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Small heat shock proteins (sHSPs) are essential ATP-independent chaperones that protect the cellular proteome. These proteins assemble into polydisperse oligomeric structures, the composition of which dramatically affects their chaperone activity. The biomolecular consequences of variations in sHSP ratios, especially inside living cells, remain elusive. Here, we study the consequences of altering the relative expression levels of HspB2 and HspB3 in HEK293T cells. These chaperones are partners in a hetero-oligomeric complex, and genetic mutations that abolish their mutual interaction are associated with myopathic disorders. HspB2 displays three distinct phenotypes when co-expressed with HspB3 at varying ratios. Expression of HspB2 alone leads to formation of liquid nuclear condensates, while shifting the stoichiometry towards HspB3 resulted in the formation of large solid-like aggregates. Only cells co-expressing HspB2 with a limited amount of HspB3 formed fully soluble complexes that were distributed homogeneously throughout the nucleus. Strikingly, both condensates and aggregates were reversible, as shifting the HspB2:HspB3 balance in situ resulted in dissolution of these structures. To uncover the molecular composition of HspB2 condensates and aggregates, we used APEX-mediated proximity labelling. Most proteins interact transiently with the condensates and were neither enriched nor depleted in these cells. In contrast, we found that HspB2:HspB3 aggregates sequestered several disordered proteins and autophagy factors, suggesting that the cell is actively attempting to clear these aggregates. This study presents a striking example of how changes in the relative expression levels of interacting proteins affects their phase behavior. Our approach could be applied to study the role of protein stoichiometry and the influence of client binding on phase behavior in other biomolecular condensates and aggregates., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Increasing evidence shows that heat shock proteins (hsp) escape the cytosol gaining access to the extracellular environment, acting as signaling agents. Since the majority of these proteins lack the information necessary for their export via the classical secretory pathway, attention has been focused on alternative releasing mechanisms. Crossing the plasma membrane is a major obstacle to the secretion of a cytosolic protein into the extracellular milieu. Several mechanisms have been proposed, including direct interaction with the plasma membrane or their release within extracellular vesicles (ECV). HSPB1 (Hsp27), which belongs to the small hsp family, was detected within the membrane of ECV released from stressed HepG2 cells. To further investigate this finding, we studied the interaction of HSPB1 with lipid membranes using liposomes. We found that HSPB1 interacted with liposomes made of palmitoyl oleoyl phosphatidylserine (POPS), palmitoyl oleoyl phosphatidylcholine (POPC), and palmitoyl oleoyl phosphatidylglycerol (POPG), with different characteristics. Another member of the small hsp family, HSPB5 (αB-crystallin), has also been detected within ECV released from HeLa cells transfected with this gene. This protein was found to interact with liposomes as well, but differently than HSPB1. To address the regions interacting with the membrane, proteoliposomes were digested with proteinase K and the protected domains within the liposomes were identified by mass spectroscopy. We observed that large parts of HSPB1 and HSPB5 were embedded within the liposomes, particularly the alpha-crystallin domain. These observations suggest that the interaction with lipid membranes may be part of the mechanisms of export of these proteins.

Small heat shock proteins (sHSPs) are present in all kingdoms of life and play fundamental roles in cell biology. sHSPs are key components of the cellular protein quality control system, acting as the first line of defense against conditions that affect protein homeostasis and proteome stability, from bacteria to plants to humans. sHSPs have the ability to bind to a large subset of substrates and to maintain them in a state competent for refolding or clearance with the assistance of the HSP70 machinery. sHSPs participate in a number of biological processes, from the cell cycle, to cell differentiation, from adaptation to stressful conditions, to apoptosis, and, even, to the transformation of a cell into a malignant state. As a consequence, sHSP malfunction has been implicated in abnormal placental development and preterm deliveries, in the prognosis of several types of cancer, and in the development of neurological diseases. Moreover, mutations in the genes encoding several mammalian sHSPs result in neurological, muscular, or cardiac age-related diseases in humans. Loss of protein homeostasis due to protein aggregation is typical of many age-related neurodegenerative and neuromuscular diseases. In light of the role of sHSPs in the clearance of un/misfolded aggregation-prone substrates, pharmacological modulation of sHSP expression or function and rescue of defective sHSPs represent possible routes to alleviate or cure protein conformation diseases. Here, we report the latest news and views on sHSPs discussed by many of the world's experts in the sHSP field during a dedicated workshop organized in Italy (Bertinoro, CEUB, October 12-15, 2016).

