The carboxy-terminus,a key regulator of protein function

All proteins end with a carboxyl terminus that has unique biophysical properties and is often disordered. Although there are examples of important C-termini functions, a more global role for the C-terminus is not yet established. In this review, we summarize research on C-termini, a unique region in proteins that cells exploit. Alternative splicing and proteolysis increase the diversity of proteins and peptides in cells with unique C-termini. The C-termini of proteins contain minimotifs, short peptides with an encoded function generally characterized as binding, posttranslational modifications, and trafficking. Many of these activities are specific to minimotifs on the C-terminus. Approximately 13% of C-termini in the human proteome have a known minimotif, and the majority, if not all of the remaining termini have conserved motifs inferring a function that remains to be discovered. C-termini, their predictions, and their functions are collated in the C-terminome, Proteus, and Terminus Oriented Protein Function INferred Database (TopFIND) database/web systems. Many C-termini are well conserved, and some have a known role in health and disease. We envision that this summary of C-termini will guide future investigation of their biochemical and physiological significance.     

.HECT类泛素连接酶对p53家族的调控作用

p53家族成员在细胞生长、组织发育及肿瘤形成等方面都具有十分重要的生物学功能,其自身受到严格调控,泛素化修饰就是其中非常重要的方式之一,作为泛素化过程中决定底物特异性的泛素连接酶E3作用则更加突出.泛素连接酶E3可以分为两类：RING（really interesting new gene）类和HECT（homologous to E6AP C-terminus）类E3近年来,HECT类E3对p53家族的调控效应不断得到揭示.本文综述了HECT类E3在调控p53家族转录活性、稳定 性方面的重要作用、分子机制以及其作用对生物体肿瘤形成和生长发育等产生的影响,为进 一步完善p53家族调控网络,揭示HECT类E3在肿瘤发生发展及防治中的作用提供参考.     

Caveolin-1 activates Rab5 and enhances endocytosis through direct interaction

Caveolin-1, a constitutive protein of the caveolae, is implicated in processes of vesicular transport during caveolae-mediated endocytosis. However, the molecular mechanisms of caveolae-mediated endocytosis are not yet clearly defined. Here, we show the physiological role of the Rab5-caveolin-1 interaction during caveolae-mediated endocytosis. Rab5 was found in caveolae-enriched fractions and Rab5 directly bound to caveolin-1. Furthermore, binding sites of Rab5 to caveolin-1 were identified in the scaffold (SD), transmembrane (TM), and C-terminus (CC) domains, and the Rab5 binding domain of caveolin-1 was required for CTXB uptake. Subsequently, we performed a GST-R5BD pull-down assay to determine whether the Rab5 binding domain of caveolin-1 is involved in Rab5 activity or not. The results showed that overexpression of the Rab5 binding domain of caveolin-1 increase the amount of Rab5-GTP in Cos-1 cells. These findings imply that caveolin-1 controls the Rab5 activity during the caveolae-mediated endocytosis.     

Viral Membrane Protein Topology Is Dictated by Multiple Determinants in Its Sequence

The targeting, insertion, and topology of membrane proteins have been extensively studied in both prokaryotes and eukaryotes. However, the mechanisms used by viral membrane proteins to generate the correct topology within cellular membranes are less well understood. Here, the effect of flanking charges and the hydrophobicity of the N-terminal hydrophobic segment on viral membrane protein topogenesis are examined systematically. Experimental data reveal that the classical topological determinants have only a minor effect on the overall topology of p9, a plant viral movement protein. Since only a few individual sequence alterations cause an inversion of p9 topology, its topological stability is robust. This result further indicates that the protein has multiple, and perhaps redundant, structural features that ensure that it always adopts the same topology. These critical topogenic sequences appear to be recognized and acted upon from the initial stages of protein biosynthesis, even before the ribosome ends protein translation.     

Studies on the regioselectivity and kinetics of the action of trypsin on proinsulin and its derivatives using mass spectrometry

Human M-proinsulin was cleaved by trypsin at the R³¹R³²–E³³ and K⁶⁴R⁶⁵–G⁶⁶ bonds (B/C and C/A junctions), showing the same cleavage specificity as exhibited by prohormone convertases 1 and 2 respectively. Buffalo/bovine M-proinsulin was also cleaved by trypsin at the K⁵⁹R⁶⁰–G⁶¹ bond but at the B/C junction cleavage occurred at the R³¹R³²–E³³ as well as the R³¹–R³²E³³ bond. Thus, the human isoform in the native state, with a 31 residue connecting C-peptide, seems to have a unique structure around the B/C and C/A junctions and cleavage at these sites is predominantly governed by the structure of the proinsulin itself. In the case of both the proinsulin species the cleavage at the B/C junction was preferred (65%) over that at the C/A junction (35%) supporting the earlier suggestion of the presence of some form of secondary structure at the C/A junction. Proinsulin and its derivatives, as natural substrates for trypsin, were used and mass spectrometric analysis showed that the k_cat./K_m values for the cleavage were most favourable for the scission of the bonds at the two junctions (1.02 ± 0.08 × 10⁵ s^− 1 M^− 1) and the cleavage of the K²⁹–T³⁰ bond of M-insulin-RR (1.3 ± 0.07 × 10⁵ s^− 1 M^− 1). However, the K²⁹–T³⁰ bond in M-insulin, insulin as well as M-proinsulin was shielded from attack by trypsin (k_cat./K_m values around 1000 s^− 1 M^− 1). Hence, as the biosynthetic path follows the sequence; proinsulin → insulin-RR → insulin, the K²⁹–T³⁰ bond becomes shielded, exposed then shielded again respectively.     

