The interaction of Escherichia coli O157 :H7 and Salmonella Typhimurium flagella with host cell membranes and cytoskeletal components.

Bacterial flagella have many established roles beyond swimming motility. Despite clear evidence of flagella-dependent adherence, the specificity of the ligands and mechanisms of binding are still debated. In this study, the molecular basis of Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium flagella binding to epithelial cell cultures was investigated. Flagella interactions with host cell surfaces were intimate and crossed cellular boundaries as demarcated by actin and membrane labelling. Scanning electron microscopy revealed flagella disappearing into cellular surfaces and transmission electron microscopy of S. Typhiumurium indicated host membrane deformation and disruption in proximity to flagella. Motor mutants of E. coli O157:H7 and S. Typhimurium caused reduced haemolysis compared to wild-type, indicating that membrane disruption was in part due to flagella rotation. Flagella from E. coli O157 (H7), EPEC O127 (H6) and S. Typhimurium (P1 and P2 flagella) were shown to bind to purified intracellular components of the actin cytoskeleton and directly increase in vitro actin polymerization rates. We propose that flagella interactions with host cell membranes and cytoskeletal components may help prime intimate attachment and invasion for E. coli O157:H7 and S. Typhimurium, respectively.

A chicken bioreactor for efficient production of functional cytokines.

BACKGROUND: The global market for protein drugs has the highest compound annual growth rate of any pharmaceutical class but their availability, especially outside of the US market, is compromised by the high cost of manufacture and validation compared to traditional chemical drugs. Improvements in transgenic technologies allow valuable proteins to be produced by genetically-modified animals; several therapeutic proteins from such animal bioreactors are already on the market after successful clinical trials and regulatory approval. Chickens have lagged behind mammals in bioreactor development, despite a number of potential advantages, due to the historic difficulty in producing transgenic birds, but the production of therapeutic proteins in egg white of transgenic chickens would substantially lower costs across the entire production cycle compared to traditional cell culture-based production systems. This could lead to more affordable treatments and wider markets, including in developing countries and for animal health applications. RESULTS: Here we report the efficient generation of new transgenic chicken lines to optimize protein production in eggs. As proof-of-concept, we describe the expression, purification and functional characterization of three pharmaceutical proteins, the human cytokine interferon α2a and two species-specific Fc fusions of the cytokine CSF1. CONCLUSION: Our work optimizes and validates a transgenic chicken system for the cost-effective production of pure, high quality, biologically active protein for therapeutics and other applications.

A recurrent de novo mutation in ACTG1 causes isolated ocular coloboma.

Ocular coloboma (OC) is a defect in optic fissure closure and is a common cause of severe congenital visual impairment. Bilateral OC is primarily genetically determined and shows marked locus heterogeneity. Whole-exome sequencing (WES) was used to analyze 12 trios (child affected with OC and both unaffected parents). This identified de novo mutations in 10 different genes in eight probands. Three of these genes encoded proteins associated with actin cytoskeleton dynamics: ACTG1, TWF1, and LCP1. Proband-only WES identified a second unrelated individual with isolated OC carrying the same ACTG1 allele, encoding p.(Pro70Leu). Both individuals have normal neurodevelopment with no extra-ocular signs of Baraitser-Winter syndrome. We found this mutant protein to be incapable of incorporation into F-actin. The LCP1 and TWF1 variants each resulted in only minor disturbance of actin interactions, and no further plausibly causative variants were identified in these genes on resequencing 380 unrelated individuals with OC.

Microbiota in Exhaled Breath Condensate and the Lung.