The fibrillization of α-synuclein (α-syn) is a key event in the pathogenesis of α-synucleinopathies. Mutant α-syn (A53T, A30P, or E46K), each linked to familial Parkinson's disease, has altered aggregation properties, fibril morphologies, and fibrillization kinetics. Besides α-syn, Lewy bodies also contain several associated proteins including small heat shock proteins (sHsps). Since α-syn accumulates intracellularly, molecular chaperones like sHsps may regulate α-syn folding and aggregation. Therefore, we investigated if the sHsps αB-crystallin, Hsp27, Hsp20, HspB8, and HspB2B3 bind to α-syn and affect α-syn aggregation. We demonstrate that all sHsps bind to the various α-syns, although the binding kinetics suggests a weak and transient interaction only. Despite this transient interaction, the various sHsps inhibited mature α-syn fibril formation as shown by a Thioflavin T assay and atomic force microscopy. Interestingly, HspB8 was the most potent sHsp in inhibiting mature fibril formation of both wild-type and mutant α-syn. In conclusion, sHsps may regulate α-syn aggregation and, therefore, optimization of the interaction between sHsps and α-syn may be an interesting target for therapeutic intervention in the pathogenesis of α-synucleinopathies., (Copyright © 2011 Wiley-Liss, Inc.)

More than 80% of Alzheimer's disease (AD) patients have some degree of cerebral amyloid angiopathy (CAA). In addition to arteries and veins, capillaries can also be affected. Capillary CAA (capCAA), rather than CAA in larger vessels, is associated with flame-like amyloid-beta (Aβ) deposits that may extend beyond the vessel wall and radiate into the neuropil, a phenomenon also known as "dyshoric angiopathy." Aβ deposits in AD, parenchymal as well as (cap)CAA and dyshoric angiopathy, are associated with a local inflammatory reaction, including activation of microglial cells and astrocytes that, among others, produce cytokines and reactive oxygen species. This neuroinflammatory reaction may account for at least part of the cognitive decline. In previous studies we observed that small heat shock proteins (sHsps) are associated with Aβ deposits in AD. In this study the molecular chaperones Hsp20, HspB8 and HspB2B3 were found to colocalize with CAA and capCAA in AD brains. In addition, Hsp20, HspB8 and HspB2B3 colocalized with intercellular adhesion molecule 1 (ICAM-1) in capCAA-associated dyshoric angiopathy. Furthermore, we demonstrated that Hsp20, HspB8 and HspB2B3 induced production of interleukin 8, soluble ICAM-1 and monocyte chemoattractant protein 1 by human leptomeningeal smooth muscle cells and human brain astrocytes in vitro and that Hsp27 inhibited production of transforming growth factor beta 1 and CD40 ligand. Our results suggest a central role for sHsps in the neuroinflammatory reaction in AD and CAA and thus in contributing to cognitive decline.

Small heat shock proteins (sHsps) are oligomers that perform a protective function by binding denatured proteins. Although ubiquitous, they are of variable sequence except for a C-terminal approximately 90-residue "alpha-crystallin domain". Unlike larger stress response chaperones, sHsps are ATP-independent and generally form polydisperse assemblies. One proposed mechanism of action involves these assemblies breaking into smaller subunits in response to stress, before binding unfolding substrate and reforming into larger complexes. Two previously solved non-metazoan sHsp multimers are built from dimers formed by domain swapping between the alpha-crystallin domains, adding to evidence that the smaller subunits are dimers. Here, the 2.5A resolution structure of an sHsp from the parasitic flatworm Taenia saginata Tsp36, the first metazoan crystal structure, shows a new mode of dimerization involving N-terminal regions, which differs from that seen for non-metazoan sHsps. Sequence differences in the alpha-crystallin domains between metazoans and non-metazoans are critical to the different mechanism of dimerization, suggesting that some structural features seen for Tsp36 may be generalized to other metazoan sHsps. The structure also indicates scope for flexible assembly of subunits, supporting the proposed process of oligomer breakdown, substrate binding and reassembly as the chaperone mechanism. It further shows how sHsps can bind coil and secondary structural elements by wrapping them around the alpha-crystallin domain. The structure also illustrates possible roles for conserved residues associated with disease, and suggests a mechanism for the sHsp-related pathogenicity of some flatworm infections. Tsp36, like other flatworm sHsps, possesses two divergent sHsp repeats per monomer. Together with the two previously solved structures, a total of four alpha-crystallin domain structures are now available, giving a better definition of domain boundaries for sHsps.