N type rapid inactivation in human Kv1.4 channels: functional role of a putative C-terminal helix

Voltage gated potassium channels are tetrameric membrane proteins, which have a central role in cellular excitability. Human Kv1.4 channels open on membrane depolarization and inactivate rapidly by a ‘ball and chain’ mechanism whose molecular determinants have been mapped to the cytoplasmic N terminus of the channel. Here we show that the other terminal end of the channel also plays a role in channel inactivation. Swapping the C-terminal residues of hKv1.4 with those from two non-inactivating channels (hKv1.1 and hKv1.2) affects the rates of inactivation, as well as the recovery of the channel from the inactivated state. Secondary structure predictions of the hKv1.4 sequence reveal a helical structure at its distal C-terminal. Complete removal or partial disruption of this helical region results in channels with remarkably slowed inactivation kinetics. The ionic selectivity and voltage-dependence of channel opening were similar to hKv1.4, indicative of an unperturbed channel pore. These results demonstrate that fast inactivation is modulated by structural elements in the C-terminus, suggesting that the process involves the concerted action of the N- and C-termini.     

基于生物质谱的蛋白质C末端研究进展

蛋白质的C末端在蛋白质进行各项生命活动过程中都起着极其重要的作用。它不仅标志着DNA转录翻译成蛋白质过程的初步完成,更是参与和调控了蛋白质的各种生理功能。研究蛋白质的C末端不仅有利于完整蛋白质的鉴定,对于在分子水平理解蛋白质的信号传导和生化功能是十分必要的。文中结合我们的研究工作,综述了近年来基于生物质谱的蛋白质C末端研究的相关进展,包括了C末端的识别、鉴定以及蛋白质C末端肽段富集的新方法和新技术。     

Pore positioning: Current concepts in Pannexin channel trafficking

Pannexins (Panxs) are a multifaceted family of ion and metabolite channels that play key roles in a number of physiological and pathophysiological settings. These single membrane large-pore channels exhibit a variety of tissue, cell type, and subcellular distributions. The lifecycles of Panxs are complex, yet must be understood to accurately target these proteins for future therapeutic use. Here we review the basics of Panx function and localization, and then analyze the recent advances in knowledge regarding Panx trafficking. We examine several intrinsic features of Panxs including specific post-translational modifications, the divergent C-termini, and oligomerization, all of which contribute to Panx anterograde transport pathways. Further, we examine the potential influence of extrinsic factors, such as protein-protein interactions, on Panx trafficking. Finally, we highlight what is currently known with respect to Panx internalization and retrograde transport, and present new data illustrating Panx1 internalization following an activating stimulus.     

Active site closure facilitates juxtaposition of reactant atoms for initiation of catalysis by human dUTPase

Human dUTPase, essential for DNA integrity, is an important survival factor for cancer cells. We determined the crystal structure of the enzyme:alpha,beta-imino-dUTP:Mg complex and performed equilibrium binding experiments in solution. Ordering of the C-terminus upon the active site induces close juxtaposition of the incoming nucleophile attacker water oxygen and the alpha-phosphorus of the substrate, decreasing their distance below the van der Waals limit. Complex interactions of the C-terminus with both substrate and product were observed via a specifically designed tryptophan sensor, suitable for further detailed kinetic and ligand binding studies. Results explain the key functional role of the C-terminus.     

Mutations in the hydrophobic core of ubiquitin differentially affect its recognition by receptor proteins

Ubiquitin (Ub) is one of the most highly conserved signaling proteins in eukaryotes. In carrying out its myriad functions, Ub conjugated to substrate proteins interacts with dozens of receptor proteins that link the Ub signal to various biological outcomes. Here we report mutations in conserved residues of Ub's hydrophobic core that have surprisingly potent and specific effects on molecular recognition. Mutant Ubs bind tightly to the Ub-associated domain of the receptor proteins Rad23 and hHR23A but fail to bind the Ub-interacting motif present in the receptors Rpn10 and S5a. Moreover, chains assembled on target substrates with mutant Ubs are unable to support substrate degradation by the proteasome in vitro or sustain viability of yeast cells. The mutations have relatively little effect on Ub's overall structure but reduce its rigidity and cause a slight displacement of the C-terminal β-sheet, thereby compromising association with Ub-interacting motif but not with Ub-associated domains. These studies emphasize an unexpected role for Ub's core in molecular recognition and suggest that the diversity of protein-protein interactions in which Ub engages placed enormous constraints on its evolvability.