessive juvenile parkin-sonism(AR-JP)via Parkin mutations and that the Parkin protein is the core expression product of the Parkin gene,which itself belongs to an E3 ubiquitin ligase.Since the discovery of the Parkin gene in the late 1990s,researchers in many countries have begun extensive research on this gene and found that in addition to AR-JP,the Parkin gene is associated with many diseases,including type 2 diabetes,leprosy,Alzheimer’s,autism,and cancer.Recent studies have found that the loss or dysfunction of Parkin has a certain relationship with tumorigenesis.In general,the Parkin gene,a well-established tumor suppressor,is deficient and mutated in a variety of malignancies.Parkin overexpres-sion inhibits tumor cell growth and promotes apoptosis.However,the functions of Parkin in tumorigenesis and its regulatory mechanisms are still not fully understood.This article describes the structure,functions,and post-transla-tional modifications of Parkin,and summarizes the recent advances in the tumor suppressive function of Parkin and its underlying mechanisms.

Role of post-translational modification of the Y box binding protein 1 in human cancers

Y box binding protein-1(YBX1)belongs to a DNA-and RNA-binding family of transcription factors,containing the highly conserved cold shock domain(CSD).YBX1 is involved in a number of cellular functions including transcription,translation,DNA damage repair etc.,and it is upregulated during times of environmental stress.YBX1 is localized in both the cytoplasm and the nucleus.There,its nuclear translocation is observed in a number of cancers and is associated with poor prognosis and disease progression.Additionally,YBX1 expression is upregulated in a variety of cancers,pointing towards its role as a potential oncogene.Under certain circumstances,YBX1 also promotes the expression of multidrug resistance 1(MDR1)gene,which is involved in the development of drug resistance.Thus,it is critical to understand the mechanism of YBX1 regulation and its downstream effects on promoting cancer development.A number of recent studies have highlighted the mechanisms of YBX1 regulation.Mass spectrometric analyses have reported several post-translational modifications that possibly play an important role in modulating YBX1 function.Phosphorylation is the most widely occurring post-translational modification in YBX1.In vivo analyses of sites like S102 and more recently,S165 illustrate the relationship of post-translational regulation of YBX1 in promoting cell proliferation and tumor growth.This review provides a comprehensive and up-to-date account of post-translational modifications identified in YBX1.This knowledge is a key in allowing us to better understand the mechanism of YBX1 regulation,which will aid in development of novel therapeutic strategies to target YBX1 in many types of cancer in the future.

Diagnostic and Prognostic Value of Protein Post-translational Modifications in Hepatocellular Carcinoma

Hepatocellular carcinoma(HCC)is a common malignant tumor with high incidence and cancer mortality worldwide.Post-translational modifications(PTMs)of proteins have a great impact on protein function.Almost all proteins can undergo PTMs,including phosphorylation,acetylation,methylation,glycosylation,ubiquitination,and so on.Many studies have shown that PTMs are related to the occurrence and development of cancers.The findings provide novel therapeutic targets for cancers,such as glypican-3 and mucin-1.Other clinical implications are also found in the studies of PTMs.Diagnostic or prognostic value,and response to therapy have been identified.In HCC,it has been shown that glycosylated alpha-fetoprotein(AFP)has a higher detection rate for early liver cancer than conventional AFP.In this review,we mainly focused on the diagnostic and prognostic value of PTM,in order to provide new insights into the clinical implication of PTM in HCC.

All Roads Lead to Auxin: Post-translational Regulation of Auxin Transport by Multiple Hormonal Pathways

Auxin is a key hormonal regulator,that governs plant growth and development in concert with other hormonal pathways.The unique feature of auxin is its polar,cell-to-cell transport that leads to the formation of local auxin maxima and gradients,which coordinate initiation and patterning of plant organs.The molecular machinery mediating polar auxin transport is one of the important points of interaction with other hormones.Multiple hormonal pathways converge at the regulation of auxin transport and form a regulatory network that integrates various developmental and environmental inputs to steer plant development.In this review,we discuss recent advances in understanding the mechanisms that underlie regulation of polar auxin transport by multiple hormonal pathways.Specifically,we focus on the post-translational mechanisms that contribute to fine-tuning of the abundance and polarity of auxin transporters at the plasma membrane and thereby enable rapid modification of the auxin flow to coordinate plant growth and development.

Post-translational O-GlcNAcylation is essential for nuclear pore integrity and maintenance of the pore selectivity filter

O-glycosylation of the nuclear pore complex(NPC)by O-linked N-acetylglucosamine(O-GlcNAc)is conserved within metazoans.Many nucleoporins(Nups)comprising the NPC are constitutively O-GlcNAcylated,but the functional role of this modification remains enigmatic.Weshowthat loss ofO-GlcNAc,induced by either inhibition ofO-GlcNAc transferase(OGT)or deletion of the gene encoding OGT,leads to decreased cellular levels of a number of natively O-GlcNAcylated Nups.Loss of O-GlcNAc enables increased ubiquitination of these Nups and their increased proteasomal degradation.The decreased half-life of these deglycosylated Nups manifests in their gradual loss from the NPC and a downstream malfunction of the nuclear pore selective permeability barrier in both dividing and post-mitotic cells.These findings define a critical role of O-GlcNAc modification of the NPC in maintaining its composition and the function of the selectivity filter.The results implicate NPC glycosylation as a regulator of NPC function and reveal the role of conserved glycosylation of the NPC among metazoans.