Martin Deeg

Contribution of Proteasomal β-Subunits to the Cleavage of Peptide Substrates Analyzed with Yeast Mutants

Proteasomes generate peptides that can be presented by major histocompatibility complex (MHC) class I molecules in vertebrate cells. Using yeast 20 S proteasomes carrying different inactivated β-subunits, we investigated the specificities and contributions of the different β-subunits to the degradation of polypeptide substrates containing MHC class I ligands and addressed the question of additional proteolytically active sites apart from the active β-subunits. We found a clear correlation between the contribution of the different subunits to the cleavage of fluorogenic and long peptide substrates, with β5/Pre2 cleaving after hydrophobic, β2/Pup1 after basic, and β1/Pre3 after acidic residues, but with the exception that β2/Pup1 and β1/Pre3 can also cleave after some hydrophobic residues. All proteolytic activities including the “branched chain amino acid-preferring” component are associated with β5/Pre2, β1/Pre3, or β2/Pup1, arguing against additional proteolytic sites. Because of the high homology between yeast and mammalian 20 S proteasomes in sequence and subunit topology and the conservation of cleavage specificity between mammalian and yeast proteasomes, our results can be expected to also describe most of the proteolytic activity of mammalian 20 S proteasomes leading to the generation of MHC class I ligands.

Serum protein profiling by SELDI mass spectrometry: detection of multiple variants of serum amyloid alpha in renal cancer patients

The molecular analysis of serum is an important field for the definition of potential diagnostic markers or disease-related protein alterations. Novel proteomic technologies such as the mass spectrometric-based surface-enhanced laser desorption/ionization (SELDI) ProteinChip® technique facilitate a rapid and reproducible analysis of such protein mixtures and affords the researcher a new dimension in the search for biomarkers of disease. Here, we have applied this technology to the study of a cohort of serum samples from well-characterized renal cell carcinoma patients for the identification of such proteins by comparison to healthy controls. We detected and characterized haptoglobin 1 α and serum amyloid α-1 (SAA-1) as disease related, in addition to an as-yet-unidentified marker of 10.84 kDa. Of particular note is the detection of multiple variants of SAA-1 in multiplex that have not been described in the sera of cancer patients. SAA-1 is detected as full-length protein, des-Arginine and des-Arginine/des-Serine variants at the N terminus by SELDI. In addition, we could also detect a low-abundant variant minus the first five N-terminal amino acids. Such variants may impact the function of the protein. We conclude the technique to be a reproducible, fast and simple mode for the discovery and analysis of marker proteins of disease in serum.

Human α-Defensins HNPs-1, -2, and -3 in Renal Cell Carcinoma : Influences on Tumor Cell Proliferation

The α-defensins human neutrophil peptides (HNPs)-1, -2, and -3 have been described as cytotoxic peptides with restricted expression in neutrophils and in some lymphocytes. In this study we report that HNPs-1, -2, and -3 are also expressed in renal cell carcinomas (RCCs). Several RCC lines were found to express mRNA as well as the specific peptides of HNP-1, -2, and -3 demonstrated by reverse transcriptase-polymerase chain reaction, mass spectrometric, and flow cytometric analyses. At physiological concentrations HNPs-1, -2, and -3 stimulated cell proliferation of selected RCC lines in vitro but at high concentrations were cytotoxic for all RCC lines tested. As in RCC lines, α-defensins were also detected in vivo in malignant epithelial cells of 31 RCC tissues in addition to their expected presence in neutrophils. In most RCC cases randomly, patchy immunostaining of α-defensins on epithelial cells surrounding neutrophils was seen, but in six tumors of higher grade malignancy all tumor cells were diffusely stained. Cellular necrosis observed in RCC tissues in association with extensive patches of HNP-1, -2, and -3, seemed to be related to high concentrations of α-defensins. The in vitro and in vivo findings suggest that α-defensins are frequent peptide constituents of malignant epithelial cells in RCC with a possible direct influence on tumor proliferation.

Cathepsin S and an asparagine-specific endoprotease dominate the proteolytic processing of human myelin basic protein in vitro

The biochemical characterization of antigen degradation is an important basis for a better understanding of both the immune response and autoimmune diseases mediated by MHC class II molecules.In this study we used high‐performance liquid chromatography and mass spectrometry to analyze the processing of myelin basic protein (MBP), a potential autoantigen implicated in the pathogenesis ofmultiple sclerosis. We resolved the kinetics of MBP processing by lysosomal extracts or purified endocytic proteases, identified the major cleavage sites during this process and assigned them to the activity of proteolytic enzymes. Proteolytic processing of MBP is mostly guided along the hydrophobic regions of the protein. It is initiated by two proteolytic steps (after N92 and S110) that are performed by an asparagine‐specific endopeptidase (AEP) and by cathepsin (Cat) S, respectively. The resulting processing intermediates are converted into more than 60 different species of 20–40‐mers due to the activity of endopeptidases including CatS, D and L. The fragments thus generated are subsequently degraded by C‐ or N‐terminal trimming. Strikingly, the initial cleavages during MBP processing affect two immunodominant regions of the potential autoantigen [MBP(85–99) and MBP(111‐129)] in an inverse manner. CatS directly generates the N terminus of the epitope MBP(111–129)in large quantities during the initial phase of processing, which might explain the immunogenicity of this region in spite of its relatively poor binding to HLA‐DR4. In contrast, the dominant cleavage by AEP mediates the destruction of MBP(85–99) unless the epitope is protected, e.g. by binding to HLA‐DR. Our results thus characterize the proteolytic events during processing of MBP on a molecular level and suggest a biochemical basis for the immunogenicity of the immunodominant epitopes, which could serve as a guideline for future therapeutic strategies.

