The document summarizes bacterial proteasomes. It discusses the structure and assembly of bacterial 20S core particles, which consist of stacked rings of α and β subunits. Proteasomal activation can occur via ATP-dependent or ATP-independent mechanisms. ATP-dependent activation involves proteasomal ATPases like Mpa and ARC that recognize pupylated substrates and unfold proteins. Pupylation is analogous to ubiquitylation in eukaryotes. Proteasomes contribute to bacterial physiology through roles in stress responses and nutrient recycling.

2. Bacterial Proteasomes: Mechanistic and
Functional Insights
M. RAVEENDRA REDDY
PALB 5033

3. Flow of presentation:
• Introduction
• Proteasome core particle
• Structure and assembly
• Catalytic activity
• Gating
• ATP-dependent proteasome activation
• ATP-independent proteasomal activation
• Contributions of proteasomes to bacterial physiology
• Conclusion

4. Introduction
Proteolysis is a fundamental process in all cells,
playing essential roles in posttranslational regulation,
responses to environmental changes and intracellular stress, and
removal of unfolded proteins.

5. In Eukaryotes, a small protein called ubiquitin (Ub) tags proteins
for ATP-dependent proteasomal degradation
Three classes of enzymes carry out ubiquitylation:
1. E1 - Ub-activating enzyme (E1) uses ATP to adenylate the C-
terminal glycine (Gly) of Ub, priming it for attack by a cysteine
(Cys) in the E1 enzyme to form a Ub-E1 thioester bond.

6. 2. E2 - Ub is then transferred to a Ub-conjugating enzyme (E2).
3. E3 - Ub ligase (E3) catalyzes the formation of an isopeptide
bond between the Ub C terminus and a substrate lysine (Lys)
residue.

7. Proteasomal genes present in archaea, Actinomycetales and
Nitrospirales.
Ex: Mycobacterium tuberculosis, Streptomyces coelicolor.
 Proteasomes perform critical physiological functions in all
proteasome-bearing bacteria characterized to date, they are not
absolutely essential as in eukaryotes.

8. The proteasome, a proteolytic machine present in all three domains
of life, is a barrel-shaped protease complex that performs both
ATP-dependent and ATP - independent proteolysis
Bacterial Proteasome

9. In Eukaryotes, 19S regulatory particle (RP), binds to both ends
of the 20S CP to form the 26S proteasome .
19S RP have the base and the lid, each composed of several
unique polypeptide subunits.
The lid - removal of ubiquitin and recognition of proteins.
The base – unfolds proteins and AAA ATPases (ATPases
associated with diverse cellular activities)

10. Modular Architecture of ATP Dependent Cytosolic
Proteases Found in the Three Domains of Life

11. Pupylation
Bacteria employ a mechanism similar to ubiquitylation,
called pupylation.
pupylation has several roles in bacterial physiology
Ex: The degradation of pupylated substrates is required for the
full virulence of M. tuberculosis.

12. 20S Core Particles are 28-subunit
complexes consist of four stacked rings.
Two identical outer rings, each constituting
a heptamer of α-subunits.
Two identical inner rings, each a heptamer
of β-subunits.
20S PROTEASOME CORE PARTICLES
Structure and Assembly
α-subunits - entry point for substrate delivery
β-subunits - active site of protease

13. Bacterial 20S CP similar to eukaryotic and archaeal complexes.
The core protease is composed of four heptameric rings, which
stack on one another to form a barrel of ∼170A° length and
∼115A°diameter.
Each outer α ring is composed of seven identical PrcA subunits,
and each inner β ring is composed of seven identical PrcB
subunits to give an overall organization of α7β7β7α7.

14. Bacterial Proteasome
α7β7β7α7

15.  Pore size of 23A° in the α ring (13A°opening in
archaea), that runs through the center of the entire
complex.
Connects three large cavities, one at each α-ring/β-ring
interface and one between the two β rings that contain the
proteolytic active sites.

16. The β - subunits belong to the amino (N)-terminal nucleophilic
hydrolase family of proteins.
Eukaryotic 20S CPs are formed from 14 unique gene products,
while archaea and proteasome-bearing bacteria encode only a
single type each of the α - and β-subunits.

17. Rhodococcus erythropolis (strain
NI86/21): encodes two functionally
interchangeable variants of each
subunit.
Two proteasome populations -
α1, β1 and α2, β2

18. The genes encoding 20S CP subunits and proteasomal cofactors
are found in operons on bacterial chromosomes.
The bacterial 20S CP is composed of two subunits, α and
β, encoded by proteasome core A ( prcA) and proteasome core B
( prcB) and are in an operon with pup (prokaryotic ubiquitin
like protein).
Core Particle Genes and Structure

19. Several other genes are also found near this locus,
dop (deamidase of Pup),
pafA (proteasome accessory factor A),
 mpa (mycobacterial proteasome ATPase),
pafB and pafC are cotranscribed with pafA but do not appear to
contribute to proteasome function .
Rhodococcus species have two 20S CP operons, each
encoding a distinct set of prcBA genes; the biological
significance of this remains unknown.

