The endosomal escape vehicle platform enhances delivery of oligonucleotides in preclinical models of neuromuscular disorders.

Biological therapeutic agents are highly targeted and potent but limited in their ability to reach intracellular targets. These limitations often necessitate high therapeutic doses and can be associated with less-than-optimal therapeutic activity. One promising solution for therapeutic agent delivery is use of cell-penetrating peptides.

Mitochondrial Permeability Uncouples Elevated Autophagy and Lifespan Extension.

Autophagy is required in diverse paradigms of lifespan extension, leading to the prevailing notion that autophagy is beneficial for longevity. However, why autophagy is harmful in certain contexts remains unexplained. Here, we show that mitochondrial permeability defines the impact of autophagy on aging.

Crystal structure of MICU2 and comparison with MICU1 reveal insights into the uniporter gating mechanism.

The mitochondrial uniporter is a Ca-channel complex resident within the organelle's inner membrane. In mammalian cells the uniporter's activity is regulated by Ca due to concerted action of MICU1 and MICU2, two paralogous, but functionally distinct, EF-hand Ca-binding proteins. Here we present the X-ray structure of the apo form of MICU2 at 2.

MICU1 imparts the mitochondrial uniporter with the ability to discriminate between Ca and Mn.

The mitochondrial uniporter is a Ca-activated Ca channel complex that displays exceptionally high conductance and selectivity. Here, we report cellular metal toxicity screens highlighting the uniporter's role in Mn toxicity. Cells lacking the pore-forming uniporter subunit, MCU, are more resistant to Mn toxicity, while cells lacking the Ca-sensing inhibitory subunit, MICU1, are more sensitive than the wild type.

Cardiovascular homeostasis dependence on MICU2, a regulatory subunit of the mitochondrial calcium uniporter.

Comparative analyses of transcriptional profiles from humans and mice with cardiovascular pathologies revealed consistently elevated expression of , a regulatory subunit of the mitochondrial calcium uniporter complex. To determine if expression was cardioprotective, we produced and characterized mice. Mutant mice had left atrial enlargement and cardiomyocytes had delayed sarcomere relaxation and cytosolic calcium reuptake kinetics, indicating diastolic dysfunction.

High-affinity cooperative Ca binding by MICU1-MICU2 serves as an on-off switch for the uniporter.

The mitochondrial calcium uniporter is a Ca-activated Ca channel that is essential for dynamic modulation of mitochondrial function in response to cellular Ca signals. It is regulated by two paralogous EF-hand proteins-MICU1 and MICU2, but the mechanism is unknown. Here, we demonstrate that both MICU1 and MICU2 are stabilized by Ca We reconstitute the MICU1-MICU2 heterodimer and demonstrate that it binds Ca cooperatively with high affinity.

Comparative Analysis of Mitochondrial N-Termini from Mouse, Human, and Yeast.

The majority of mitochondrial proteins are encoded in the nuclear genome, translated in the cytoplasm, and directed to the mitochondria by an N-terminal presequence that is cleaved upon import. Recently, N-proteome catalogs have been generated for mitochondria from yeast and from human U937 cells. Here, we applied the subtiligase method to determine N-termini for 327 proteins in mitochondria isolated from mouse liver and kidney.

Homozygous deletion in MICU1 presenting with fatigue and lethargy in childhood.

Objective: To define the mechanism responsible for fatigue, lethargy, and weakness in 2 cousins who had a normal muscle biopsy.

Methods: Exome sequencing, long-range PCR, and Sanger sequencing to identify the pathogenic mutation. Functional analysis in the patient fibroblasts included oxygen consumption measurements, extracellular acidification studies, Western blotting, and calcium imaging, followed by overexpression of the wild-type protein.

The molecular era of the mitochondrial calcium uniporter.

The mitochondrial calcium uniporter is an evolutionarily conserved calcium channel, and its biophysical properties and relevance to cell death, bioenergetics and signalling have been investigated for decades. However, the genes encoding this channel have only recently been discovered, opening up a new 'molecular era' in the study of its biology. We now know that the uniporter is not a single protein but rather a macromolecular complex consisting of pore-forming and regulatory subunits.

4-ketoproline: An electrophilic proline analog for bioconjugation.

Installing an electrophilic amino-acid residue can engender a peptide or protein with chemoselective reactivity. Such a modification to collagen, which is the most abundant protein in animals, could facilitate the development of new biomaterials. Collagen has an abundance of proline-like residues.

Directed evolution of APEX2 for electron microscopy and proximity labeling.

APEX is an engineered peroxidase that functions as an electron microscopy tag and a promiscuous labeling enzyme for live-cell proteomics. Because limited sensitivity precludes applications requiring low APEX expression, we used yeast-display evolution to improve its catalytic efficiency. APEX2 is far more active in cells, enabling the use of electron microscopy to resolve the submitochondrial localization of calcium uptake regulatory protein MICU1.

Reconstitution of the mitochondrial calcium uniporter in yeast.

The mitochondrial calcium uniporter is a highly selective calcium channel distributed broadly across eukaryotes but absent in the yeast Saccharomyces cerevisiae. The molecular components of the human uniporter holocomplex (uniplex) have been identified recently. The uniplex consists of three membrane-spanning subunits--mitochondrial calcium uniporter (MCU), its paralog MCUb, and essential MCU regulator (EMRE)--and two soluble regulatory components--MICU1 and its paralog MICU2.

