Within the European Immunogenicity Platform (EIP) (http://www.e-i-p.eu), the Protein Characterization Subcommittee (EIP-PCS) has been established to discuss and exchange experience of protein characterization in relation to unwanted immunogenicity. In this commentary, we, as representatives of EIP-PCS, review the current state of methods for analysis of protein aggregates. Moreover, we elaborate on why these methods should be used during product development and make recommendations to the biotech community with regard to strategies for their application during the development of protein therapeutics.

␣B-Crystallin is a small heat-shock protein in which three serine residues (positions 19, 45, and 59) can be phosphorylated under various conditions. We describe here the interaction of ␣B-crystallin with FBX4, an Fbox-containing protein that is a component of the ubiquitin-protein isopeptide ligase SCF (SKP1/CUL1/F-box). The interaction with FBX4 was enhanced by mimicking phosphorylation of ␣B-crystallin at both Ser-19 and Ser-45 (S19D/S45D), but not at other combinations. Ser-19 and Ser-45 are preferentially phosphorylated during the mitotic phase of the cell cycle. Also ␣B-crystallin R120G, a mutant found to co-segregate with a desmin-related myopathy, displayed increased interaction with FBX4. Both ␣B-crystallin S19D/S45D and R120G specifically translocated FBX4 to the detergentinsoluble fraction and stimulated the ubiquitination of one or a few yet unknown proteins. These findings implicate the involvement of ␣B-crystallin in the ubiquitin/ proteasome pathway in a phosphorylation-and cell cycledependent manner and may provide new insights into the ␣B-crystallin-induced aggregation in desmin-related myopathy.

Phosphorylation modulates the functioning of ␣B-crystallin as a molecular chaperone. We here explore the role of phosphorylation in the nuclear import and cellular localization of ␣B-crystallin in HeLa cells. Inhibition of nuclear export demonstrated that phosphorylation of ␣B-crystallin is required for import into the nucleus. As revealed by mutant analysis, phosphorylation at Ser-59 is crucial for nuclear import, and phosphorylation at Ser-45 is required for speckle localization. Co-immunoprecipitation experiments suggested that the import of ␣B-crystallin is possibly regulated by its phosphorylation-dependent interaction with the survival motor neuron (SMN) protein, an important factor in small nuclear ribonucleoprotein nuclear import and assembly. This interaction was supported by co-localization of endogenous phosphorylated ␣B-crystallin with SMN in nuclear structures. The cardiomyopathy-causing ␣B-crystallin mutant R120G was found to be excessively phosphorylated, which disturbed SMN interaction and nuclear import, and resulted in the formation of cytoplasmic inclusions. Like for other protein aggregation disorders, hyperphosphorylation appears as an important aspect of the pathogenicity of ␣B-crystallin R120G.␣B-crystallin, one of the most prominent human small heat-shock proteins (1-3), has its highest expression in eye lens and muscle cells (4). This molecular chaperone has an important role in the protection and modulation of the cytoskeleton (5-7), but is also involved in other processes, such as apoptosis (8, 9), protein degradation (10 -12), and Golgi reorganization (13). The properties of ␣B-crystallin are modulated by differential phosphorylation at . Phosphorylation varies during the cell cycle and clearly relates to the cellular localization of ␣B-crystallin. In mitotic cells, phosphorylation is enhanced at Ser-19 and especially at Ser-45, whereas phosphorylation at Ser-59 is reduced (15). Phosphorylation at Ser-59 is associated with the localization of ␣B-crystallin in the centrosomes of dividing cells (17). It has recently been observed that, in the nucleus ␣B-crystallin localizes in specific nuclear structures, identified as SC35 speckles (22-24), in a phosphorylation-dependent manner (24). These nuclear speckles, also known as interchromatin granule clusters, are thought to be sites for storage and recycling of splicing factors (25).The mutation R120G in ␣B-crystallin is associated with a desminrelated myopathy, characterized by the presence of aggregates containing ␣B-crystallin and desmin (26). This mutant causes cardiac hypertrophy and heart failure upon cardiac-restricted transgenic expression in mice (27). ␣B-Crystallin R120G has altered structural properties and a diminished chaperoning activity, both in vitro (28 -31) and in vivo (32). The inclusion bodies in which ␣B-crystallin R120G accumulates upon transfection (32, 33) and transgenesis (34) have the characteristics of aggresomes, cellular structures to sequester aggregated proteins. ␣B-Crystallin R120G might be responsible for agg...