21. Assembly
In eukaryotes, this is accomplished with the aid of proteasomal
chaperones bind to 20S CP and facilitate their assembly
Bacterial CPs, are capable of self-assembly.
Coproduction of Rhodococcus PrcA and PrcB in E. coli
results in the formation of active 20S CPs, without the need for
Rhodococcus-specific factors.

22. First half-proteasome is formed which consists of an α ring and
a β ring, that forms spontaneously on coproduction of PrcA and
PrcB.
Two half-proteasomes come together to form a
preholoproteasome to become proteolytically active.
Amino (N)-terminal propeptides in PrcB are removed and
leaving N-terminal threonines (Thr1) that act as the catalytic
nucleophiles of the mature holoproteasome

23. Catalytic Activities
The active sites of bacterial 20S CPs are similar to archaea and
eukaryotes.
The hydroxyl group of PrcB Thr1 is the nucleophile, responsible
for the proteolytic activity of the proteasome.
The amino group of PrcB Thr1 (Thr1N) acts as a proton acceptor
that allows the side chain oxygen (Thr1γO) to attack an
electrophilic center on a substrate.

24. Aspartate 17 (Asp17) forms a salt bridge to a lysine (Lys33), and
the Lys33 side chain amino group is protonated and forms a
hydrogen bond to Thr1γO.
Thr1γO, Thr1N, Lys33, and Asp17 form a catalytic tetrad to
promote nucleophilic attack by Thr1γO.

26. In 20 CPs, a binding pocket formed by the β rings , provides
substrate specificity.
 Eukaryotic 20 CPs have the three active β-subunits each have a
unique specificity for hydrophobic, basic or acidic sequences.
 Bacteria lack β – subunit diversity, carry a single allele each for
the α- and β-subunits.
Rhodococcus and Thermoplasma – hydrophobic
M. tuberculosis - have both hydrophobic and hydrophilic surfaces.

27. Gating
α-subunits of 20S CPs have an N-terminal extensions to
prevents substrates, including most peptides, from freely entering
the central protease chamber.
In M. tuberculosis PrcA, consists of several hydrophobic
residues, and the N termini of PrcA monomers stack atop one
another to occlude the CP entrance.

28. In bacteria, they perform three essential functions:
 specific recognition of substrates,
 ATPase-driven unfolding, and
 engagement with 20S CPs to induce gate opening.
ATP-DEPENDENT PROTEASOME ACTIVATION
Proteasomal ATPases

29. The two best characterized bacterial ATPase activators
M. tuberculosis Mpa (mycobacterial proteasome ATPase),
R. erythropolis ARC (AAA ATPase forming ringshaped
complexes).
Mpa and ARC form homohexameric rings, highly similar to
the previously characterized archaeal proteasome activator PAN
(proteasome-activating nucleotidase) .

30. Mpa and ARC consist three domains in an hexamer - form a
channel: an N-terminal coiled-coil domain, an interdomain, and
an ATPase domain.

31. Coiled-coil domain is a conserved feature of proteasomal
ATPases and is absent from non proteasomal protease activators.
CC domain for substrate recognition.
Crystal structure of an Mpa cc domain

32. X-ray crystal structure CC
and OB domains of Mpa
The interdomain of Mpa and ARC have two
oligosaccharide/oligonucleotide binding- fold (OB) sub domains.
The only known function of the interdomain is to promote
hexamer assembly and stability.

33. Mpa and ARC are representative of AAA ATPases (ATPases
Associated with various cellular Activities).
Role of AAA ATPases in protein degradation performed on
ATPases of non proteasomal bacterial proteases (ClpP, HslV
ClpQ)

34. Both nonproteasomal and proteasomal activators have mutually
beneficial and carry out similar mechanisms of protein unfolding and
translocation.
AAA ATPase (ATPase Associated with various Activities) has a
conserved aromatic-hydrophobic-glycine (Ar-ψ) motif (pore loop).

35. PAN (archaea) and several eukaryotic activators also have C-
terminal extensions to open proteasome gates for proteolysis.
The C-terminal have three amino acids - hydrophobic amino acid,
tyrosine, and any amino acid (HbYX motif) .
Mpa/ARC hexamers have C-terminal extensions, ending
with the sequence glycine-glutamine-tyrosine-leucine (GQYL).
By sequence and location, the GQYL motif is similar to the
HbYX motif.