Generation of the vesicular stomatitis virus nucleoprotein cytotoxic T lymphocyte epitope requires proteasome-dependent and -independent proteolytic activities.

The proteasome is involved in the generation of most of the MHC class I antigenic epitopes. However, it is not known if the proteasome generates the exact cytotoxic T lymphocyte (CTL) epitope or only epitope precursors which require further modification by additional proteases. Digestion of the extended vesicular stomatitis virus nucleoprotein epitope 52 – 59 (RGYVYQGL) by the 20S proteasome in vitro shows that the proteasome is capable of generating the correct C terminus but not the exact N terminus of the CTL epitope. This finding suggests that proteolytic activity in addition to the proteasome is required for generation of the CTL epitope. By using the proteasome inhibitor lactacystin we were able to confirm this finding in vivo. Lactacystin prevented the processing of N‐ and C‐terminally extended epitopes, whereas the processing of only N‐terminally extended epitopes was unaffected. Thus, the proteasome is necessary and sufficient for the generation of the exact C terminus of this CTL epitope, whereas the exact N terminus seems to be generated by a different protease.

Naturally Processed Dermcidin-Derived Peptides Do Not Permeabilize Bacterial Membranes and Kill Microorganisms Irrespective of Their Charge

ABSTRACT Dermcidin (DCD) is a recently described antimicrobial peptide, which is constitutively expressed in eccrine sweat glands and transported via sweat to the epidermal surface. By postsecretory proteolytic processing in sweat the dermcidin protein gives rise to several truncated DCD peptides which differ in length and net charge. In order to understand the mechanism of antimicrobial activity, we analyzed the spectrum of activity of several naturally processed dermcidin-derived peptides, the secondary structure in different solvents, and the ability of these peptides to interact with or permeabilize the bacterial membrane. Interestingly, although all naturally processed DCD peptides can adopt an α-helical conformation in solvents, they have a diverse and partially overlapping spectrum of activity against gram-positive and gram-negative bacteria. This indicates that the net charge and the secondary structure of the peptides are not important for the toxic activity. Furthermore, using carboxyfluorescein-loaded liposomes, membrane permeability studies and electron microscopy we investigated whether DCD peptides are able to permeabilize bacterial membranes. The data convincingly show that irrespective of charge the different DCD peptides are not able to permeabilize bacterial membranes. However, bacterial mutants lacking specific cell envelope modifications exhibited different susceptibilities to killing by DCD peptides than wild-type bacterial strains. Finally, immunoelectron microscopy studies indicated that DCD peptides are able to bind to the bacterial surface; however, signs of membrane perturbation were not observed. These studies indicate that DCD peptides do not exert their activity by permeabilizing bacterial membranes.

Pro- and anti-inflammatory cytokine production by autoimmune T cells against preproinsulin in HLA-DRB1*04, DQ8 Type 1 diabetes

Aims/hypothesisPreproinsulin is a target T cell autoantigen in human Type 1 diabetes. This study analyses the phenotype and epitope recognition of preproinsulin reactive T cells in subjects with a high genetic risk of diabetes [HLA-DRB1*04, DQ8 with Ab+ (autoantibody-positive) or without islet autoantibodies (control subjects)], and in HLA-matched diabetic patients.MethodsA preproinsulin peptide library approach was used to screen for cytokine profiles and epitope specificities in human peripheral blood lymphocytes, and CD4+CD45RA− and CD4+CD45RA+ T cell subfractions, representing memory and naive and recently primed T cells respectively.ResultsIn CD4+ T cell subsets we identified immunodominant epitopes and cytokine production patterns that differed profoundly between patients, Ab+ subjects and non-diabetic HLA-matched control subjects. In Ab+ subjects, a C-peptide epitope C13–29 and insulin B-chain epitope B11–27 were preferentially recognised, whereas insulin-treated Type 1 diabetic patients reacted to native insulin and B-chain epitope B1–16. In peripheral blood lymphocytes of Ab+ subjects, an increase in T helper (Th) 1 (IFNγ, IL-2) and Th2 (IL-4) cytokines was detectable, wheras in CD45RA+ and CD45RA− subsets, IL-4 and IL-10 phenotypes dominated, compatible with the contribution of non-CD4 cells to IFNγ content. In insulin-treated Type 1 diabetic patients, naive and recently primed CD4+ cells were characterised by increasd IFNγ, TNFα, and IL-5.Conclusions/interpretationOur data show that T cell reactivity to preproinsulin in CD45RA subsets is Th2-dominant in Ab+ subjects, challenging the Th1 paradigm in Type 1 diabetes. Characteristic immunodominant epitopes and cytokine patterns distinguish diabetic patients and Ab+ subjects from HLA-matched healthy individuals. This could prove useful in monitoring of T-cell immunity in clinical diabetes intervention trials.