The lung microbiota is commonly sampled using relatively invasive bronchoscopic procedures. Exhaled breath condensate (EBC) collection potentially offers a less invasive alternative for lung microbiota sampling. We compared lung microbiota samples retrieved by protected specimen brushings (PSB) and exhaled breath condensate collection. We also sought to assess whether aerosolized antibiotic treatment would influence the lung microbiota and whether this change could be detected in EBC. EBC was collected from 6 conscious sheep and then from the same anesthetized sheep during mechanical ventilation. Following the latter EBC collection, PSB samples were collected from separate sites within each sheep lung. On the subsequent day, each sheep was then treated with nebulized colistimethate sodium. Two days after nebulization, EBC and PSB samples were again collected. Bacterial DNA was quantified using 16S rRNA gene quantitative PCR. The V2-V3 region of the 16S rRNA gene was amplified by PCR and sequenced using Illumina MiSeq. Quality control and operational taxonomic unit (OTU) clustering were performed with mothur. The EBC samples contained significantly less bacterial DNA than the PSB samples. The EBC samples from anesthetized animals clustered separately by their bacterial community compositions in comparison to the PSB samples, and 37 bacterial OTUs were identified as differentially abundant between the two sample types. Despite only low concentrations of colistin being detected in bronchoalveolar lavage fluid, PSB samples were found to differ by their bacterial compositions before and after colistimethate sodium treatment. Our findings indicate that microbiota in EBC samples and PSB samples are not equivalent.IMPORTANCE Sampling of the lung microbiota usually necessitates performing bronchoscopic procedures that involve a hospital visit for human participants and the use of trained staff. The inconvenience and perceived discomfort of participating in this kind of research may deter healthy volunteers and may not be a safe option for patients with advanced lung disease. This study set out to evaluate a less invasive method for collecting lung microbiota samples by comparing samples taken via protected specimen brushings (PSB) to those taken via exhaled breath condensate (EBC) collection. We found that there was less bacterial DNA in EBC samples compared with that in PSB samples and that there were differences between the bacterial communities in the two sample types. We conclude that while EBC and PSB samples do not produce equivalent microbiota samples, the study of the EBC microbiota may still be of interest.

PrP aggregation can be seeded by pre-formed recombinant PrP amyloid fibrils without the replication of infectious prions.

Mammalian prions are unusual infectious agents, as they are thought to consist solely of aggregates of misfolded prion protein (PrP). Generation of synthetic prions, composed of recombinant PrP (recPrP) refolded into fibrils, has been utilised to address whether PrP aggregates are, indeed, infectious prions. In several reports, neurological disease similar to transmissible spongiform encephalopathy (TSE) has been described following inoculation and passage of various forms of fibrils in transgenic mice and hamsters. However, in studies described here, we show that inoculation of recPrP fibrils does not cause TSE disease, but, instead, seeds the formation of PrP amyloid plaques in PrP-P101L knock-in transgenic mice (101LL). Importantly, both WT-recPrP fibrils and 101L-recPrP fibrils can seed plaque formation, indicating that the fibrillar conformation, and not the primary sequence of PrP in the inoculum, is important in initiating seeding. No replication of infectious prions or TSE disease was observed following both primary inoculation and subsequent subpassage. These data, therefore, argue against recPrP fibrils being infectious prions and, instead, indicate that these pre-formed seeds are acting to accelerate the formation of PrP amyloid plaques in 101LL Tg mice. In addition, these data reproduce a phenotype which was previously observed in 101LL mice following inoculation with brain extract containing in vivo-generated PrP amyloid fibrils, which has not been shown for other synthetic prion models. These data are reminiscent of the "prion-like" spread of aggregated forms of the beta-amyloid peptide (Aß), α-synuclein and tau observed following inoculation of transgenic mice with pre-formed seeds of each misfolded protein. Hence, even when the protein is PrP, misfolding and aggregation do not reproduce the full clinicopathological phenotype of disease. The initiation and spread of protein aggregation in transgenic mouse lines following inoculation with pre-formed fibrils may, therefore, more closely resemble a seeded proteinopathy than an infectious TSE disease.

The PrP(C) C1 fragment derived from the ovine A136R154R171PRNP allele is highly abundant in sheep brain and inhibits fibrillisation of full-length PrP(C) protein in vitro.

Expression of the cellular prion protein (PrP(C)) is crucial for the development of prion diseases. Resistance to prion diseases can result from reduced availability of the prion protein or from amino acid changes in the prion protein sequence. We propose here that increased production of a natural PrP α-cleavage fragment, C1, is also associated with resistance to disease. We show, in brain tissue, that ARR homozygous sheep, associated with resistance to disease, produced PrP(C) comprised of 25% more C1 fragment than PrP(C) from the disease-susceptible ARQ homozygous and highly susceptible VRQ homozygous animals. Only the C1 fragment derived from the ARR allele inhibits in-vitro fibrillisation of other allelic PrP(C) variants. We propose that the increased α-cleavage of ovine ARR PrP(C) contributes to a dominant negative effect of this polymorphism on disease susceptibility. Furthermore, the significant reduction in PrP(C) ß-cleavage product C2 in sheep of the ARR/ARR genotype compared to ARQ/ARQ and VRQ/VRQ genotypes, may add to the complexity of genetic determinants of prion disease susceptibility.

