Permissive Conformations of a Transmembrane Helix Allow Intramembrane Proteolysis by γ-Secretase

Ortner, Martin; Guschtschin-Schmidt, Nadja; Stelzer, Walter; Muhle‐Goll, Claudia; Langosch, Dieter

doi:10.1016/j.jmb.2023.168218

Cited by 4 publications

(19 citation statements)

References 67 publications

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“…A mutational screen of positions G1753, V1754, and especially S1757 revealed that exchanges against bulky and/or large hydrophobic residues were detrimental to cleavage. 28 But again, other substrate TMDs indicate that a destabilized cleavage site is not mandatory, as the TMDs of APP, APLP2, and that of ErbB4 were highly stable within their respective cleavage site region (compare Figures 1C, 2B, 4B, and S8). Yet, the neighboring basic lysines clearly ended the stable helical part.…”

Section: ■ Discussionmentioning

confidence: 92%

“…One should note that Notch1 TMD does not contain a double glycine motif, but a stretch of four consecutive alanines (Scheme 1) could allow for some flexibility that was proven to occur at least in a TFE/water environment. 70 Ortner et al and our lab recently compared the structure of Notch1 TMD with two mutants that addressed the role of the central four alanines 28 (Scheme 1). Stabilizing the Notch1 TMD helix by exchanging the four alanines with leucines (Notch1 L1740−1743) diminished cleavage severely, whereas destabilizing it by replacing them with glycines (Notch1 G1740−1743) led to the opposite effect.…”

Section: ■ Notch1mentioning

confidence: 99%

“…Contrary to APP TMD where the initial ε-cleavage site was among the most stable parts of TMD-C, the S3 cleavage site showed signs of destabilization in the H/D exchange starting with G1751 with respect to the central part of the TMD. 28 C1752 and G1753 in the WT exchanged in H/D experiments too fast to determine a k cat value for these two residues. This correlates with helix unravelling in the cryo-EM structure 23 and was already observed in the bicelle Notch1 structure, where V1754 had increased 15 N relaxations rates.…”

Section: ■ Notch1mentioning

confidence: 99%

“…To get an impression of whether the different conformational repertoires might add to our understanding of the different PSEN cleavage efficiencies, we again superimposed the structural bundles of Notch1 and its mutants onto the cryo-EM structure of γ-secretase in complex with Notch1 TMD 28 (Figure 3C−F). The Notch1 TMD bundle fitted reasonably well into the space between the TMDs 6, 7, and 9 of PSEN (12/40 colliding structures).…”

Section: ■ Notch1mentioning

confidence: 99%

“…A proline at the beginning of the TMD renders the TMD of Notch3 one helical turn shorter than that of Notch1 based on chemical shift analysis. 28 The alanine tetrad is replaced by an AGAV motif in Notch3 with slightly reduced helicity and stability (Figure 3A,B), but the structural bundle is not markedly bent. The overall stability of Notch3 TM-N is comparable to that of Notch1, whereas the residues preceding the S3 cleavage site were more stable, with a significantly lower H/D exchange rate.…”

Section: ■ Notch1mentioning

confidence: 99%

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Substrate Selection Criteria in Regulated Intramembrane Proteolysis

Moser,

Guschtschin-Schmidt,

Silber

et al. 2024

ACS Chem. Neurosci.

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Alzheimer's disease is the most common form of dementia encountered in an aging population. Characteristic amyloid deposits of Aβ peptides in the brain are generated through cleavage of amyloid precursor protein (APP) by γ-secretase, an intramembrane protease. Cryo-EM structures of substrate γ-secretase complexes revealed details of the process, but how substrates are recognized and enter the catalytic site is still largely ignored. γ-Secretase cleaves a diverse range of substrate sequences without a common consensus sequence, but strikingly, single point mutations within the transmembrane domain (TMD) of specific substrates may greatly affect cleavage efficiencies. Previously, conformational flexibility was hypothesized to be the main criterion for substrate selection. Here we review the 3D structure and dynamics of several γ-secretase substrate TMDs and compare them with mutants shown to affect the cleavage efficiency. In addition, we present structural and dynamic data on ITGB1, a known nonsubstrate of γsecretase. A comparison of biophysical details between these TMDs and changes generated by introducing crucial mutations allowed us to unravel common principles that differ between substrates and nonsubstrates. We identified three motifs in the investigated substrates: a highly flexible transmembrane domain, a destabilization of the cleavage region, and a basic signature at the end of the transmembrane helix. None of these appears to be exclusive. While conformational flexibility on its own may increase cleavage efficiency in well-known substrates like APP or Notch1, our data suggest that the three motifs seem to be rather variably combined to determine whether a transmembrane helix is efficiently recognized as a γ-secretase substrate.

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Section: ■ Discussionmentioning

confidence: 92%

Section: ■ Notch1mentioning

confidence: 99%

Section: ■ Notch1mentioning

confidence: 99%

Section: ■ Notch1mentioning

confidence: 99%

Section: ■ Notch1mentioning

confidence: 99%

See 3 more Smart Citations

Substrate Selection Criteria in Regulated Intramembrane Proteolysis

Moser,

Guschtschin-Schmidt,

Silber

et al. 2024

ACS Chem. Neurosci.

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C-terminal amino acids in the type I transmembrane domain of L-type lectin VIP36 affect γ-secretase susceptibility

Hua,

Watanabe,

Fukunaga

et al. 2024

Biochemical and Biophysical Research Communications

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Cleavage efficiency of the intramembrane protease γ‐secretase is reduced by the palmitoylation of a substrate's transmembrane domain

Aßfalg,

Güner,

Müller

et al. 2024

The FASEB Journal

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The intramembrane protease γ‐secretase has broad physiological functions, but also contributes to Notch‐dependent tumors and Alzheimer's disease. While γ‐secretase cleaves numerous membrane proteins, only few nonsubstrates are known. Thus, a fundamental open question is how γ‐secretase distinguishes substrates from nonsubstrates and whether sequence‐based features or post‐translational modifications of membrane proteins contribute to substrate recognition. Using mass spectrometry‐based proteomics, we identified several type I membrane proteins with short ectodomains that were inefficiently or not cleaved by γ‐secretase, including ‘pituitary tumor‐transforming gene 1‐interacting protein’ (PTTG1IP). To analyze the mechanism preventing cleavage of these putative nonsubstrates, we used the validated substrate FN14 as a backbone and replaced its transmembrane domain (TMD), where γ‐cleavage occurs, with the one of nonsubstrates. Surprisingly, some nonsubstrate TMDs were efficiently cleaved in the FN14 backbone, demonstrating that a cleavable TMD is necessary, but not sufficient for cleavage by γ‐secretase. Cleavage efficiencies varied by up to 200‐fold. Other TMDs, including that of PTTG1IP, were still barely cleaved within the FN14 backbone. Pharmacological and mutational experiments revealed that the PTTG1IP TMD is palmitoylated, which prevented cleavage by γ‐secretase. We conclude that the TMD sequence of a membrane protein and its palmitoylation can be key factors determining substrate recognition and cleavage efficiency by γ‐secretase.

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Permissive Conformations of a Transmembrane Helix Allow Intramembrane Proteolysis by γ-Secretase

Cited by 4 publications

References 67 publications

Substrate Selection Criteria in Regulated Intramembrane Proteolysis

Substrate Selection Criteria in Regulated Intramembrane Proteolysis

C-terminal amino acids in the type I transmembrane domain of L-type lectin VIP36 affect γ-secretase susceptibility

Cleavage efficiency of the intramembrane protease γ‐secretase is reduced by the palmitoylation of a substrate's transmembrane domain

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