Detection of dermcidin-derived peptides in sweat by ProteinChip® Technology

Recently, a novel antimicrobial peptide DCD-1, derived from the Dermcidin (DCD) gene and secreted by sweat glands, has been described by Schittek et al. [Nat. Immunol. 2 (2001) 1133.]. Here we describe the application of the surface-enhanced laser desorption/ionisation (SELDI) technology for the detection of DCD-1 and other dermcidin-derived peptides directly from microlitre amounts of human sweat. The advantages of the technique are as follows: (a) it can be carried out with ease and rapidity; (b) multiple samples can be processed simultaneously; (c) prior purification is not required; and (d) only a limited sample volume is necessary for both protein profiling and semiquantitation. Profiling of human sweat from various donors revealed that in addition to DCD-1, other DCD-derived peptide species were also present in significant quantities. Four of five identified peptides were DCD-1 related, while the fifth corresponded to a portion of the DCD protein outside the DCD-1 core. This provides clues as to how the novel protein is processed to its active form, though further work remains to elucidate this fully. Thus, we have demonstrated the applicability of such technology to the detection of DCD-1 and for the protein profiling of sweat in general. Such studies could reveal valuable new biomarkers for diagnosis and treatment of skin and sweat gland disorders.

Dermcidin-Derived Peptides Show a Different Mode of Action than the Cathelicidin LL-37 against Staphylococcus aureus

ABSTRACT Dermcidin (DCD) is an antimicrobial peptide which is constitutively expressed in eccrine sweat glands. By postsecretory proteolytic processing in sweat, the DCD protein gives rise to anionic and cationic DCD peptides with a broad spectrum of antimicrobial activity. Many antimicrobial peptides induce membrane permeabilization as part of their killing mechanism, which is accompanied by a loss of the bacterial membrane potential. In this study we show that there is a time-dependent bactericidal activity of anionic and cationic DCD-derived peptides which is followed by bacterial membrane depolarization. However, DCD-derived peptides do not induce pore formation in the membranes of gram-negative and gram-positive bacteria. This is in contrast to the mode of action of the cathelicidin LL-37. Interestingly, LL-37 as well as DCD-derived peptides inhibit bacterial macromolecular synthesis, especially RNA and protein synthesis, without binding to microbial DNA or RNA. Binding studies with components of the cell envelope of gram-positive and gram-negative bacteria and with model membranes indicated that DCD-derived peptides bind to the bacterial envelope but show only a weak binding to lipopolysaccharide (LPS) from gram-negative bacteria or to peptidoglycan, lipoteichoic acid, and wall teichoic acid, isolated from Staphylococcus aureus. In contrast, LL-37 binds strongly in a dose-dependent fashion to these components. Altogether, these data indicate that the mode of action of DCD-derived peptides is different from that of the cathelicidin LL-37 and that components of the bacterial cell envelope play a role in the antimicrobial activity of DCD.

Identification of an in vitro insulin receptor substrate-1 phosphorylation site by negative-ion μLC/ES-API-CID-MS hybrid scan technique

Recently, we reported a fast on-line alkaline micro-liquid chromatography/electrospray-atmospheric pressure ionization/collision-induced dissociation/mass spectrometric approach for sensitive phosphopeptide screening of a tryptic digested protein and subsequent characterization of the identified phosphopeptide. Based on this study, we now applied an improved method for the identification of phosphorylation sites in insulin receptor substrate 1, an important mediator in insulin signal transduction which was phosphorylated in vitro by protein kinase C-ζ. The approach consists of an on-line alkaline negative-ion micro-liquid chromatography/electrospray-atmospheric pressure ionization/collision-induced dissociation/mass spectrometric hybrid scan experiment using a triple-quadrupole mass spectrometer with fractionation and subsequent off-line nanoES-MS (ion trap) analysis of the phosphopeptide-containing fractions. During the liquid chromatography (LC)/ES-MS experiment, the phosphopeptides of the enzymatic digest mixture of the studied insulin receptor substrate 1 fragment were detected under high skimmer potential (API-CID) using phosphorylation-specific m/z 79 marker ions as well as the intact m/z-values of the peptides which were recorded under low skimmer potential. Subsequently, the targeted fractions were analyzed by off-line nanoES-MS/MS and MS3. Using this approach, serine 318 was clearly identified as a major in vitro protein kinase C-ζ phosphorylation site in the insulin receptor substrate −1 fragment. Together, our results indicate that the applied strategy is useful for unequivocal and fast analysis of phosphorylation sites in low abundant signaling proteins.