Hyperactivation of the proteasome core particle in Caenorhabditis elegans protects against proteotoxic stress and extends lifespan

Many age-related diseases (ARDs) including virtually all neurodegenerative diseases (NDs) are characterized by the accumulation of proteins that are thought to significantly contribute to disease pathogenesis. One of the cell’s primary systems for the degradation of misfolded/damaged proteins is the Ubiquitin Proteasome System (UPS), and its impairment is implicated in essentially all NDs. Thus, upregulating this system to combat NDs has garnered a great deal of interest in recent years. Various animal models have focused on increasing the total proteasome levels, but thus far, none have focused on intrinsic activation of the proteasome itself. With this in mind, we constructed a, first to our knowledge, animal model that endogenously expresses a hyperactive open-gate proteasome in Caenorhabditis elegans (C. elegans). The gate-destabilizing mutation introduced into the nematode germline created a viable nematode population with substantially enhanced proteasomal peptidase and unstructured protein degradation activity. These CRISPR edited nematodes showed a significantly increased lifespan and substantial resistance to oxidative/proteotoxic stress with surprisingly mild consequential phenotypes. These results show that introducing a constitutively active proteasome into a multicellular organism is feasible and suggests targeting the proteasome gating mechanism as a valid approach for future ARD research efforts in mammals.

p27Kip1, an Intrinsically Unstructured Protein with Scaffold Properties

The Cyclin-dependent kinase (CDK) regulator p27Kip1 is a gatekeeper of G1/S transition. It also regulates G2/M progression and cytokinesis completion, via CDK-dependent or -independent mechanisms. Recently, other important p27Kip1 functions have been described, including the regulation of cell motility and migration, the control of cell differentiation program and the activation of apoptosis/autophagy. Several factors modulate p27Kip1 activities, including its level, cellular localization and post-translational modifications. As a matter of fact, the protein is phosphorylated, ubiquitinated, SUMOylated, O-linked N-acetylglicosylated and acetylated on different residues. p27Kip1 belongs to the family of the intrinsically unstructured proteins and thus it is endowed with a large flexibility and numerous interactors, only partially identified. In this review, we look at p27Kip1 properties and ascribe part of its heterogeneous functions to the ability to act as an anchor or scaffold capable to participate in the construction of different platforms for modulating cell response to extracellular signals and allowing adaptation to environmental changes.

Analysis of the SNARE Stx8 recycling reveals that the retromer-sorting motif has undergone evolutionary divergence

Fsv1/Stx8 is a Schizosaccharomyces pombe protein similar to mammalian syntaxin 8. stx8Δ cells are sensitive to salts, and the prevacuolar endosome (PVE) is altered in stx8Δ cells. These defects depend on the SNARE domain, data that confirm the conserved function of syntaxin8 and Stx8 in vesicle fusion at the PVE. Stx8 localizes at the trans-Golgi network (TGN) and the prevacuolar endosome (PVE), and its recycling depends on the retromer component Vps35, and on the sorting nexins Vps5, Vps17, and Snx3. Several experimental approaches demonstrate that Stx8 is a cargo of the Snx3-retromer. Using extensive truncation and alanine scanning mutagenesis, we identified the Stx8 sorting signal. This signal is an IEMeaM sequence that is located in an unstructured protein region, must be distant from the transmembrane (TM) helix, and where the 133I, 134E, 135M, and 138M residues are all essential for recycling. This sorting motif is different from those described for most retromer cargoes, which include aromatic residues, and resembles the sorting motif of mammalian polycystin-2 (PC2). Comparison of Stx8 and PC2 motifs leads to an IEMxx(I/M) consensus. Computer-assisted screening for this and for a loose Ψ(E/D)ΨXXΨ motif (where Ψ is a hydrophobic residue with large aliphatic chain) shows that syntaxin 8 and PC2 homologues from other organisms bear variation of this motif. The phylogeny of the Stx8 sorting motifs from the Schizosaccharomyces species shows that their divergence is similar to that of the genus, showing that they have undergone evolutionary divergence. A preliminary analysis of the motifs in syntaxin 8 and PC2 sequences from various organisms suggests that they might have also undergone evolutionary divergence, what suggests that the presence of almost-identical motifs in Stx8 and PC2 might be a case of convergent evolution.

