Proteomics to study the diversity and dynamics of proteasome complexes: from fundamentals to the clinic

Figures & data

Figure 1. Diversity of proteasome complexes.

Mammalian proteasome complexes result from the assembly of a 20S core complex, the 20S proteasome, with one or two regulatory particles (RPs) of identical or different protein composition. The 20S core complex is a stable entity assembled in four stacked ring-shape heptamers, α7β7β7α7. Three subunits, β1, β2 and β5, of each β ring exhibit three distinct catalytic activities. These standard β subunits can be completely or partially replaced by three immunosubunits (β1i, β2i and β5i, respectively), which leads to various forms of 20S proteasomes (immunoproteasome and intermediate-type proteasomes). The two α-rings are implicated in the recruitment of RPs at each side of the 20S core complex. In mammals, four activators have been identified: the 19S RPs, PA28αβ, PA28γ and PA200.

Figure 2. Affinity purification methods of proteasome complexes.

(1) Purification using a tagged 19S regulator subunit, (2) purification using a tagged domain recognizing a subunit of the 19S regulator, (3) purification using a tagged 20S core particle subunit and (4) purification using an antibody specifically directed against a conserved 20S proteasome subunit.

-: Not possible; +: Good; ++: Very good.

PIP: Proteasome-interacting proteins; UBL: Ubiquitin-like; UIM: Ubiquitin-interacting motif; Xlink: Proteins have been in vivo crosslinked prior to purification.

Figure 3. Targeted detection of peptides by mass spectrometry using selected reaction monitoring acquisition mode.

m/z: Mass-to-charge ratio; SRM: Selected reaction monitoring.

Table 1. Summary of the N-terminal processing and endogenous phosphorylation sites identified and localized by MS/MS on human, mouse and yeast proteasomes.

