SMD1

Amino Acid Sequence

3D Structure (/structures/AF-O60885-F1-model_v6.cif)

Function

Chromatin reader protein that recognizes and binds acetylated histones and plays a key role in transmission of epigenetic memory across cell divisions and transcription regulation. Remains associated with acetylated chromatin throughout the entire cell cycle and provides epigenetic memory for postmitotic G1 gene transcription by preserving acetylated chromatin status and maintaining high-order chromatin structure. During interphase, plays a key role in regulating the transcription of signal-inducible genes by associating with the P-TEFb complex and recruiting it to promoters. Also recruits P-TEFb complex to distal enhancers, so called anti-pause enhancers in collaboration with JMJD6. BRD4 and JMJD6 are required to form the transcriptionally active P-TEFb complex by displacing negative regulators such as HEXIM1 and 7SKsnRNA complex from P-TEFb, thereby transforming it into an active form that can then phosphorylate the C-terminal domain (CTD) of RNA polymerase II. Regulates differentiation of naive CD4 T-cells into T-helper Th17 by promoting recruitment of P-TEFb to promoters (By similarity). Promotes phosphorylation of 'Ser-2' of the C-terminal domain (CTD) of RNA polymerase II. According to a report, directly acts as an atypical protein kinase and mediates phosphorylation of 'Ser-2' of the C-terminal domain (CTD) of RNA polymerase II; these data however need additional evidences in vivo. In addition to acetylated histones, also recognizes and binds acetylated RELA, leading to further recruitment of the P-TEFb complex and subsequent activation of NF-kappa-B. Also acts as a regulator of p53/TP53-mediated transcription: following phosphorylation by CK2, recruited to p53/TP53 specific target promoters.

Mechanism

The ubiquitin-proteasome pathway (UPP) constitutes the principal pathway for protein degradation in eukaryotic cells, mediating the breakdown of target proteins through ubiquitin tagging and subsequent proteasomal degradation. In an ATP-dependent manner, the ubiquitin-activating enzyme (E1) activates ubiquitin (Ub) and conjugates it to a ubiquitin-conjugating enzyme (E2). E3 ligase subsequently catalyzes the transfer of Ub to the target protein, leading to the formation of a polyubiquitin chain that completes the ubiquitination mark. The 19S regulatory particle of the 26S proteasome recognizes this ubiquitination signal and, after deubiquitination, mediates the ATP-dependent delivery of the substrate into the 20S proteasome for degradation.

Description

Real-time quantitative polymerase chain reaction (RTqPCR) analysis showed that SMD1 did not alter BRD4 mRNA levels in MV4-11 cells (Figure 5a), validating a posttranscriptional effect. The reduction in BRD4 protein levels by SMD1 was partially blocked by either a proteasome inhibitor (carfilzomib, CFZ) or a lysosome inhibitor (bafilomycin A1, BafA1) and was totally suppressed by their cotreatment (Figure 5b and Supporting Information Figure S21). These results thus demonstrate that misfolded proteins mimicked via LACHT are removed both by the proteasome and the lysosome.41 The degradation processes depend on ubiquitination that is a common factor directing misfolded proteins to the proteasome and the lysosome for maintaining protein homeostasis,42,43 as His-ubiquitin (His-Ub) pulldown assay revealed that SMD1 induced the polyubiquitination of BRD4(Figure 5c). Also supportive of this conclusion, PYR-41, a ubiquitin-activating enzyme E1 inhibitor, completely reversed SMD1-caused BRD4 loss (Figure 5d). Collectively, the TPD mechanism of the LACHT approach can be explained as follows (Figure 5e): BRD4 was first covalently labeled with the hydrophobic Ad by SMD1, which then underwent polyubiquitination before removal by the proteasome and the lysosome.

Mechanism

The autophagy-lysosomal pathway (ALP) is capable of degrading a broader range of substrates, including longer-lived and larger misfolded proteins and damaged organelles. ALP is classified into three subtypes: microautophagy, chaperone-mediated autophagy (CMA), and macroautophagy. In microautophagy, substrates are directly sequestered and degraded by lysosomes and late endosomes via membrane protrusion and invagination. In CMA, HSPA8/HSC70 recognizes specific substrates containing KFERQ-like sequences; these substrates subsequently bind to LAMP2A on the lysosomal surface and are delivered into lysosomes for degradation. In macroautophagy, substrates are encapsulated through the formation of double-membrane autophagosomes, which subsequently fuse with lysosomes to degrade the substrates.

Description

Real-time quantitative polymerase chain reaction (RTqPCR) analysis showed that SMD1 did not alter BRD4 mRNA levels in MV4-11 cells (Figure 5a), validating a posttranscriptional effect. The reduction in BRD4 protein levels by SMD1 was partially blocked by either a proteasome inhibitor (carfilzomib, CFZ) or a lysosome inhibitor (bafilomycin A1, BafA1) and was totally suppressed by their cotreatment (Figure 5b and Supporting Information Figure S21). These results thus demonstrate that misfolded proteins mimicked via LACHT are removed both by the proteasome and the lysosome.41 The degradation processes depend on ubiquitination that is a common factor directing misfolded proteins to the proteasome and the lysosome for maintaining protein homeostasis,42,43 as His-ubiquitin (His-Ub) pulldown assay revealed that SMD1 induced the polyubiquitination of BRD4(Figure 5c). Also supportive of this conclusion, PYR-41, a ubiquitin-activating enzyme E1 inhibitor, completely reversed SMD1-caused BRD4 loss (Figure 5d). Collectively, the TPD mechanism of the LACHT approach can be explained as follows (Figure 5e): BRD4 was first covalently labeled with the hydrophobic Ad by SMD1, which then underwent polyubiquitination before removal by the proteasome and the lysosome.

Description

The half degradation concentration (DC50) of SMD1 was determined to be 3 μM (Figure 4b).

Description

A time-dependent decrease of BRD4 protein levels was observed after treatment of MV4-11 cells with 10 μM SMD1, affording a maximum degradation (Dmax) rate of 98% at 12 h (Figure 4a).