The barrel-shaped 20S proteasome is one of the two components of a larger 26S particle, the multicatalytic 2000-kDa protease complex. The proteolytic sites are located in the inner chamber of the 20S particle and are only accessible via narrow entrances. This paper reviews the current knowledge concerning proteasome formation, proteolytic activities, structural aspects and assembly. Eukaryotic proteasomes are made up by four rings each of which contains seven different subunits occurring at fixed positions. While the outer rings contain alpha-type subunits, the inner ones comprise beta-type subunits. The current assembly model for eukaryotic 20S proteasomes is based upon the detection of 13S and 16S intermediates, respectively, in addition to previous findings with archaebacterial and eubacterial proteasome assembly. The available data suggest a cooperative assembly of the alpha-type and beta-type subunits into half proteasome-like complexes followed by dimerization into proteasomes. During or after dimerization of half proteasomes, the beta-type subunits are processed. The prosequence of the beta-type subunits is essential for the assembly proves and prevents protease activity of immature proteasomes.

The eukaryotic 20 S proteasome is a barrel-shaped protease complex, made up of four seven-membered rings. The outer and inner rings contain seven different alpha and beta-type subunits, respectively, each subunit located at a defined position. Recently, we have reported that the recombinant human alpha-type subunit C8 (HsC8) assembles into a heptameric ring-like structure by itself. In the present study we show that the two naturally neighboring alpha-type subunits of HsC8, HsPROS30 and HsPROS27, do not form ring-like complexes by themselves, but only dimers. This indicates that the propensity to form homo-oligomeric rings is not a general feature among human alpha-type subunits. However, coexpression of HsC8 and either of these neighbor alpha-type subunits results in the formation of hetero-oligomeric ring complexes, resembling the HsC8 ring-like structure. The ratio between the two types of subunits in the mixed complexes is surprisingly heterogeneous, varying from very high to very low HsC8 content. The three tested alpha-type subunits thus apparently lack binding sites that selectively interact with a specific neighboring subunit. This suggests that the correct positioning of the different alpha-type subunits in the eukaryotic 20 S proteasome is not dictated by the alpha-type subunits themselves, but rather by the interaction with specific beta-type subunits.

The eukaryotic proteasome is a barrel-shaped protease complex made up of four seven-membered rings of which the outer and inner rings may contain up to seven different alpha- and beta-type subunits, respectively. The assembly of the eukaryotic proteasome is not well understood. We cloned the cDNA for HsC8, which is one of the seven known human alpha-type subunits, and produced the protein in Escherichia coli. Recombinant HsC8 protein forms a complex of about 540 kDa consisting of double ringlike structures, each ring containing seven subunits. Such a structure has not earlier been reported for any eukaryotic proteasome subunit, but is similar to the complex formed by the recombinant alpha-subunit of the archaebacterium Thermoplasma acidophilum (Zwickl, P., Kleinz, J., and Baumeister, W. (1994) Nat. Struct. Biol. 1, 765-770). The ability of HsC8 to form alpha-rings suggests that these complexes may play an important role in the initiation of proteasome assembly in eukaryotes. To test this, we used two human beta-type subunits, HsBPROS26 and HsDelta. Both these beta-type subunits, either in the proprotein or in the mature form, exist in monomers up to tetramers. In contrast to the alpha- and beta-subunit of T. acidophilum, coexpression of the human beta-type subunits with HsC8 does not result in the formation of proteasome-like particles, which would be in agreement with the notion that proteasome assembly in eukaryotes is much more complex than in archaebacteria.

In addition to their role in pre-mRNA splicing, the human spliceosomal proteins U1A and U2B" are important models of how RNP motif-containing proteins execute sequence-specific RNA binding. Genes encoding U1A and U2B" have been isolated from potato and thereby provide the only evolutionary comparison available for both proteins and represent the only full-length genes encoding plant spliceosomal proteins to have been cloned and characterized. In vitro RNA binding experiments revealed the ability of potato U2B" to interact with human U2A' to enhance sequence-specific binding and to distinguish cognate RNAs of either plant or animal origin. A comparison of the sequence of U1A and U2B" proteins indicated that multiple residues which could affect RNP motif conformation probably govern the specific distinction in RNA binding by these proteins. Since human U1A modulates polyadenylation in vertebrates, the possibility that plant U1A might be exploited in the characterization of this process in plants was examined. However, unlike vertebrate U1A, neither U1A from potato nor Arabidopsis bound their own mRNA and no evidence for binding to upstream efficiency elements in polyadenylation signals was obtained, suggesting that plant U1A is not involved in polyadenylation.

