Targeting endogenous proteins for degradation through the affinity-directed protein missile system

Targeted proteolysis of endogenous proteins is desirable as a research toolkit and in therapeutics. CRISPR/Cas9-mediated gene knockouts are irreversible and often not feasible for many genes. Similarly, RNA interference approaches necessitate prolonged treatments, can lead to incomplete knockdowns and are often associated with off-target effects. Targeted proteolysis can overcome these limitations. In this report, we describe an affinity-directed protein missile (AdPROM) system that harbours the von Hippel–Lindau (VHL) protein, the substrate receptor of the Cullin2 (CUL2) E3 ligase complex, tethered to polypeptide binders that selectively bind and recruit endogenous target proteins to the CUL2-E3 ligase complex for ubiquitination and proteasomal degradation. By using synthetic monobodies that selectively bind the protein tyrosine phosphatase SHP2 and a camelid-derived VHH nanobody that selectively binds the human ASC protein, we demonstrate highly efficient AdPROM-mediated degradation of endogenous SHP2 and ASC in human cell lines. We show that AdPROM-mediated loss of SHP2 in cells impacts SHP2 biology. This study demonstrates for the first time that small polypeptide binders that selectively recognize endogenous target proteins can be exploited for AdPROM-mediated destruction of the target proteins.</jats:p

Continuous subcutaneous insulin infusion alters microRNA expression and glycaemic variability in children with type 1 diabetes

To determine whether continuous subcutaneous insulin infusion (CSII) vs. multiple daily injections (MDI) therapy from near-diagnosis of type 1 diabetes is associated with reduced glycaemic variability (GV) and altered microRNA (miRNAs) expression. Adolescents (74% male) within 3-months of diabetes diagnosis (n = 27) were randomized to CSII (n = 12) or MDI. HbA1c, 1-5-Anhydroglucitol (1,5-AG), high sensitivity C-peptide and a custom TaqMan qPCR panel of 52 miRNAs were measured at baseline and follow-up (median (LQ-UQ); 535 (519-563) days). There were no significant differences between groups in baseline or follow-up HbA1c or C-peptide, nor baseline miRNAs. Mean +/- SD 1,5-AG improved with CSII vs. MDI (3.1 +/- 4.1 vs. - 2.2 +/- - 7.0 mg/ml respectively, P = 0.029). On follow-up 11 miRNAs associated with diabetes vascular complications had altered expression in CSII-users. Early CSII vs. MDI use is associated with lower GV and less adverse vascular-related miRNAs. Relationships with future complications are of interest

The Hyperfine Spin Splittings In Heavy Quarkonia

The hyperfine spin splittings in heavy quarkonia are studied using the recently developed renormalization group improved spin-spin potential which is independent of the scale parameter $\mu$. The calculated energy difference between the $J/\psi$ and the $\eta_c$ fits the experimental data well, while the predicted energy difference $\Delta M_p$ between the center of the gravity of $1^3P_{0,1,2}$ states and the $1^1P_1$ state of charmonium has the correct sign but is somewhat larger than the experimental data. This is not surprising since there are several other contributions to $\Delta M_p$, which we discuss, that are of comparable size ($\sim 1$ MeV) that should be included, before precise agreement with the data can be expected. The mass differences of the $\psi'-\eta_c'$, $\Upsilon(1S)-\eta_b$, $\Upsilon(2S)-\eta_b'$, and $B_c^*-B_c$ are also predicted.Comment: 17 page

Complete O(v^2) corrections to the static interquark potential from SU(3) gauge theory

For the first time, we determine the complete spin- and momentum-dependent order v^2 corrections to the static interquark potential from simulations of QCD in the valence quark approximation at inverse lattice spacings of 2-3 GeV. A new flavor dependent correction to the central potential is found. We report a 1/r^2 contribution to the long range spin-orbit potential V_1'. The other spin-dependent potentials turn out to be short ranged and can be well understood by means of perturbation theory. The momentum-dependent potentials qualitatively agree with minimal area law expectations. In view of spectrum calculations, we discuss the matching of the effective nonrelativistic theory to QCD as well as renormalization of lattice results. In a first survey of the resulting bottomonia and charmonia spectra we reproduce the experimental levels within average errors of 12.5 MeV and 22 MeV, respectively.Comment: 54 pages REVTeX with 24 encapsuled ps figure

Electronic supplementary figures and figure legends from Targeting endogenous proteins for degradation through the affinity-directed protein missile system

Targeted proteolysis of endogenous proteins is desirable as a research toolkit and in therapeutics. CRISPR/Cas9-mediated gene knockouts are irreversible and often not feasible for many genes. Similarly, RNA interference approaches necessitate prolonged treatments, can lead to incomplete knockdowns, and are often associated with off-target effects. Targeted proteolysis can overcome these limitations. In this report, we describe an Affinity-directed PROtein Missile (AdPROM) system that harbours the von Hippel–Lindau (VHL) protein, the substrate receptor of the Cullin2 (CUL2) E3 ligase complex, tethered to polypeptide binders that selectively bind and recruit endogenous target proteins to the CUL2-E3 ligase complex for ubiquitination and proteasomal degradation. By using synthetic monobodies that selectively bind the protein tyrosine phosphatase SHP2 and a camelid-derived VHH nanobody that selectively binds the human ASC protein, we demonstrate highly efficient AdPROM-mediated degradation of endogenous SHP2 and ASC in human cell lines. We show that AdPROM-mediated loss of SHP2 in cells impacts SHP2 biology. This study demonstrates for the first time that small polypeptide binders that selectively recognize endogenous target proteins can be exploited for AdPROM-mediated destruction of the target proteins