Name	Gene name (human)	Protein name (human)	Protein name (yeast)	PTM (amino acid, ^species)^†‡
20S proteasome subunits
α-type 1	PSMA1	α6 [230]	α6_sc [231]	Ac(M1^h) [98]; p(S14^y,T16^y) [232,233]
α-type 2	PSMA2	α2 [230]	α2_sc [231]	Ac(A2^h,m) [17,100]; p(S198^m) [23]; p(S13,53^y) [232]
α-type 3	PSMA3	α7 [230]	α7_sc [231]	Ac(S2^h,m) [17,74,98,100]^§; Fr(S2^h) [74]; p(S249^h) [98,100]; p(S250^m) [23,234]; p(S13^m,T186^m) [23]; p(S258, 263, 264^y) [235]; p(T278, 279^y) [232]
α-type 4	PSMA4	α3 [230]	α3_sc [231]	Ac(S2^h) [100]; p(S75^h) [98]; p(S33,75,173^m) [23]
α-type 5	PSMA5	α5 [230]	α5_sc [231]	Ac(M1^h,m) [17,74,98,100]; p(S134^m,T230^m) [23] p(S35,55,56,87,251^y) [93,232,233,236]
α-type 6	PSMA6	α1 [230]	α1_sc [231]	Ac(S2^h,y) [74,93,100]; p(S237^y) [232]
α-type 7	PSMA7	α4 [230]	α4_sc [231]	Ac(S2^h,m) [17,98,100]; p(T97^m) [23]; p(T60^y) [93,116]; p(S69^y) [93,232]
β-type 1	PSMB1	β6 [230]	β6_sc [231]	Tr(R29^h) [98,100]; p(S167^m) [23]; p(S53,209^y) [116,232]
β-type 2	PSMB2	β4 [230]	β4_sc [231]	Ac(M1^h,m) [17,74,98,100]; p(S39^m) [23]; p(S76,82^y) [232]
β-type 3	PSMB3	β3 [230]	β3_sc [231]	Ac(S2^h,m) [17,74,98,100]; p(S31,33^y) [232,236]
β-type 4	PSMB4	β7 [230]	β7_sc [231]	Tr(T46^h,m) [17,100]; p(S93^m) [23]
β-type 5	PSMB5	β5 [230]	β5_sc [231]	Tr(T60^h) [98,100]; p(T48^m) [23]; p(S51^y) [232]
β-type 6	PSMB6	β1 [230]	β1_sc [231]	Tr(T35^h,m) [17,98,100]; p(S157^m) [23]; p(T91^y) [232]
β-type 7	PSMB7	β2 [230]	β2_sc [231]	Tr(T44^h,m) [17,98,100]; p(T273^m) [23]
β-type 8	PSMB8	β5i [230]
β-type 9	PSMB9	β1i [230]		Tr(T21^m) [17]
β-type 10	PSMB10	β2i [230]
19S proteasome subunits
Regulatory subunit 4	PSMC1	S4 [237]	Rpt2 [238]	Myr(G2^h) [98]; p(S176,180,182,356^y;T178,357^y; Y181) [232,233,236]
Regulatory subunit 7	PSMC2	S7 [237]	Rpt1 [238]	Fr(P2^h,m) [74,93,98]; p(S164,231^y) [93,232,236]
Regulatory subunit 6A	PSMC3	S6a [237]	Rpt5 [238]	Ac(M1^h) [74,98]; p(S9^h) [98]; p(S181^y;T11,27,369,370^y) [93,232,236]
Regulatory subunit 6B	PSMC4	S6b [237]	Rpt3 [238]	Ac(M1^h) [74,98]; p(T8^y) [93,232]
Regulatory subunit 8	PSMC5	S8 [237]	Rpt6 [238]	Ac(A2^h) [74,98]; p(S120^h) [239]
Regulatory subunit 10B	PSMC6	S10b [237]	Rpt4 [238]	Ac(A2^h) [98]; p(S292^y) [233,236]
Non-ATPase regulatory subunit 1	PSMD1	S1 [237]	Rpn2 [238]	Ac(M1^h) [74,98]^§; Fr(M1^h) [74,98]; p(T273,311,315^h) [98,240]; p(S56,883,932^y;T801^y) [93,232]
Non-ATPase regulatory subunit 2	PSMD2	S2 [237]	Rpn1 [238]	Ac(M1^h) [98]; p(S16,361^h) [98]; p(S11,16,19,178,179,182,187^y; T24,190,640^y) [93,116,232,236]
Non-ATPase regulatory subunit 3	PSMD3	S3 [237]	Rpn3 [238]	p(S454^y) [232]
Non-ATPase regulatory subunit 4	PSMD4	S5a [237]	Rpn10 [238]	Fr(V2^h) [74,98]; p(S358,361^h) [98]; p(S259^y) [236]
Non-ATPase regulatory subunit 5	PSMD5	S5b [237]
Non-ATPase regulatory subunit 6	PSMD6	S10a [237]	Rpn7 [238]	Fr(P2^h) [74,98]; p(S8,77^y) [232]
Non-ATPase regulatory subunit 7	PSMD7	S12 [237]	Rpn8 [238]	Fr(P2^h) [74,98]; p(T186^h) [98]; p(S311,314,317,319,321^y; T327^y) [93,232,236,233]
Non-ATPase regulatory subunit 8	PSMD8	S14 [237]	Rpn12 [238]
non-ATPase regulatory subunit 9	PSMD9	S15 [237]	Rpn15 [238]
Non-ATPase regulatory subunit 10	PSMD10	Gankyrin		Ac(M1^h) [98]
Non-ATPase regulatory subunit 11	PSMD11	S9 [237]	Rpn6 [238]	Ac(A2^h) [74,98]; p(S13,78^h) [98]; p(S38^y) [93]; (T287^y) [236]
Non-ATPase regulatory subunit 12	PSMD12		Rpn5 [238]	p(S26^y) [93]
Non-ATPase regulatory subunit 13	PSMD13	S11 [237]	Rpn9 [238]	Fr(M1^h) [74,98]; p(S106,266^h) [98]
Non-ATPase regulatory subunit 14	PSMD14	S13 [237]	Rpn11 [238]	p(S224^h) [98]; p(S243^y) [93]; p(T262^y) [232]
Proteasomal ubiquitin receptor ADRM1	ADRM1	hRpn13	Rpn13 [238]	Ac(T2^h) [74,98]; Ac(S2^y) [93]; p(S132,133,135,140^y) [93,232,233,236]
Proteasomal ATPase-associated factor 1	PAAF1		Rpn14 [238]
26S proteasome complex subunit DSS1	SHFM1 DSS1	DSS1	Rpn15 [238]	Ac(S2^y) [93]; p(S12^y) [93]
Other proteasome activators
Proteasome activator complex subunit 1	PSME1	PA28α – 11Sα – REGα
Proteasome activator complex subunit 2	PSME2	PA28β – 11Sβ – REGβ
Proteasome activator complex subunit 3	PSME3	PA28γ – 11Sγ – REGγ
Proteasome activator complex subunit 4	PSME4	PA200	Blm10	p(S11,29,34,35,56,62^y;T64,66^y) [93,116,232,233,236]

^†Superscript letters in this column denote species.

^‡Determined by CID unless otherwise stated (underlined: determined by ETD and CID; underlined and bold: determined by ETD only).

^§Partial N-acetylation. The free N-terminus was also observed.

Ac: N-acetylation; CID: Collision-induced dissociation; ETD: Electron transfer dissociation; Fr: Free N-terminus; h: Human; m: Mouse; Myr: Myristoylation; P: Phosphorylation; PTM: Post-translational modification; Tr: N-terminal truncated; y: Yeast.

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