Proteasome-Generated cis-Spliced Peptides and Their Potential Role in CD8+ T Cell Tolerance

The human immune system relies on the capability of CD8+ T cells to patrol body cells, spot infected cells and eliminate them. This cytotoxic response is supposed to be limited to infected cells to avoid killing of healthy cells. To enable this, CD8+ T cells have T Cell Receptors (TCRs) which should discriminate between self and non-self through the recognition of antigenic peptides bound to Human Leukocyte Antigen class I (HLA-I) complexes—i.e., HLA-I immunopeptidomes—of patrolled cells. The majority of these antigenic peptides are produced by proteasomes through either peptide hydrolysis or peptide splicing. Proteasome-generated cis-spliced peptides derive from a given antigen, are immunogenic and frequently presented by HLA-I complexes. Theoretically, they also have a very large sequence variability, which might impinge upon our model of self/non-self discrimination and central and peripheral CD8+ T cell tolerance. Indeed, a large variety of cis-spliced epitopes might enlarge the pool of viral-human zwitter epitopes, i.e., peptides that may be generated with the exact same sequence from both self (human) and non-self (viral) antigens. Antigenic viral-human zwitter peptides may be recognized by CD8+ thymocytes and T cells, induce clonal deletion or other tolerance processes, thereby restraining CD8+ T cell response against viruses. To test this hypothesis, we computed in silico the theoretical frequency of zwitter non-spliced and cis-spliced epitope candidates derived from human proteome (self) and from the proteomes of a large pool of viruses (non-self). We considered their binding affinity to the representative HLA-A*02:01 complex, self-antigen expression in Medullary Thymic Epithelial cells (mTECs) and the relative frequency of non-spliced and cis-spliced peptides in HLA-I immunopeptidomes. Based on the present knowledge of proteasome-catalyzed peptide splicing and neglecting CD8+ TCR degeneracy, our study suggests that, despite their frequency, the portion of the cis-spliced peptides we investigated could only marginally impinge upon the variety of functional CD8+ cytotoxic T cells (CTLs) involved in anti-viral response.

Fine-Tuning of Sequence Specificity by Near Attack Conformations in Enzyme-Catalyzed Peptide Hydrolysis

The catalytic role of near attack conformations (NACs), molecular states that lie on the pathway between the ground state (GS) and transition state (TS) of a chemical reaction, is not understood completely. Using a computational approach that combines Bürgi–Dunitz theory with all-atom molecular dynamics simulations, the role of NACs in catalyzing the first stages of HIV-1 protease peptide hydrolysis was previously investigated using a substrate that represents the recognized SP1-NC cleavage site of the HIV-1 Gag polyprotein. NACs were found to confer no catalytic effect over the uncatalyzed reaction there ( Δ Δ G N ‡ ∼ 0 kcal/mol). Here, using the same approach, the role of NACs across multiple substrates that each represent a further recognized cleavage site is investigated. Overall rate enhancement varies by | Δ Δ G ‡ | ∼ 12–15 kcal/mol across this set, and although NACs contribute a small and approximately constant barrier to the uncatalyzed reaction (< Δ G N ‡ u > = 4.3 ± 0.3 kcal/mol), they are found to contribute little significant catalytic effect ( | Δ Δ G N ‡ | ∼ 0–2 kcal/mol). Furthermore, no correlation is exhibited between NAC contributions and the overall energy barrier ( R 2 = 0.01). However, these small differences in catalyzed NAC contributions enable rates to match those required for the kinetic order of processing. Therefore, NACs may offer an alternative and subtle mode compared to non-NAC contributions for fine-tuning reaction rates during complex evolutionary sequence selection processes—in this case across cleavable polyproteins whose constituents exhibit multiple functions during the virus life-cycle.

Increased temperatures alter viable microbial biomass, ammonia oxidizing bacteria and extracellular enzymatic activities in Antarctic soils

ABSTRACT The effects of temperature on microorganisms in high latitude regions, and their possible feedbacks in response to change, are unclear. Here, we assess microbial functionality and composition in response to a substantial temperature change. Total soil biomass, amoA gene sequencing, extracellular activity assays and soil physicochemistry were measured to assess a warming scenario. Soil warming to 15°C for 30 days triggered a significant decrease in microbial biomass compared to baseline soils (0°C; P &lt; 0.05) after incubations had induced an initial increase. These changes coincided with increases in extracellular enzymatic activity for peptide hydrolysis and phenolic oxidation at higher temperatures, but not for the degradation of carbon substrates. Shifts in ammonia-oxidising bacteria (AOB) community composition related most significantly to changes in soil carbon content (P &lt; 0.05), which gradually increased in microcosms exposed to a persistently elevated temperature relative to baseline incubations, while temperature did not influence AOBs. The concentration of soil ammonium (NH4+) decreased significantly at higher temperatures subsequent to an initial increase, possibly due to higher conversion rates of NH4+ to nitrate by nitrifying bacteria. We show that higher soil temperatures may reduce viable microbial biomass in cold environments but stimulate their activity over a short period.

