Peptide-size–dependent active transport in the proteasome

Abstract

We investigate the transport of proteins inside the proteasome and propose an active transport mechanism based on a spatially asymmetric interaction potential of peptide chains. The transport is driven by fluctuations which are always present in such systems. We compute the peptide-size dependent transport rate which is essential for the functioning of the proteasome. In agreement with recent experiments, varying temperature changes the transport mechanism qualitatively. [4,5,6] and to predict the cleavage results [7], however, basic principles of the proteasome operation mechanisms are still poorly understood, mainly due to the lack of experimental results.In this Letter, we focus on understanding of protein translocation inside the proteasome, leaving the mechanisms of cleavage, targeting, etc. beyond the scope. The main question is: given proteasomes are highly complex pipe-like structures of tenthousands atoms of almost perfect left-right symmetry with respect to the axis of the pipe [9], thus, operating bi-directional. To be cleaved, a protein has to enter the proteasome at one side, pass the active sites (where the cleavage occurs), which makes about 1/3 of the total length of the pipe. Then the cleaved peptides have to pass all the way through the pipe to finally reappear at the other side of the proteasome. Although there is a number of examples where protein transport in cells occurs due to diffusion, i.e. Brownian motion [8], diffusive transport may be excluded as the main mechanism for translocation in the proteasome because of the enormous cargo [10]. Also other proposed transport mechanisms, such as the power stroke model of protein translocation do not suffice to explain translocation [11]. Therefore, the question arises how the protein's motion inside the proteasome is driven?Since proteasomes are large multi-subunit structures consisting of proteins, the mechanism of the protein transport is directly related to protein-protein interaction. In [12] it has been noticed that if attachment and detachment rates are specified asymmetrically, the protein-protein binding interaction acts as a ratchet. Following this argumentation, active protein transport, based on the mechanism of a molecular ratchet, has been studied for transport through membranes [13] and has been also discussed as a mechanism for cytosolic destruction by the proteasome [14]. Moreover, maximum likelihood tests have shown that other models, e.g. the power stroke model of protein translocation, do not lead to better agreement with the experiment than the Brownian ratchet model [11]. Noteworthy, in these ratchet effects transport is possible only in a certain temperature interval, and stochasticity, intrinsically present due to fluctuations in any biochemical reaction [15,16], provides the driving mechanism.Here we propose a model for active protein translocation in the proteasome to explain the peptide size dependence of the transport velocity as well as its temperature dependence which possibly explains the mechanism of temperature r...

Modeling the in Vitro 20S Proteasome Activity: The Effect of PA28–αβ and of the Sequence and Length of Polypeptides on the Degradation Kinetics

“…20, 21 We have recently discussed that transport of substrate can be described by a stochastic model where stochastic fluctuations and interaction forces between substrate and proteasome gate subunits provide the passive mechanism to translocate polypeptides within the proteasome core particle i.e., without a requirement for external energy. 20 Experimental studies suggested this type of mechanism in many biological systems involving protein transport. 22 A stochastic approach allows us to model the degradation dynamics assuming a specific transport function, without the requirement for detailed information on the translocation mechanisms.…”

“…In order to calculate the cleavage strengths of the substrates tested in the new biochemical experiments, we used the following method: for each residue of the substrate (e.g., residue 7), we summed the amount (in pmol) of each peptide that had this residue at the C terminus (e.g., peptide 1-7) or the following residue at the N terminus (e.g., peptide [8][9][10][11][12][13][14][15][16][17][18][19][20][21]. Since the production of DCPs depends on the cleavage of both peptide ends.…”

Periodic Forces Trigger a Complex Mechanical Response in Ubiquitin

“…6,[16][17][18][19][20][21][22][23] For instance, it is vital for cellular protein unfolding machineries to periodically release substrates, allowing them to adjust their conformation and prevent entanglements. 21,40 Similarly, instrumentation has been developed to probe single-molecule protein mechanics with periodic forces.…”

“…6,14,15 As a direct consequence of these chemo-mechanical cycles, biological machines are repetitive force generators, and it is believed that forces with periodic signatures are experienced by biomolecules in many physiological contexts. [16][17][18][19][20][21][22][23] For instance, it has been postulated that an ATP-dependent 'pulling' force is utilized by proteasomes and mitochondrial import machines to unfold proteins. 16,18,[24][25][26] Similarly, optical trapping experiments revealed that the force generated by the motor protein dynein is oscillatory.…”

“…Our work is motivated by the desire to understand protein dynamics under biological repetitive forces as well as by growing theoretical interest in periodically modulated biomolecular systems. 22,[28][29][30][31] To obtain quantitative insights into dynamic ubiquitin stability, we devised periodic force MD simulations and combined them with a kinetic model. We found a complex, linkagedependent response that included asymmetric weakening of the protein, shifts in unfolding pathways, and refolding.…”