36. Pupylation Targets Proteins for ATP-Dependent Degradation
Prokaryotic ubiquitin-like protein (Pup) interacts with
Mpa
Pup covalently links to several known proteasome substrates
in mycobacteria, and this “pupylation” is essential for their
degradation.
(Pearce et al., 2008)

37. Two enzymes, Dop (deamidase of Pup) and PafA (proteasome
accessory factor A), are required to carry pupylation.
Dop catalyzes the ATP-dependent deamidation
PafA phosphorylates the C-terminal Glu (nucleophilic attack)

38. Dop and PafA are related to glutamine synthetase (GS) family
of proteins and carry condensation reactions between Glu and
amino groups.
In contrast, Dop performs deamidation of Pup without ATP
hydrolysis, rather using ATP as a cofactor.

39. Pup Interacts with Mpa/ARC To Target Substrates for
Proteolysis
In mycobacteria, substrates are targeted to the proteasome through
the interaction of Pup with the N-terminal coiled-coil domains of
Mpa.

40. Dop Is a Depupylase
 Dop might have a function beyond deamidation of Pup.
The mechanisms of deamidation and depupylation by Dop are
similar; both processes involve the hydrolysis of an amide bond at
the C terminus of Pup

41. Depupylation :
Pup can be removed from pupylated proteins,
Partial purification of pupylated proteins (the pupylome)
from Mycobacterium tuberculosis results in the rapid depupylation
of these proteins in the presence of ATP.
This led to the discovery that Dop has a second
function as a depupylase. Dop can remove Pup from a pupylated
protein, yielding Pup-Glu.

43. Mpa/Pup-Dependent Proteolysis
Regulated proteolysis requires that protease substrates be
distinguished from non substrates.
In mycobacteria, this is accomplished through an
interaction between Pup and Mpa. The N-terminal coiled coils of
an Mpa hexamer serve as a template for the C-terminal half of Pup
to fold into a helix.

44. Pupylation of PanB with an N-terminally truncated Pup
variant (Pup) produces Pup-PanB that is capable of binding to
Mpa but unable to be degraded by the proteasome in vitro.
Similarly, production of an N-terminally truncated Pup in
Mycobacterium smegmatis results in the modification of proteins
with a truncated Pup that cannot facilitate protein degradation.

45. ATP-INDEPENDENT PROTEASOME ACTIVATORS
Several phenotypes have 20S CP had Mpa/Pup-independent
functions in mycobacteria.
PafE interacted with 20S CP of Mycobacterium tuberculosis.
PafE is functional homolog of the eukaryotic 11S ATP-
independent proteasome activators.

46. It shares no sequence homology with its eukaryotic
counterparts.
 PafE forms a ringed oligomer, binds 20S CPs by using its C
terminus, and enhances proteasomal peptidase activity.
PafE stimulates the ATP-independent degradation of HspR, to
avoid sensitivity to heat stress.

47. Furthermore, PafE has C-terminal residues identical to those
of Mpa (GQYL).
Gly, Tyr, and Leu, but not Gln, are required for PafE and Mpa
function and thus constitute an essential motif for proteasomal
activation.

48. PROTEASOMES AND BACTERIAL PHYSIOLOGY
Nitric Oxide Resistance of Mycobacterium tuberculosis
Mycobacterium tuberculosis primarily resides in macrophages.
Macrophages produce NO, which can form a variety of reactive
nitrogen intermediates leading to the damage of proteins, nucleic
acids, and lipids.
 Nitric oxide synthase (iNOS), catalyzes the production of NO in
macrophages to give resistance against M.tuberculosis.

49. PafA and Mpa as essential for NO resistance in
Mycobacterium tuberculosis
M. tuberculosis mutants that lack the mycobacterial
proteasome ATPase (Mpa) or the Pup ligase PafA are susceptible
to RNI (Reactive Nitrogen intermediates)

50. Copper Resistance of Mycobacterium tuberculosis
Microarray analysis identified a novel copper-responsive regulon
that is repressed in both mpa and pafA mutants when compared with
wild-type Mycobacterium tuberculosis.
Metallothioneins are small, usually cysteine-rich proteins that bind
metals to protect against toxicity.

51. Survival Under Nutrient-Limiting Conditions of M. smegmatis
Mycobacterium smegmatis pup prcBA mutant has a survival defect
under nitrogen starvation conditions, which suggests that the
proteasome is required for amino acid recycling.

52. Proteotoxic Stress Response

53. • ATP-independent proteasomal
degradation was established with
M. tuberculosis PafE-proteasome
substrate HspR
• HspR is a repressor of several
genes - DnaK (Hsp70), ClpB, and
Hsp (Acr2)

54. Conclusion
The physiological role of ATP-independent protein degradation in
eukaryotes has remained elusive.
The study of proteasome-dependent degradation has also revealed
insights into the challenges faced by bacterial pathogens, such as M.
tuberculosis, during infections.
Finally, much remains to be determined about the biological roles
of pupylation, with or without proteasomes.