To obtain information on the role of proteasomes in the immune system, we examined the effect of a major immunomodulatory cytokine, gamma interferon (IFN-gamma), on the expressions, structures, and functions of proteasomes. IFN-gamma greatly increased the levels of the mRNAs encoding LMP2 and LMP7, putative immuno-proteasome subunits encoded by genes within the class II MHC region, and these two subunits synthesized were assembled completely into the proteasomal multi-subunit complex in various types of human cells. The subunit organization of proteasome changed in response to IFN-gamma stimulation, due to assembly of newly synthesized subunits through up- and down-expressions of at least 6 proteasome genes including LMP2/LMP7 without change in the structure of pre-existing proteasomes. Interestingly, IFN-gamma dramatically stimulated the trypsin-like and chymotrypsin-like activities of the multifunctional proteasome and depressed the peptidylglutamyl-peptide-hydrolyzing activity, without affecting the activity for ATP-, ubiquitin-dependent proteolysis. These results indicate that IFN-gamma modifies not only the structural organization of the proteasome, but also its functions. Based on these findings, we discuss the role in the antigen processing/presentation pathway of proteasomes with functional diversity acquired through alteration of their subunit assembly in response to IFN-gamma stimulation.

Proteasomes are the proteolytic complex responsible for major histocompatibility complex (MHC) class I-restricted antigen presentation. Interferon gamma treatment increases expression MHC-encoded LMP2 and LMP7 subunits of the proteasome and decreases expression of two proteasome subunits, named X and Y, which alters the proteolytic specificity of proteasomes. Molecular cloning of complementary DNAs encoding X and Y showed that their proteins are proteasomal subunits with high amino acid similarity to LMP7 and LMP2, respectively. Thus, interferon gamma may induce subunit replacements of X and Y by LMP7 and LMP2, respectively, producing proteasomes perhaps more appropriate for the immunological processing of endogenous antigens.

Proteasomes catalyze the non-lysosomal, An-dependent selective breakdown of ubiquitinated proteins and are thought to be responsible for MHC class I-restricted antigen presentation. Recently, we reported that gamma interferon (IFN-y) induced not only marked synthesis of the MHC-encoded proteasome subunits LMP2 and LMP7, but also ahnost complete loss of two unidentified proteasome subunits tentatively designated as X and Y in various human cells. Here, we show that subunit X is a new proteasomai subunit highly homologous to LMW, and that subunit Y is identical to the LM~-related proteasomal subunit delta. Thus, IFN-y appears to induce subunit replacements of X and Y by LMP7 and LMPZ, respectively, producing 'Juno-prot~omes' with the functional diversity responsible for processing of endogenous antigens.

The primary structures of two proteins that comprise PA28, an activator of the 20S proteasome, have been determined by cDNA cloning and sequencing. These protein subunits, termed PA28a and PA28JB, are about 50% identical to one another and are highly conserved between rat and human. PA28a and PA28~ are homologous to a previously described protein, Ki antigen, whose function is unknown. PA28c~, but neither PA28~ nor Ki antigen, contains a 'KEKE motif', which has been postulated to promote the binding of proteins having this structural feature. PA28a and PA2818 were coordinately regulated by y-interferon, which greatly induced mRNA levels of both proteins in cultured cells. The mRNA level of the Ki antigen also increased in response to "y-interferon treatment, but the magnitude of the increase was less than that for the PA28s, and the effect was transient. These results demonstrate the existence of a new protein family, at least two of whose members are involved in proteasome activation. They also provide the basis for future structure/function studies of PA28 subunits and the determination of their relative physiological roles in the regulation of proteasome activity.

To examine whether renal cell carcinoma displays altered CD44 expression we performed reverse transcription‐polymerase chain reaction (RT‐PCR) analysis of CD44 in 38 specimens from renal cancer, normal kidney and metastases of 19 patients and 6 renal cancer cell lines. To detect the CD44 variants, we utilized the RT‐PCR Southern blot method. One out of 19 (5.3%) renal cancer specimens expressed a larger molecular weight band than 1 kb by RT‐PCR analysis, in contrast to previous findings in colon and breast cancer. The band patterns in RT‐PCR were different in 14/17 (82.4%) cases between normal kidney and tumors, and a band of about 700 bp was especially marked in 12/17 (70.6%) tumor specimens and 4/6 (66.7%) cell lines. By cloning and sequencing of the 700 bp band, we found that this variant is identical to the CD44 variant sharing only exon v10. Examination by Northern blot analysis has revealed that all tumors express a higher level of CD44 mRNA than paired normal kidneys. These findings suggested that the CD44 variants sharing exon v10 play some role in renal cancer.