Significant differences in incubation times in sheep infected with bovine spongiform encephalopathy result from variation at codon 141 in the PRNP gene.

The susceptibility of sheep to prion infection is linked to variation in the PRNP gene, which encodes the prion protein. Common polymorphisms occur at codons 136, 154 and 171. Sheep which are homozygous for the A(136)R(154)Q(171) allele are the most susceptible to bovine spongiform encephalopathy (BSE). The effect of other polymorphisms on BSE susceptibility is unknown. We orally infected ARQ/ARQ Cheviot sheep with equal amounts of BSE brain homogenate and a range of incubation periods was observed. When we segregated sheep according to the amino acid (L or F) encoded at codon 141 of the PRNP gene, the shortest incubation period was observed in LL(141) sheep, whilst incubation periods in FF(141) and LF(141) sheep were significantly longer. No statistically significant differences existed in the expression of total prion protein or the disease-associated isoform in BSE-infected sheep within each genotype subgroup. This suggested that the amino acid encoded at codon 141 probably affects incubation times through direct effects on protein misfolding rates.

Low density subcellular fractions enhance disease-specific prion protein misfolding.

The production of prion particles in vitro by amplification with or without exogenous seed typically results in infectivity titers less than those associated with PrP(Sc) isolated ex vivo and highlights the potential role of co-factors that can catalyze disease-specific prion protein misfolding in vivo. We used a cell-free conversion assay previously shown to replicate many aspects of transmissible spongiform encephalopathy disease to investigate the cellular location of disease-specific co-factors using fractions derived from gradient centrifugation of a scrapie-susceptible cell line. Fractions from the low density region of the gradient doubled the efficiency of conversion of recombinant PrP. These fractions contain plasma membrane and cytoplasmic proteins, and conversion enhancement can be achieved using PrP(Sc) derived from two different strains of mouse-passaged scrapie as seed. Equivalent fractions from a second scrapie-susceptible cell line also stimulate conversion. We also show that subcellular fractions enhancing disease-specific prion protein conversion prevent in vitro fibrillization of recombinant prion protein, suggesting the existence of separate, competing mechanisms of disease-specific and nonspecific misfolding in vivo.

Statistical Evidence for a Helical Nascent Chain.

We investigate the hypothesis that protein folding is a kinetic, non-equilibrium process, in which the structure of the nascent chain is crucial. We compare actual amino acid frequencies in loops, α-helices and ß-sheets with the frequencies that would arise in the absence of any amino acid bias for those secondary structures. The novel analysis suggests that while specific amino acids exist to drive the formation of loops and sheets, none stand out as drivers for α-helices. This favours the idea that the α-helix is the initial structure of most proteins before the folding process begins.

Inverse correlation of thermal lability and conversion efficiency for five prion protein polymorphic variants.

Transmissible spongiform encephalopathies (TSEs) are associated with the accumulation of deposits of an abnormal form, PrP(Sc), of the host-encoded prion protein, PrP(C). Amino acid substitutions in PrP(C) have long been known to affect TSE disease outcome. In extreme cases in humans, various mutations appear to cause disease. In animals, polymorphisms are associated with variations in disease susceptibility and, in sheep, several polymorphisms have been identified that are known to affect susceptibility of carriers to disease. The mechanisms of polymorphism-mediated modulation of disease susceptibility remain elusive, and we have been studying the effect of various amino acid substitutions at PrP codon 164 (mouse numbering), in the beta2-alpha2 loop region of the prion protein, to attempt to decipher how polymorphisms may affect disease susceptibility. Combined in vitro approaches suggest that there exists a correlation between the ability of protein variants to convert to abnormal isoforms in seeded conversion assays versus the thermal stability of the protein variants, as judged by both thermal denaturation and an unseeded in vitro oligomerization assay. We have performed molecular dynamics simulations to give an indication of backbone conformational changes as a result of amino acid changes and found that alteration of a single residue in PrP can result in local changes in structure that may affect global conformation and stability. Our results are consistent with modulation of disease susceptibility through differential protein stability leading to enhanced generic misfolding of TSE resistance-associated protein variants.

Effect of enzymatic deimination on the conformation of recombinant prion protein.