Despite the great sequence similarity between U1A and U2B", both proteins do have a difference in RNA binding specificity and in the way they bind to their cognate RNAs. The U1A protein is able to bind in vitro U1 RNA independently of other factors. The U2B" protein binds specifically to U2 RNA in the presence of the U2A' protein only. We have compared the effect on RNA binding of multiple double point mutations at analogous positions in the U1A and U2B" protein. The results obtained show that amino acids at almost all of the analogous positions tested in U1A and U2B" have a comparable qualitative effect on RNA binding although the quantitative effect of mutations on U2B" is more severe than on U1A. Using U1A mutants with internal duplications a distinct area of the RNP motif of the U1A protein was identified which appears not to be directly involved in U1 RNA binding. In addition, roles of the highly conserved RNP1 and RNP2 sequences of the N-terminal RNP motif of the U1A protein, are investigated by replacing them with the analogous U1-70K sequences.

By the use of hybrids between a U1 small nuclear ribonucleoprotein (snRNP: U1A) and a U2 snRNP (U2B") we have identified regions containing 29 U1A-specific amino acid residues scattered throughout the 117 N-terminal residues of the protein, which are involved in binding to U1 RNA. The U1A-specific amino acid residues have been arbitrarily divided into seven contiguous groups. None of these groups is sufficient for U1 binding when transferred singly into the U2B" context, and none of the groups is essential for U1 binding in U1A. Several different combinations of two or more groups can, however, confer the ability to bind U1 RNA to U2B", suggesting that most or all of the U1A-specific amino acid residues contribute incrementally to the strength of the specific binding interaction. Further evidence for the importance of the U1A-specific amino acid residues, some of which lie outside the region previously shown to be sufficient for U1 RNA binding, is obtained by comparison of the sequence of human and Xenopus laevis U1A cDNAs. These are extremely similar (94.4% identical) between amino acid residues 7 and 114 but much less conserved immediately upstream and downstream from this region.

The U2 snRNP complex contains two specific proteins, U2B" and U2A'. We have analysed the interaction of U2A' with U2B" and with U2 RNA. U2A' can form an weak but detectable RNA-protein complex with U2 RNA and a stable protein complex with U2B". This protein-protein complex binds efficiently and specifically to U2 RNA. Binding experiments with mutant forms of U2A' shows that the region of U2A' essential for binding to U2B" is extensive, being located between amino acid position 1-164. The behaviour of the wild type U2A' protein, and in particular of a mutant version of the protein in which amino acids 3, 4 and 5 are mutated, suggests that U2A' forms a weak interaction with U2 RNA which helps to stabilize the U2A'-U2B"-U2 RNA complex. Mutants of U2 RNA were used to localize the region of U2 RNA important for interaction with U2A'. The results show that U2A' interacts with the stem of hairpin IV.

The U2 snRNP contains two specific proteins, U2B'' and U2A'. Neither of these proteins, on its own, is capable of specific interactions with U2 RNA. Here, a complex between U2B'' and U2A' that forms in the absence of RNA is identified. Analysis of mutant forms of U2B'' shows that the smallest fragment able to bind specifically U2 RNA (amino acids 1-88) is also the minimal region required for complex formation with U2A', and implies that this region must be largely structurally intact for U2A' interaction. Although this truncated U2B'' fragment is capable of making specific protein--RNA and protein-protein interactions its structure, as measured by the ability to bind to U2A'', appears to depend on the rest of the protein. Hybrids between U2B'' and the closely related U1A protein are used to localize U2B'' specific amino acids involved in protein-protein interaction. These can be divided into two functional groups. U2A' interaction with U2B'' amino acids 37-46 permits binding to U2 RNA whereas interaction with U2B'' specific amino acids between positions 14 and 25 reduces non-specific binding to U1 RNA. These two proteins may serve as a general example of how RNA binding may be modulated by protein-protein interaction in the assembly of RNPs, particularly since the region of U2'' involved in interaction with U2A' consists mainly of a conserved RNP motif.

The basis of the specificity of interaction of U1 and U2 small nuclear (sn)RNAs and their cognate binding proteins, U1A and U2B'', has been examined. The U1A protein recognizes U1 snRNA on its own, whereas U2B'' binds specifically to U2 snRNA only in the presence of a second protein, U2A'. Exchange of two nucleotides between the two RNAs or of eight amino acids between the two proteins reverses binding specificity.

