Evolution in the structure and function of carboxyl proteases

Summary A model for the structure and function of extracellular carboxyl (acid) proteases can be established from three amino acid sequences and four crystal structures of these enzymes. The carboxyl proteases from gastric and fungal origins are very homologous in both primary and tertiary structures. The molecules consist of about 320 residues organized with a secondary structure which is primarily comprised of -strands and very similar tertiary structures. An apparent binding cleft, which can accommodate a substrate with about eight amino acid residues, contains near its midpoint the active center residues Asp-215, Asp-32, and Ser-35. These three residues are hydrogen bonded to each other.An intracellular carboxyl protease, cathepsin D, is very homologous to the extracellular enzymes in N-terminal amino acid sequence and primary structure location of active center residues. The tertiary structure of cathepsin D is probably similar, as well. However, cathepsin D contains a unique hydrophobic tail made up of about 100 residues added on the C-terminal side. Cathepsin D precursor is over 100,000 daltons in molecular weights, as contrasted to the gastric carboxyl protease zymogens, which are about 40,000 daltons.     

Photochemical activation of Rana pipiens tyrosinase

Purified tyrosinase from $\text{Rana pipiens}$ is activated by light. An action spectrum for the process indicates that there are two absorption bands responsible for the activation (290nm and 334nm). The kinetics of the photochemical process show an initial activation followed by inhibition. Molecular oxygen is required. The ability of the protein to be photoactivated and the absorbancy of the protein at 334nm can be extracted with 50% acetone/water.     

Mechanism of intramolecular activation of pepsinogen. Evidence for an intermediate delta and the involvement of the active site of pepsin in the intramolecular activation of pepsinogen.

Intramolecular pepsinogen activation is inhibited either by pepstatin, a potent pepsin inhibitor, or by purified globin from hemoglobin, a good pepsin substrate. Also, pepsinogen at pH 2 can be bound to a pepstatin-Sepharose column and recovered as native zymogen upon elution in pH 8 buffer. Kinetic studies of the globin inhibition of pepsinogen activation show that globin binds to a pepsinogen intermediate. This interaction gives rise to competitive inhibition of intramolecular pepsinogen activation. The evidence presented in this paper suggests that pepsinogen is converted rapidly upon acidification to the pepsinogen intermediate delta. In the absence of an inhibitor, the intermediate undergoes conformational change to bind the activation peptide portion of this same pepsinogen molecule in the active center to form an intramolecular enzyme-substrate complex (intermediate theta). This is followed by the intramolecular hydrolysis of the peptide bond between residues 44 and 45 of the pepsinogen molecule and the dissociation of the activation peptide from the pepsin. Intermediate delta apparently does not activate another pepsinogen molecule via an intermolecular process. Neither does intermediate delta hydrolyze globin substrate.     

以结核分枝杆菌丙氨酸消旋酶为靶点的抗结核药物筛选及其与D-环丝氨酸的共结晶

【目的】阐释结核分枝杆菌丙氨酸消旋酶原有抑制剂D-环丝氨酸的作用机制,建立丙氨酸消旋酶抑制剂的高通量筛选模型,并用此模型筛选到新的抑制剂分子。【方法】将结核分枝杆菌中丙氨酸消旋酶基因克隆到pET-28a表达载体中,在大肠杆菌BL21(DE3)菌株中得到可溶性的大量表达。表达后的蛋白质通过镍离子亲和层析和阴离子交换层析得到纯化,一方面将纯化后的蛋白和D-环丝氨酸共结晶以解析其抑制剂作用的分子机制。另一方面建立并优化丙氨酸消旋酶抑制剂的高通量筛选体系,用D-环丝氨酸验证体系的可行性并用该体系筛选本实验室药物库中的384种小分子片段、792种化合物及2 200种中药样品。【结果】得到的共晶晶体衍射能力2.50?,晶体空间群为P41212,晶胞参数a=b=163.92?,c=57.44?。结构分析表明,D-环丝氨酸进入活性位点之后与磷酸吡哆醛相互作用形成磷酸吡哆胺,使得磷酸吡哆醛的C4?原子与K42之间的相互作用被破坏,从而改变了结核分枝杆菌丙氨酸消旋酶的活性中心氢键网络。同时经D-环丝氨酸验证建立的抑制剂筛选体系可行,获得阳性化合物分子2个。【结论】依据我们所建立的高通量筛选体系可以有效地为结核分枝杆菌丙氨酸消旋酶筛选到可信的抑制剂分子。     

The main driver of soil organic carbon differs greatly between topsoil and subsoil in a grazing steppe

Soil organic carbon (SOC) dynamics is regulated by a complex interplay of factors such as climate and potential anthropogenic activities. Livestocks play a key role in regulating the C cycle in grasslands. However, the interrelationship between SOC and these drivers remains unclear at different soil layers, and their potential relationships network have rarely been quantitatively assessed. Here, we completed a six‐year manipulation experiment of grazing exclusion (no grazing: NG) and increasing grazing intensity (light grazing: LG, medium grazing: MG, heavy grazing: HG). We tested light fraction organic carbon (LFOC) and heavy fraction organic carbon (HFOC) in 12 plots along grazing intensity in three soil layers (topsoil: 0–10 cm, mid‐soil: 10–30 cm, subsoil: 30–50 cm) to assess the drivers of SOC. Grazing significantly reduced SOC of the soil profile, but with significant depth and time dependencies. (1) SOC and SOC stability of the topsoil is primarily regulated by grazing duration (years). Specifically, grazing duration and grazing intensity increased the SOC lability of topsoil due to an increase in LFOC. (2) Grazing intensity was the major factor affecting the mid‐soil SOC dynamics, among which MG had significantly lower SOC than did NG. (3) Subsoil organic carbon dynamics were mainly regulated by climatic factors. The increase in mean annual temperature (MAT) may have promoted the turnover of LFOC to HFOC in the subsoil. Synthesis and applications. When evaluating the impacts of grazing on soil organic fraction, we need to consider the differences in sampling depth and the duration of grazing years. Our results highlight that the key factors influencing SOC dynamics differ among soil layers. Climatic and grazing factors have different roles in determining SOC in each soil layer.     

Oxidative stress induces meiotic defects of oocytes in a mouse psoriasis model

Psoriasis, an immune-mediated inflammatory disease, is associated with poor pregnancy outcomes. Emerging evidence indicates that these defects are likely attributed to compromised oocyte competence. Nevertheless, little is known about the underlying associated mechanisms between psoriasis and poor oocyte quality. In this study, we construct an imiquimod-induced chronic psoriasis-like mouse model to review the effects of psoriasis on oocyte quality. We discover that oocytes from psoriasis-like mice display spindle/chromosome disorganization, kinetochore-microtubule mis-attachment, and aneuploidy. Importantly, our results show that melatonin supplement in vitro and in vivo not only increases the rate of matured oocytes but also significantly attenuates oxidative stress and meiotic defects by restoring mitochondrial function in oocytes from psoriasis-like mice. Altogether, our data uncover the adverse effects of psoriasis symptoms on oocytes, and melatonin supplement ameliorates oxidative stress and meiotic defects of oocytes from psoriatic mice.Subject terms: Meiosis, Immunology     

Single-cell transcriptomic atlas of primate cardiopulmonary aging

Aging is a major risk factor for many diseases,especially in highly prevalent cardiopulmonary comorbidities and infectious diseases including Coronavirus Diseas...