Biology subjects

Sahu, I.

Publications and source records attributed to Sahu, I..

4 recordsLinked to original sources

bioRxiv2025

USP15: the fourth Proteasome-associated DUB

The human genome encodes approximately 100 deubiquitinating enzymes (DUBs), but only three are considered proteasome-associated DUBs (pDUBs): PSMD14/Rpn11, USP14, and UCHL5. Among these, only PSMD14 is an integral 19S subunit, whereas USP14 and UCHL5 bind transiently to specific proteasomal subunits. Given the dynamic nature of proteasome composition, we searched for additional pDUBs. USP15 was found to be associated with 26S proteasomes purified from cultured cells. In proteasome preparations from erythrocytes, USP15 was identified as the most abundant transient pDUB. It was even feasible to separate proteasomes containing USP15 from those containing USP14. Although USP15 utilizes an internal ubiquitin-like (UBL) domain for positioning itself at the proteasome, it did not compete with the UBL-containing USP14. USP15 facilitated substrate selection at the proteasome by efficiently disassembling short polyubiquitin (polyUb) chains, while sparing K48-linked tetra-ubiquitin conjugates from deubiquitination. This feature may aid the proteasome in differentiating between substrates to be rescued from those committed to proteolysis. Identification of a fourth pDUB encourages the continued search for additional proteasome-interacting proteins that modulate its substrate specificity in a context-specific manner.

biochemistry↗

bioRxiv2023

Activity-Guided Proteomic Profiling of Proteasomes Uncovers a Variety of Active (And Inactive) Proteasome species

Proteasomes are multi-subunit, multi-catalytic protein complexes present in eukaryotic cells that degrade misfolded, damaged, or unstructured proteins. In this study, we used an activity-guided proteomic methodology based on a fluorogenic peptide substrate to characterize the composition of proteasome complexes in WT yeast, and the changes these complexes undergo upon the deletion of Pre9 ({Delta}3) or of Sem1 ({Delta}Sem1). A comparison of whole-cell proteomic analysis to activity-guided proteasome profiling indicates that the amounts of proteasomal proteins and proteasome interacting proteins in the assembled active proteasomes differ significantly from their total amounts in the cell as a whole. Using this activity-guided approach, we characterized the changes in the abundance of subunits of various active proteasome species in different strains, quantified the relative abundance of active proteasomes across these strains, and charted the overall distribution of different proteasome species within each strain. The distributions obtained by our mass spectrometry-based quantification were markedly higher for some proteasome species than those obtained by activity-based quantification alone, suggesting that the activity of some of these species is impaired. The impaired activity appeared mostly among 20SBlm10 proteasome species which account for 20% of the active proteasomes in WT. To identify the factors behind this impaired activity, we mapped and quantified known proteasome-interacting proteins. Our results suggested that some of the reduced activity might be due to the association of the proteasome inhibitor Fub1. Additionally, we provide novel evidence for the presence of non-mature and therefore inactive proteasome protease subunits {beta}2 and {beta}5 in the fully assembled proteasomes. Significance StatementProteasomes, essential protein complexes in eukaryotic cells, degrade misfolded, damaged, or unstructured proteins. Here we present an activity-guided proteomic method to characterize the composition and abundance of proteasomes. When applied to yeast proteasomes, this method revealed discrepancies between proteasome distributions determined by mass spectrometry and peptidase activity. This implies that a substantial portion of the proteasomes may exhibit reduced activity. Our findings indicate that these changes in proteasome activity could be linked to proteasome inhibition by Fub1. Furthermore, we identified signature peptides that indicate incomplete maturation of some of the {beta}2 and {beta}5 proteolytic subunits in fully assembled proteasomes, suggesting that proteasome core particle assembly can proceed even without the complete maturation of all {beta} subunits.

biochemistry↗

bioRxiv2023

A role for the proteasome alpha2 subunit N-tail in substrate processing

The proteolytic active sites of the 26S proteasome are sequestered within the catalytic chamber of its 20S core particle (CP). Access to this chamber is through a narrow channel defined by the outer subunits. Free 20S CP is found in a latent state in which the N-termini of neighboring subunits form a gate blocking access to the channel. Entry of substrates can be facilitated by the attachment of the activators or regulatory particles, which can rearrange the blocking subunit N-terminal residues. In order to determine the specific physiological role of individual elements working in concert within the gate, we constructed a set of truncations or single-site mutations in each of the participating N-terminal tails. We report herein that whereas only a few N-termini are important for maintaining a closed gate, all seven N-termini participate in the open gate. Specifically, an invariant tyrosine (Y) in each subunit forms a hydrogen bond with a conserved aspartate (D) in the N-terminal tail of its counterclockwise neighbor, with the exception of the 1-2 pair leaving a gap in the ring circumference. A third residue (X) of this YD(X) motif aligns the open channel; specifically, the phenylalanine (F) at this position of the 2 subunit comes in direct contact with the translocating substrate. Consequently, deletion of this 2 N-terminal tail slows down proteolysis despite the appearance of an open gate state. We conclude that the YD(X) motif in N-terminal tail of subunits plays an important role in gating the proteasome and in processing the substrate.

biochemistry↗

bioRxiv2019

Signature activities of 20S proteasome include degradation of the ubiquitin-tag with the protein under hypoxia

Careful removal of unwanted proteins is necessary for cell survival. The primary constitutive intracellular protease is the 26S proteasome complex, often found in equilibrium with its free catalytic subcomplex- the 20S core particle. Protein degradation by 26S is tightly regulated by prior ubiquitination of substrates, whereas 20S is amenable to substrates with an unstructured segment. Differentiating their contributions to intracellular proteolysis is challenging due to their common catalytic sites. Here, by chemically synthesizing a synoptic set of homogenous ubiquitinated proteins, we ascribe signature features to 20S function and demonstrate a unique property: degrading the ubiquitin-tag along with the target protein. Cryo-EM confirms that a ubiquitinated substrate can induce asymmetric conformational changes to 20S. Mass-spectrometry of intracellular peptidome under hypoxia and in human failing heart identifies the signature properties of 20S in cells. Moreover, the ability of 20S proteasome to clear toxic proteins rapidly, contributes to better survival under these conditions.

biochemistry↗