Structural basis for recruitment of the CHK1 DNA damage kinase by the CLASPIN scaffold protein

ABSTRACTCHK1 is a protein kinase that functions downstream of activated ATR to phosphorylate multiple targets as part of intra-S and G2/M DNA damage checkpoints. Its role in allowing cells to survive replicative stress has made it an important target for anti-cancer drug discovery. Activation of CHK1 by ATR depends on their mutual interaction with CLASPIN – a natively unstructured protein that interacts with CHK1 through a cluster of phosphorylation sites in its C-terminal half. We have now determined the crystal structure of the kinase domain of CHK1 bound to a high-affinity motif from CLASPIN. Our data show that CLASPIN engages a conserved site on CHK1 adjacent to the substrate-binding cleft, involved in phosphate sensing in other kinases. The CLASPIN motif is not phosphorylated by CHK1, nor does it affect phosphorylation of a CDC25 substrate peptide, suggesting that it functions purely as a scaffold for CHK1 activation by ATR.

Advantages and Disadvantages of His-Tagged Beta-Galactosidase

β-Galactosidase is one of the most important biotechnological enzyme used in the dairy industry, pharmacology and in molecular biology. In our laboratory we have overexpressed a recombinant β-galactosidase in Escherichia coli (E. coli). This enzyme differs from its native version (β-GalWT) in that 6 histidine residues have been added to the carboxyl terminus in the primary sequence (β-GalHis), which allows its purification by immobilized metal affinity chromatography (IMAC). In this work we compared the functionality and structure of both proteins and evaluated their catalytic behavior on the kinetics of lactose hydrolysis. We observed a significant reduction in the enzymatic activity of β-GalHis with respect to β-GalWT. Although, both enzymes showed a similar catalytic profile as a function of temperature, β-GalHis presented a higher resistance to the thermal inactivation and evidenced greater half-life time compared to β-GalWT. At room temperature, β-GalHis showed a fluorescence spectrum compatible with a partially unstructured protein however, it exhibited a lower tendency to the thermal-induced unfolding with respect to β-GalWT. Analytical ultracentrifugation experiments demonstrated that the population of β-GalHis molecules exhibited a higher proportion of monomers and a lower proportion of tetrameric species with respect to the His-tag free protein. The impairment of tetramerization may would explain the negative effect of the presence of His-tag on the enzymatic activity. In addition, the present results, analyzed in the context of the available literature, suggest that the effect of the His-tag is protein-specific.

Fusion Speed of Biomolecular Condensates

Biomolecular condensates formed through phase separation have a tendency to fuse. The speed with which fusion occurs is a direct indicator of condensate liquidity, which is key to both cellular functions and diseases. Using a dual-trap optical tweezers setup, we found the fusion speeds of four types of condensates to differ by two orders of magnitude. The order of fusion speed correlates with the fluorescence of Thioflavin T, which in turn reflects the macromolecular packing density inside condensates. Unstructured protein or polymer chains pack loosely and readily rearrange, leading to fast fusion. In contrast, structured protein domains pack more closely and have to break extensive contacts before rearrangement, corresponding to slower fusion. This molecular interpretation for disparate fusion speeds portends a unified understanding of the underlying physicochemical determinants.Entry for the Table of ContentsThe tendency of biomolecular condensates to fuse is key to cellular function and diseases. Using optical tweezers, fluorescence microscopy, and theoretical modeling, Ghosh and Zhou have begun to unravel the molecular origin for disparate fusion speeds among different biomolecular condensates. They found that fusion speed is dictated by macromolecular packing density inside condensates, which can be reported by ThT fluorescence.

CDK-Independent and PCNA-Dependent Functions of p21 in DNA Replication

p21Waf/CIP1 is a small unstructured protein that binds and inactivates cyclin-dependent kinases (CDKs). To this end, p21 levels increase following the activation of the p53 tumor suppressor. CDK inhibition by p21 triggers cell-cycle arrest in the G1 and G2 phases of the cell cycle. In the absence of exogenous insults causing replication stress, only residual p21 levels are prevalent that are insufficient to inhibit CDKs. However, research from different laboratories has demonstrated that these residual p21 levels in the S phase control DNA replication speed and origin firing to preserve genomic stability. Such an S-phase function of p21 depends fully on its ability to displace partners from chromatin-bound proliferating cell nuclear antigen (PCNA). Vice versa, PCNA also regulates p21 by preventing its upregulation in the S phase, even in the context of robust p21 induction by γ irradiation. Such a tight regulation of p21 in the S phase unveils the potential that CDK-independent functions of p21 may have for the improvement of cancer treatments.