We have shown previously that the putative oncogene pim-1 is frequently activated by provirus insertion in murine leukemia virus-induced T cell lymphomas. Here we describe the structure of the pim-1 gene as determined by sequencing genomic and cDNA clones. The gene has an open reading frame, encoding a protein of 313 amino acids, extending over six exons and preceded and followed by stop codons in all reading frames. Proviruses always integrate outside the protein-encoding domain, showing a high preference for a small region in the 3'-terminal exon; integration in the 3' exon results in relatively high levels of pim-1 mRNA. Computer search reveals homology between pim-1 and protein kinases: all the domains characteristic of protein kinases are conserved in the pim-1 amino acid sequence. The highest homologies were observed with the protein-serine kinases.

A number of mink cell focus-forming (MCF) proviruses was molecularly cloned from mouse lymphoma DNA. From each clone, flanking probes were prepared to detect common integration regions in other MuLV-induced lymphomas. One clone frequently revealed variations in the molecular structure of the corresponding region (Pim-1) in other lymphomas. The results show the following. Changes in the Pim region are seen in 24 out of 93 lymphomas tested. Over 50% of the early T-cell lymphomas show integration in the Pim-1 region. The alterations are seen in different mouse strains and with various MuLVs. The observed variations are caused by the integration of predominantly MCF genomes. All integrations occur in a region spanning less than 20 kb and are associated with the transcriptional activation of a distinct region within the Pim-1 domain. The activated region does not show any homology with 13 known and three putative oncogenes.

AKV and AKR mink cell focus-forming virus-specific probes from the envelope and long terminal repeat (LTR) regions were prepared for study of the structure of recombinant proviruses in tumor tissues of AKR mice. The results showed that (i) all somatically acquired proviruses possessed, besides a recombinant gp70 gene, an altered U3 LTR; (ii) in a substantial portion of the somatically acquired AKR mink cell focus-forming proviruses, the LTR comprised sequences derived from the same xenotropic-like provirus; (iii) this U3 LTR donating parental provirus (Xeno-dL) was present only once per genome equivalent in several mouse strains; (iv) in the strains containing the Xeno-dL provirus, the provirus was present in the same chromosomal site; (v) restriction analysis of the Xeno-dL revealed that the mink cell focus-forming gp70 sequences were derived from a parental provirus, different from Xeno-dL. Therefore, at least two non-ecotropic parents participate in the generation of leukemogenic AKR mink cell focus-forming viruses: a xenotropic-like virus, Xeno-dL, donating U3 LTR sequences, and another xenotropic-like virus or viruses providing gp70 sequences.

The interaction between the U1 snRNP-specific U1 A protein and U1 snRNA has been analysed. The binding site for the protein on the RNA is shown to be in hairpin II, which extends from positions 48 to 91 in the RNA. Within this hairpin the evolutionarily conserved loop sequence is crucial for interaction with U1 A protein. U1 A protein can also bind the loop sequence when it is part of an artificial RNA which cannot form a stable hairpin structure. The region of the protein required to bind to U1 snRNA consists of a conserved 80 amino acid motif, previously identified in many ribonucleoprotein (RNP) proteins, together with (maximally) 11 N-terminal and 10 C-terminal flanking amino acids. Point mutations introduced into two of the most highly conserved regions of this motif abolish RNA binding. U1 snRNA mutants from which the U1 A binding site has been deleted are shown to be capable of assembly into RNP particles which are immunoprecipitable by patient antisera which recognize U1 A protein. The role of RNA-protein and protein-protein interactions in U snRNP assembly are discussed.

The 3' end of an AKR-MCF provirus (MCFr35) was cloned and found to be biologically active. Comparison of the nucleotide sequence of MCFr35 with the sequence of other MuLVs revealed that the MCFr35 was most likely derived from the same xenotropic and ecotropic parents, which were involved in the generation of AKR-MCF247. Ecotropic sequences are present around the XbaI site at position 7.9 on the genomic map, and in the long terminal repeat. Most of the T1 oligonucleotide sequences, characteristic for the leukemogenic "class I" MCFs, are also present in MCFr35, with the exception of T1 oligonucleotides 108 and 18. The MCFr35 LTR contains a duplicated enhancer sequence from a xenotropic-like provirus, which is present only once per haploid genome equivalent. The 3' end of MCFr35 consist predominantly of nonecotropic sequences, thereby delimiting the positions of recombination in various MCF viruses.