MICU1 and MICU2 play nonredundant roles in the regulation of the mitochondrial calcium uniporter.

The mitochondrial uniporter is a selective Ca(2+) channel regulated by MICU1, an EF hand-containing protein in the organelle's intermembrane space. MICU1 physically associates with and is co-expressed with a paralog, MICU2. To clarify the function of MICU1 and its relationship to MICU2, we used gene knockout (KO) technology.

EMRE is an essential component of the mitochondrial calcium uniporter complex.

The mitochondrial uniporter is a highly selective calcium channel in the organelle's inner membrane. Its molecular components include the EF-hand-containing calcium-binding proteins mitochondrial calcium uptake 1 (MICU1) and MICU2 and the pore-forming subunit mitochondrial calcium uniporter (MCU). We sought to achieve a full molecular characterization of the uniporter holocomplex (uniplex).

An n→π* interaction reduces the electrophilicity of the acceptor carbonyl group.

Carbonyl-carbonyl (C=O···C'=O') interactions are ubiquitous in both small and large molecular systems. This interaction involves delocalization of a lone pair (n) of a donor oxygen into the antibonding orbital (π*) of an acceptor carbonyl group. Analyses of high-resolution protein structures suggest that these carbonyl-carbonyl interactions prefer to occur in pairs, that is, one donor per acceptor.

MICU1 controls both the threshold and cooperative activation of the mitochondrial Ca²⁺ uniporter.

Mitochondrial Ca(2+) uptake via the uniporter is central to cell metabolism, signaling, and survival. Recent studies identified MCU as the uniporter's likely pore and MICU1, an EF-hand protein, as its critical regulator. How this complex decodes dynamic cytoplasmic [Ca(2+)] ([Ca(2+)]c) signals, to tune out small [Ca(2+)]c increases yet permit pulse transmission, remains unknown.

MICU2, a paralog of MICU1, resides within the mitochondrial uniporter complex to regulate calcium handling.

Mitochondrial calcium uptake is present in nearly all vertebrate tissues and is believed to be critical in shaping calcium signaling, regulating ATP synthesis and controlling cell death. Calcium uptake occurs through a channel called the uniporter that resides in the inner mitochondrial membrane. Recently, we used comparative genomics to identify MICU1 and MCU as the key regulatory and putative pore-forming subunits of this channel, respectively.

Intimate interactions with carbonyl groups: dipole-dipole or n→π*?

Amide carbonyl groups in proteins can engage in C═O···C═O and C-X···C═O interactions, where X is a halogen. The putative involvement of four poles suggests that these interactions are primarily dipolar. Our survey of crystal structures with a C-X···C═O contact that is short (i.

A conserved interaction with the chromophore of fluorescent proteins.

The chromophore of fluorescent proteins, including the green fluorescent protein (GFP), contains a highly conjugated imidazolidinone ring. In many fluorescent proteins, the carbonyl group of the imidazolidinone ring engages in a hydrogen bond with the side chain of an arginine residue. Prior studies have indicated that such an electrophilic carbonyl group in a protein often accepts electron density from a main-chain oxygen.

An n→π* interaction in aspirin: implications for structure and reactivity.

Stereoelectronic effects modulate molecular structure, reactivity, and conformation. We find that the interaction between the ester and carboxyl moieties of aspirin has a previously unappreciated quantum mechanical character that arises from the delocalization of an electron pair (n) of a donor group into the antibonding orbital (π*) of an acceptor group. This interaction affects the physicochemical attributes of aspirin and could have implications for its pharmacology.

Adrenocorticotropic hormone and corticosterone responses to acute hypoxia in the neonatal rat: effects of body temperature maintenance.

The corticosterone response to acute hypoxia in neonatal rats develops in the 1st wk of life, with a shift from ACTH independence to ACTH dependence. Acute hypoxia also leads to hypothermia, which may be protective. There is little information about the endocrine effects of body temperature maintenance during periods of neonatal hypoxia.

A stereoelectronic effect in prebiotic nucleotide synthesis.

A plausible route for the spontaneous synthesis of an activated ribonucleotide that is poised for polymerization has been put forth (Powner et al. (2009) Nature, 459, 239-242). A key step in this route necessitates the regioselective phosphorylation of the secondary alcohol on C(3') of an anhydroarabinonucleoside in the presence of the primary alcohol on C(5').

Origin of the stability conferred upon collagen by fluorination.

According to a prevailing theory, (2S,4R)-4-hydroxyproline (Hyp) residues stabilize the collagen triple helix via a stereoelectronic effect that preorganizes appropriate backbone torsion angles for triple-helix formation. This theory is consistent with the marked stability that results from replacing the hydroxyl group with the more electron-withdrawing fluoro group, as in (2S,4R)-4-fluoroproline (Flp). Nonetheless, the hyperstability of triple helices containing Flp has been attributed by others to the hydrophobic effect rather than a stereoelectronic effect.

Development of the ACTH and corticosterone response to acute hypoxia in the neonatal rat.

Acute episodes of severe hypoxia are among the most common stressors in neonates. An understanding of the development of the physiological response to acute hypoxia will help improve clinical interventions. The present study measured ACTH and corticosterone responses to acute, severe hypoxia (8% inspired O(2) for 4 h) in neonatal rats at postnatal days (PD) 2, 5, and 8.