A characteristic feature of neoplastic transformation is a perpetual activation of oncogenic proteins. Here, we studied signal transducers and activators of transcription (STAT) in patients with mycosis fungoides (MF)/cutaneous T-cell lymphoma (CTCL). Malignant lymphocytes in dermal infiltrates of CTCL tumors showed frequent and intense nuclear staining with anti-PY-STAT3 antibody, indicating a constitutive activation of STAT3 in vivo in tumor stages. In contrast, only sporadic and faint staining was observed in indolent lesions of patch and plaque stages of MF. Moreover, neoplastic lymphocytes in the epidermal Pautrier abscesses associated with early stages of MF did not express activated STAT3. To address the role of STAT3 in survival/apoptosis, CTCL tumor cells from an advanced skin tumor were transfected with either wild-type STAT3 (STAT3wt) or dominant-negative STAT3 (STAT3D). Forced inducible expression of STAT3D triggered a significant increase in tumor cells undergoing apoptosis, whereas forced expression of STAT3wt or empty vector had no effect. In conclusion, a profound in vivo activation of STAT3 is observed in MF tumors but not in the early stages of MF. Moreover, STAT3 protects tumor cells from apoptosis in vitro. Taken together, these findings suggest that STAT3 is a malignancy factor in CTCL.

Interleukin-2 (IL-2

Beta(2)-microglobulin (beta(2)m) is the amyloidogenic protein in dialysis-related amyloidosis, but the mechanisms underlying beta(2)m fibrillogenesis in vivo are largely unknown. We study a structural variant of beta(2)m that has been linked to cancer and inflammation and may be present in the circulation of dialysis patients. This beta(2)m variant, DeltaK58-beta(2)m, is a disulfide-linked two-chain molecule consisting of amino acid residues 1-57 and 59-99 of intact beta(2)m, and we here demonstrate and characterize its decreased conformational stability as compared to wild-type (wt) beta(2)m. Using amide hydrogen/deuterium exchange monitored by mass spectrometry, we show that DeltaK58-beta(2)m has increased unfolding rates compared to wt-beta(2)m and that unfolding is highly temperature dependent. The unfolding rate is 1 order of magnitude faster in DeltaK58-beta(2)m than in wt-beta(2)m, and at 37 degrees C the half-time for unfolding is more than 170-fold faster than at 15 degrees C. Conformational changes are also reflected by a very prominent Congo red binding of DeltaK58-beta(2)m at 37 degrees C, by the evolution of thioflavin T fluorescence, and by changes in intrinsic fluorescence. After a few days at 37 degrees C, in contrast to wt-beta(2)m, DeltaK58-beta(2)m forms well-defined high molecular weight aggregates that are detected by size-exclusion chromatography. Atomic force microscopy after seeding with amyloid-beta(2)m fibrils under conditions that induce minimal fibrillation in wt-beta(2)m shows extensive amyloid fibrillation in DeltaK58-beta(2)m samples. The results highlight the instability and amyloidogenicity under near physiological conditions of a slightly modified beta(2)m variant generated by limited proteolysis and illustrate stages of amyloid formation from early conformational variants to overt fibrillation.

Many different assays for measuring peptide-MHC interactions have been suggested over the years. Yet, there is no generally accepted standard method available. We have recently generated preoxidized recombinant MHC class I molecules (MHC-I) which can be purified to homogeneity under denaturing conditions (i.e., in the absence of any contaminating peptides). Such denatured MHC-I molecules are functional equivalents of "empty molecules". When diluted into aqueous buffer containing beta-2 microglobulin (beta2m) and the appropriate peptide, they fold rapidly and efficiently in an entirely peptide dependent manner. Here, we exploit the availability of these molecules to generate a quantitative ELISA-based assay capable of measuring the affinity of the interaction between peptide and MHC-I. This assay is simple and sensitive, and one can easily envisage that the necessary reagents, standards and protocols could be made generally available to the scientific community.