Various mammalian small heat-shock proteins (sHSPs) can interact with one another to form large polydisperse assemblies. In muscle cells, HSPB2/MKBP (myotonic dystrophy protein kinase-binding protein) and HSPB3 have been shown to form an independent complex. To date, the biochemical properties of this complex have not been thoroughly characterized. In this study, we show that recombinant HSPB2 and HSPB3 can be successfully purified from E.coli cells co-expressing both proteins. Nanoelectrospray ionization mass spectrometry and sedimentation velocity analytical ultracentrifugation analysis showed that HSPB2/B3 forms a series of well defined hetero-oligomers, consisting of 4,8,12,16,20 and 24 subunits, each maintaining a strict 3:1 HSPB2:HSPB3 subunit ratio. These complexes are thermally stable up to 40 °C, as determined by far-UV circular dichroism spectroscopy. Surprisingly, HSPB2/B3 exerted a poor chaperone-like and thermoprotective activity, which is likely related to the low surface hydrophobicity, as revealed by its interaction with the hydrophobic probe 1-anilino-8-naphthalenesulfonic acid. Co-immunoprecipitation experiments demonstrated that the HSPB2/B3 oligomer cannot interact with HSP20, HSP27 or αB-crystallin, whereas the homomeric form of HSPB2, thus not in complex with HSPB3, could efficiently associate with HSP20. Taken altogether, this study brings evidence that despite the high sequence homology within the sHSP family, the biochemical properties of the HSPB2/B3 complex are distinctly different from other sHSPs, indicating that the HSPB2/B3 assembly likely possesses other cellular functions than its family members. Various mammalian small heat shock proteins (sHSPs) can interact with one another to form large polydisperse assemblies. In muscle cells, HSPB2/MKBP (myotonic dystrophy protein kinase-binding protein) and HSPB3 have been shown to form an independent complex. To date, the biochemical properties of this complex have not been thoroughly characterized. In this study, we show that recombinant HSPB2 and HSPB3 can be successfully purified from E.coli cells co-expressing both proteins. Nanoelectrospray ionization mass spectrometry and sedimentation velocity analytical ultracentrifugation analysis showed that HSPB2/B3 forms a series of well defined hetero-oligomers, consisting of 4, 8, 12, 16, 20 and 24 subunits, each maintains a strict 3:1 HSPB2:HSPB3 subunit ratio. Analyzing the thermal stability of the HSPB2/B3 assembly by far-UV circular dichroism spectroscopy revealed subtle structural changes, occurring slightly above 40 °C, and an unfolding curve with an inflection point at approximately 56 °C. Furthermore, HSPB2/B3 exerted poor chaperone-like and thermoprotective activity, which is likely related to the low surface hydrophobicity, as revealed by its interaction with the hydrophobic probe 1-anilino-8-naphthalenesulfonic acid. Finally, coimmunoprecipitation experiments demonstrated that the HSPB2/B3 oligomer does not interact with HSP20, HSP27 or αB-crystallin. However, HSPB2 that ...

The mammalian small heat shock protein aB-crystallin can be phosphorylated at three different sites, Ser19, Ser45 and Ser59. We compared the intracellular distribution of wildtype, nonphosphorylatable and all possible pseudophosphorylation mutants of aB-crystallin by immunoblot and immunocytochemical analyses of stable and transiently transfected cells. We observed that pseudophosphorylation at two (especially S19D/S45D) or all three (S19D/S45D/ S59D) sites induced the partial translocation of aB-crystallin from the detergent-soluble to the detergent-insoluble fraction. Double immunofluorescence studies showed that the pseudophosphorylation mutants localized in nuclear speckles containing the splicing factor SC35. The aB-crystallin mutants in these speckles were resistant to mild detergent treatment, and also to DNase I or RNase A digestion, indicating a stable interaction with one or more speckle proteins, not dependent on intact DNA or RNA. We further found that FBX4, an adaptor protein of the ubiquitin-protein isopeptide ligase SKP1/CUL1/F-box known to interact with pseudophosphorylated aB-crystallin, was also recruited to SC35 speckles when cotransfected with the pseudophosphorylation mutants. Because SC35 speckles also react with an antibody against aB-crystallin endogenously phosphorylated at Ser45, our findings suggest that aB-crystallin has a phosphorylation-dependent role in the ubiquitination of a component of SC35 speckles.