Deimination is the post-translational conversion of arginine residues to citrulline. It has been implicated as a causative factor in autoimmune diseases such as multiple sclerosis and rheumatoid arthritis and more recently, as a marker of neurodegeneration. We have investigated the effect of the post-translational modification of arginine residues on the structure of recombinant ovine prion protein. Deiminated prion protein exhibited biophysical properties characteristic of the scrapie-associated conformer of prion protein viz. an increased beta-sheet secondary structure, congophilic structures indicative of amyloid and proteinase K resistance which could be templated onto normal unmodified prion protein. In the light of these findings, a potential role of post-translational modifications to prion protein in disease initiation or propagation is discussed.

Staphylococcus aureus Lipase 1 Enhances Influenza A Virus Replication.

Influenza A virus (IAV) causes annual epidemics of respiratory disease in humans, often complicated by secondary coinfection with bacterial pathogens such as Staphylococcus aureus Here, we report that the S. aureus secreted protein lipase 1 enhances IAV replication in vitro in primary cells, including human lung fibroblasts. The proviral activity of lipase 1 is dependent on its enzymatic function, acts late in the viral life cycle, and results in increased infectivity through positive modulation of virus budding. Furthermore, the proviral effect of lipase 1 on IAV is exhibited during in vivo infection of embryonated hen's eggs and, importantly, increases the yield of a vaccine strain of IAV by approximately 5-fold. Thus, we have identified the first S. aureus protein to enhance IAV replication, suggesting a potential role in coinfection. Importantly, this activity may be harnessed to address global shortages of influenza vaccines.IMPORTANCE Influenza A virus (IAV) causes annual epidemics and sporadic pandemics of respiratory disease. Secondary bacterial coinfection by organisms such as Staphylococcus aureus is the most common complication of primary IAV infection and is associated with high levels of morbidity and mortality. Here, we report the first identified S. aureus factor (lipase 1) that enhances IAV replication during infection via positive modulation of virus budding. The effect is observed in vivo in embryonated hen's eggs and greatly enhances the yield of a vaccine strain, a finding that could be applied to address global shortages of influenza vaccines.

Investigation of relationship between vitamin D status and reproductive fitness in Scottish hill sheep.

There is a growing interest in the influence of vitamin D on ovine non-skeletal health. The aim of this study was to explore the relationship between pre-mating vitamin D status, as assessed by serum concentrations of 25-Hydroxyvitamin D [25(OH)D; comprising D2 and D3] and subsequent reproductive performance of genetically unimproved Scottish Blackface (UBF), genetically improved Scottish Blackface (IBF) and Lleyn ewes kept under Scottish hill conditions. 25-Hydroxyvitamin D2 (25(OH)D2) and 25-Hydroxyvitamin D3 (25(OH)D3) concentrations were determined in serum samples harvested in November from ewes grazed outdoors. There were no significant differences in 25(OH)D2concentrations amongst the 3 genotypes. Lleyn ewes had significantly higher 25(OH)D3 and 25(OH)D concentrations than both Scottish Blackface ewe genotypes, whereas these vitamin D parameters did not differ significantly between the UBF and IBF ewes. Concentrations of 25(OH)D3 and 25(OH)D were positively associated with subsequent birth weights of singleton and of twin lamb litters. No significant associations between vitamin D status and number of lambs born or weaned per ewe were found. This study demonstrates that concentrations of cutaneously-derived 25(OH)D3, but not of orally consumed 25(OH)D2, differed between breeds. The positive association between ewe vitamin D status and offspring birth weight highlights the need for further investigations.

Effects of post-translational modifications on prion protein aggregation and the propagation of scrapie-like characteristics in vitro.

Prion diseases, or transmissible spongiform encephalopathies (TSEs) are typically characterised by CNS accumulation of PrP(Sc), an aberrant conformer of a normal cellular protein PrP(C). It is thought PrP(Sc) is itself infectious and the causative agent of such diseases. To date, no chemical modifications of PrP(Sc), or a sub-population thereof, have been reported. In this study we have investigated whether chemical modification of amino acids within PrP might cause this protein to exhibit aberrant properties and whether these properties can be propagated onto unmodified prion protein. Of particular interest were post-translational modifications resulting from physiological conditions shown to be associated with TSE disease. Here we report that in vitro exposure of recombinant PrP to conditions that imitate the end effects of oxidative/nitrative stress in TSE-infected mouse brains cause the protein to adopt many of the physical characteristics of PrP(Sc). Most interestingly, these properties could be propagated onto unmodified PrP protein when the modified protein was used as a template. These data suggest that post-translational modifications of PrP might contribute to the initiation and/or propagation of prion protein-associated plaques in vivo during prion disease, thereby high-lighting novel biochemical pathways as possible therapeutic targets for these conditions.