No abstract

Aggregation and fibrillation of ␤ 2 -microglobulin are hallmarks of dialysis-related amyloidosis. We characterize perturbations of the native conformation of ␤ 2 -microglobulin that may precede fibril formation. For a ␤ 2 -microglobulin variant cleaved at lysine 58, we show using capillary electrophoresis that two conformers spontaneously exist in aqueous buffers at neutral pH. Upon treatment of wild-type ␤ 2 -microglobulin with acetonitrile or trifluoroethanol, two conformations were also observed. These conformations were in equilibrium dependent on the sample temperature and the percentage of organic solvent present. Circular dichroism showed a loss of ␤-structures and gain of ␣-helices. Reversal to the native conformation occurred when removing the organics. Affinity capillary electrophoresis experiments showed increased specific interactions of the nonnative ␤ 2 -microglobulin conformation with the dyes 8-anilino-1-naphthalene sulfonic acid and Congo red. The observations may relate to early folding events prior to amyloid fibrillation and facilitate the development of methods to detect and inhibit pro-amyloid protein and peptide conformations.Abnormal protein folding leads to amyloid deposition in a number of different chronic disorders including Alzheimer's disease, senile systemic amyloidosis, transmissible spongiform encephalopathies, and dialysis-related amyloidosis (1). Different proteins and polypeptides can acquire the abnormal conformational changes that lead to amyloid formation, and at least 19 different proteins are now known to be involved in the generation of amyloid in vivo (1). In the case of dialysis-related amyloidosis the protein involved is ␤ 2 -microglobulin (␤ 2 m), 1 which aggregates as amyloid fibrils in joints and tissues in ϳ20% of patients with kidney failure within 2 years after the onset of hemodialysis treatment (2). This type of amyloidosis does not seem to require a mutated, processed, or chemically modified precursor protein. Although the cause of the abnormal folding in vivo is unknown, it has been reported recently that ␤ 2 m can be induced to generate amyloid fibrils in vitro at low pH and high salt conditions (3) and that fibrillar ␤ 2 m can be refolded back in vitro (4).Similar pre-amyloid folding states have now been demonstrated for a number of amyloidosis-related proteins (5-9) and also in other proteins and peptides without known amyloid disease associations (10 -13). ␤ 2 m is an attractive protein for studying amyloid-promoting folding events because it is small (M r ϭ 11,729), nonglycosylated, nonpolymorphic, and has a simple native structure (14), i.e. it is a one-chain all ␤-protein with two antiparallel pleated sheets of ␤-strands linked together by a disulfide bridge (15). It is structurally homologous to constant domains of immunoglobulins (14). In chronic renal failure ␤ 2 m serum concentrations may increase 10 -70 times despite dialysis, but there is no simple relationship between ␤ 2 m serum concentrations and the extent of amyloidosis (16 The Lys 58 -␤ 2 ...

The function of major histocompatibility complex (MHC) class I molecules is to sample peptides derived from intracellular proteins and to present these peptides to CD8+ cytotoxic T lymphocytes. In this paper, biochemical assays addressing MHC class I binding of both peptide and beta 2-microglobulin (beta 2m) have been used to examine the assembly of the trimolecular MHC class I/beta 2m/peptide complex. Recombinant human beta 2m and mouse beta 2ma have been generated to compare the binding of the two beta 2m to mouse class I. It is frequently assumed that human beta 2m binds to mouse class I heavy chain with a much higher affinity than mouse beta 2m itself. We find that human beta 2m only binds to mouse class I heavy chain with slightly (about 3-fold) higher affinity than mouse beta 2m. In addition, we compared the effect of the two beta 2m upon peptide binding to mouse class I. The ability of human beta 2m to support peptide binding correlated well with its ability to saturate mouse class I heavy chains. Surprisingly, mouse beta 2m only facilitated peptide binding when mouse beta 2m was used in excess (about 20-fold) of what was needed to saturate the class I heavy chains. The inefficiency of mouse beta 2m to support peptide binding could not be attributed to a reduced affinity of mouse beta 2m/MHC class I complexes for peptides or to a reduction in the fraction of mouse beta 2m/MHC class I molecules participating in peptide binding. We have previously shown that only a minor fraction of class I molecules are involved in peptide binding, whereas most of class I molecules are involved in beta 2m binding. We propose that mouse beta 2m interacts with the minor peptide binding (i.e. the "empty") fraction with a lower affinity than human beta 2m does, whereas mouse and human beta 2m interact with the major peptide-occupied fraction with almost similar affinities. This would explain why mouse beta 2m is less efficient than human beta 2m in generating the peptide binding moiety, and identifies the empty MHC class I heavy chain as the molecule that binds human beta 2m preferentially.

RPE cells responded to exposure to T-cell-derived cytokines by upregulating expression of multiple chemokines related to microglial, T-cell, and monocyte chemotaxis and activation. This inflammatory stress response may have implications for immune homeostasis in the retina, and for the further understanding of inflammatory ocular diseases such as uveitis and AMD.