Proteins containing ubiquitin-like (Ubl) domains not only bind to 26S proteasomes but also induce their activation

During protein degradation by the ubiquitin–proteasome pathway, latent 26S proteasomes in the cytosol must assume an active form. Proteasomes are activated when ubiquitylated substrates bind to them and interact with the proteasome-bound deubiquitylase Usp14/Ubp6. The resulting increase in the proteasome’s degradative activity was recently shown to be mediated by Usp14’s ubiquitin-like (Ubl) domain, which, by itself, can trigger proteasome activation. Many other proteins with diverse cellular functions also contain Ubl domains and can associate with 26S proteasomes. We therefore tested if various Ubl-containing proteins that have important roles in protein homeostasis or disease also activate 26S proteasomes. All seven Ubl-containing proteins tested—the shuttling factors Rad23A, Rad23B, and Ddi2; the deubiquitylase Usp7, the ubiquitin ligase Parkin, the cochaperone Bag6, and the protein phosphatase UBLCP1—stimulated peptide hydrolysis two- to fivefold. Rather than enhancing already active proteasomes, Rad23B and its Ubl domain activated previously latent 26S particles. Also, Ubl-containing proteins (if present with an unfolded protein) increased proteasomal adenosine 5′-triphosphate (ATP) hydrolysis, the step which commits substrates to degradation. Surprisingly, some of these proteins also could stimulate peptide hydrolysis even when their Ubl domains were deleted. However, their Ubl domains were required for the increased ATPase activity. Thus, upon binding to proteasomes, Ubl-containing proteins not only deliver substrates (e.g., the shuttling factors) or provide additional enzymatic activities (e.g., Parkin) to proteasomes, but also increase their capacity for proteolysis.

A Probiotic Preparation Hydrolyzes Gliadin and Protects Intestinal Cells from the Toxicity of Pro-Inflammatory Peptides

Celiac disease (CD) is an autoimmune enteropathy caused by an intolerance to gluten proteins. It has been hypothesized that probiotic bacteria may exert beneficial effects by modulating inflammatory processes and by sustaining peptide hydrolysis at the intestinal level. This study aims at evaluating the capacity of a probiotic mixture (two different strains of lactobacilli and three of bifidobacteria) to hydrolyze gluten peptides following simulated gastrointestinal digestion of gliadin (PT-gliadin). The capacity of bacterial hydrolysates to counteract the toxic effects of gliadin-derived peptides in Caco-2 cells was also assessed. The protein and peptide mixtures, untreated or proteolyzed with the probiotic preparation, were analyzed before and after each proteolytic step with different techniques (SDS-PAGE, reverse phase HPLC, filtration on different molecular cut-off membranes). These experiments demonstrated that PT-gliadin can be further digested by bacteria into lower molecular weight peptides. PT-gliadin, untreated or digested with the probiotics, was then used to evaluate oxidative stress, IL-6 cytokine production and expression of tight junctions’ proteins—such as occludin and zonulin—in Caco-2 cells. PT-gliadin induced IL-6 production and modulation and redistribution of zonulin and occludin, while digestion with the probiotic strains reversed these effects. Our data indicate that this probiotic mixture may exert a protective role in CD.

A novel binuclear Pd(ii) complex displaying synergic peptide cleavage behaviour

A novel binuclear Pd(ii) complex promotes His- and Met-orientated peptide hydrolysis in an internuclear synergic manner but not Cys-orientated hydrolysis.

Purification and Identification of Antioxidant Peptides from Hydrolysates of Large Yellow Croaker (Pseudosciaena crocea) Scales

HighlightsThree antioxidant peptides were efficiently obtained from large yellow croaker scales.The molecular weights of the three peptides were 879.4, 952.5, and 1070.4 Da, respectively.The large yellow croaker scales’ hydrolysates are a good source of natural antioxidants.Abstract. The scales of large yellow croaker () are typically discarded as waste during processing. In this study, three peptides with high antioxidant properties were purified from the scales via ultrafiltration and column chromatography. Through matrix-assisted laser desorption ionization/time-of-flight mass spectrometry, the sequences of the three peptides were identified as Gln-Arg-Pro-Pro-Glu-Pro-Arg (M1N4-1, 879.4 Da), Glu-Lys-Val-Trp-Lys-Tyr-Cys-Asp (M1N4-2, 1070.4 Da), and Val-Gly-Leu-Pro-Gly-Leu-Ser-Gly-Pro-Val-Gly (M2N2-1, 952.5 Da). The antioxidant activity of the peptides was directly determined through 1-diphenyl-2-picrylhydrazyl scavenging assay. Results suggested that large yellow croaker scales are a good source of natural antioxidants. Keywords: Antioxidant peptide, Hydrolysis, Pseudosciaena crocea, Scale.

Combined co-solvent and pressure effect on kinetics of a peptide hydrolysis: an activity-based approach

An activity-based approach to predict combined influence of pressure and co-solvent on enzymatic reaction kinetics is presented.

UBL domain of Usp14 and other proteins stimulates proteasome activities and protein degradation in cells

The best-known function of ubiquitin-like (UBL) domains in proteins is to enable their binding to 26S proteasomes. The proteasome-associated deubiquitinating enzyme Usp14/UBP6 contains an N-terminal UBL domain and is an important regulator of proteasomal activity. Usp14 by itself represses multiple proteasomal activities but, upon binding a ubiquitin chain, Usp14 stimulates these activities and promotes ubiquitin-conjugate degradation. Here, we demonstrate that Usp14’s UBL domain alone mimics this activation of proteasomes by ubiquitin chains. Addition of this UBL domain to purified 26S proteasomes stimulated the same activities inhibited by Usp14: peptide entry and hydrolysis, protein-dependent ATP hydrolysis, deubiquitination by Rpn11, and the degradation of ubiquitinated and nonubiquitinated proteins. Thus, the binding of Usp14’s UBL (apparently to Rpn1’s T2 site) seems to mediate the activation of proteasomes by ubiquitinated substrates. However, the stimulation of these various activities was greater in proteasomes lacking Usp14 than in wild-type particles and thus is a general response that does not involve some displacement of Usp14. Furthermore, the UBL domains from hHR23 and hPLIC1/UBQLN1 also stimulated peptide hydrolysis, and the expression of hHR23A’s UBL domain in HeLa cells stimulated overall protein degradation. Therefore, many UBL-containing proteins that bind to proteasomes may also enhance allosterically its proteolytic activity.