Application of a novel in vitro selection technique to isolate and characterise high affinity DNA aptamers binding mammalian prion proteins.

Clinical diagnosis and research into transmissible spongiform encephalopathies are hampered by the lack of sufficiently sensitive and specific reagents able to adequately detect the normal cellular form of the prion protein, PrP(C), and the pathological isoform, PrP(Sc). In order to provide such reagents, we applied Systematic Evolution of Ligands by EXponential enrichment (SELEX) against a recombinant murine prion protein, to select single-stranded DNA ligands (aptamers) of high affinity. The SELEX protocol and subsequent aptamer characterisation employed protein immobilisation/partitioning using nickel-complexed magnetic particles and a novel SYBR Green-mediated quantitative real-time PCR technique. Following eight rounds of selection, the enriched aptamer pool was cloned and 24 clones sequenced. Seven of these were 'orphan' clones and the remainder were grouped into three separate T-rich families. All but four of the aptamer clones exhibited specific binding to the murine prion protein and the majority also bound to human and ovine prion proteins. Dissociation constants (K(d)) ranged from 18 to 79 nM. Flow cytometry with fluorescein-labelled aptamers confirmed that binding to cells was dependent on the expression of PrP(C). Preliminary studies also indicate that a trivalent aptamer pool is capable of binding the pathological isoform PrP(Sc) following guanidinium denaturation.

The cellular and pathologic prion protein.

The cellular prion protein, PrPC, is a small, cell surface glycoprotein with a function that is currently somewhat ill defined. It is also the key molecule involved in the family of neurodegenerative disorders called transmissible spongiform encephalopathies, which are also known as prion diseases. The misfolding of PrPC to a conformationally altered isoform, designated PrPTSE, is the main molecular process involved in pathogenesis and appears to precede many other pathologic and clinical manifestations of disease, including neuronal loss, astrogliosis, and cognitive loss. PrPTSE is also believed to be the major component of the infectious "prion," the agent responsible for disease transmission, and preparations of this protein can cause prion disease when inoculated into a naïve host. Thus, understanding the biochemical and biophysical properties of both PrPC and PrPTSE, and ultimately the mechanisms of their interconversion, is critical if we are to understand prion disease biology. Although entire books could be devoted to research pertaining to the protein, herein we briefly review the state of knowledge of prion biochemistry, including consideration of prion protein structure, function, misfolding, and dysfunction.

Sex-dependent changes in neuroactive steroid concentrations in the rat brain following acute swim stress.

Sex differences in hypothalamic-pituitary-adrenal (HPA) axis activity are well established in rodents. In addition to glucocorticoids, stress also stimulates the secretion of progesterone and deoxycorticosterone (DOC) from the adrenal gland. Neuroactive steroid metabolites of these precursors can modulate HPA axis function; however, it is not known whether levels of these steroids differ between male and females following stress. In the present study, we aimed to establish whether neuroactive steroid concentrations in the brain display sex- and/or region-specific differences under basal conditions and following exposure to acute stress. Brains were collected from male and female rats killed under nonstress conditions or following exposure to forced swimming. Liquid chromatography-mass spectrometry was used to quantify eight steroids: corticosterone, DOC, dihydrodeoxycorticosterone (DHDOC), pregnenolone, progesterone, dihydroprogesterone (DHP), allopregnanolone and testosterone in plasma, and in five brain regions (frontal cortex, hypothalamus, hippocampus, amygdala and brainstem). Corticosterone, DOC and progesterone concentrations were significantly greater in the plasma and brain of both sexes following stress; however, the responses in plasma were greater in females compared to males. This sex difference was also observed in the majority of brain regions for DOC and progesterone but not for corticosterone. Despite observing no stress-induced changes in circulating concentrations of pregnenolone, DHDOC or DHP, concentrations were significantly greater in the brain and this effect was more pronounced in females than males. Basal plasma and brain concentrations of allopregnanolone were significantly higher in females; moreover, stress had a greater impact on central allopregnanolone concentrations in females. Stress had no effect on circulating or brain concentrations of testosterone in males. These data indicate the existence of sex and regional differences in the generation of neuroactive steroids in the brain following acute stress, especially for the 5α-reduced steroids, and further suggest a sex-specific expression of steroidogenic enzymes in the brain. Thus, differential neurosteroidogenesis may contribute to sex differences in HPA axis responses to stress.

Effects of mutations in the effector domain of influenza A virus NS1 protein.

OBJECTIVE: The multifunctional NS1 protein of influenza A virus has roles in antagonising cellular innate immune responses and promoting viral gene expression. To better understand the interplay between these functions, we tested the effects of NS1 effector domain mutations known to affect homo-dimerisation or interactions with cellular PI3 kinase or Trim25 on NS1 ability to promote nuclear export of viral mRNAs. RESULTS: The NS1 dimerisation mutant W187R retained the functions of binding cellular NXF1 as well as stabilising NXF1 interaction with viral segment 7 mRNAs and promoting their nuclear export. Two PI3K-binding mutants, NS1 Y89F and Y89A still bound NXF1 but no longer promoted NXF1 interactions with segment 7 mRNA or its nuclear export. The Trim25-binding mutant NS1 E96A/E97A bound NXF1 and supported NXF1 interactions with segment 7 mRNA but no longer supported mRNA nuclear export. Analysis of WT and mutant NS1 interaction partners identified hsp70 as specifically binding to NS1 E96A/E97A. Whilst these data suggest the possibility of functional links between NS1's effects on intracellular signalling and its role in viral mRNA nuclear export, they also indicate potential pleiotropic effects of the NS1 mutations; in the case of E96A/E97A possibly via disrupted protein folding leading to chaperone recruitment.

Effects of anti-inflammatory drugs on the expression of tryptophan-metabolism genes by human macrophages.

Several lines of evidence link macrophage activation and inflammation with (monoaminergic) nervous systems in the etiology of depression. IFN treatment is associated with depressive symptoms, whereas anti-TNFα therapies elicit positive mood. This study describes the actions of 2 monoaminergic antidepressants (escitalopram, nortriptyline) and 3 anti-inflammatory drugs (indomethacin, prednisolone, and anti-TNFα antibody) on the response of human monocyte-derived macrophages (MDMs) from 6 individuals to LPS or IFN-α. Expression profiling revealed robust changes in the MDM transcriptome (3294 genes at P < 0.001) following LPS challenge, whereas a more limited subset of genes (499) responded to IFNα. Contrary to published reports, administered at nontoxic doses, neither monoaminergic antidepressant significantly modulated the transcriptional response to either inflammatory challenge. Each anti-inflammatory drug had a distinct impact on the expression of inflammatory cytokines and on the profile of inducible gene expression-notably on the regulation of enzymes involved in metabolism of tryptophan. Inter alia, the effect of anti-TNFα antibody confirmed a predicted autocrine stimulatory loop in human macrophages. The transcriptional changes were predictive of tryptophan availability and kynurenine synthesis, as analyzed by targeted metabolomic studies on cellular supernatants. We suggest that inflammatory processes in the brain or periphery could impact on depression by altering the availability of tryptophan for serotonin synthesis and/or by increasing production of neurotoxic kynurenine.

Lysine-Directed Post-translational Modifications of Tau Protein in Alzheimer's Disease and Related Tauopathies.

Tau is a microtubule-associated protein responsible mainly for stabilizing the neuronal microtubule network in the brain. Under normal conditions, tau is highly soluble and adopts an "unfolded" conformation. However, it undergoes conformational changes resulting in a less soluble form with weakened microtubule stabilizing properties. Altered tau forms characteristic pathogenic inclusions in Alzheimer's disease and related tauopathies. Although, tau hyperphosphorylation is widely considered to be the major trigger of tau malfunction, tau undergoes several post-translational modifications at lysine residues including acetylation, methylation, ubiquitylation, SUMOylation, and glycation. We are only beginning to define the site-specific impact of each type of lysine modification on tau biology as well as the possible interplay between them, but, like phosphorylation, these modifications are likely to play critical roles in tau's normal and pathobiology. This review summarizes the latest findings focusing on lysine post-translational modifications that occur at both endogenous tau protein and pathological tau forms in AD and other tauopathies. In addition, it highlights the significance of a site-dependent approach of studying tau post-translational modifications under normal and pathological conditions.